Developing an insect-nematode Drosophila- Abbreviata

hastaspicula model to study the effects of two natural

compounds on anti-aging

Chloe King

BSc

The Marshall Centre for Infectious Diseases Research and Training

School of Pathology and Laboratory Medicine

The University of Western Australia

This thesis is presented for the degree of Doctor of Philosophy of

The University of Western Australia

2017

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SUMMARY

The nematode genus Abbreviata (Spirurida: Physalopteridae: Physalopterinae) is the

predominant gastric metazoan parasite in Australian reptiles and requires an arthropod

intermediate host to complete its life-cycle. Only one life cycle of Abbreviata is known

from Australia. Genetic and environmental regulations can postpone aging-related

degenerative changes in lifespan and health span. Pharmacological and nutraceutical

regulation may result in a profoundly extended lifespan, slow aging, and help to prevent

degenerative diseases such as osteoporosis, osteoarthritis and Alzheimer. The four

papers that comprise this thesis attempt to find out whether intervention of a natural

compound (resveratrol analogues and gastrodin) can control senescence in higher

organisms using a new insect-nematode model. Upon understanding the biology of

Abbreviata hastaspicula, a new aging model of A. hastapicula (spirurid nematodes) is

established in addition to the traditional model system of fruit fly Drosophila

melanogaster and Drosophila simulans (one of the most studied organisms in aging

research).

In order to elucidate the complete life cycle of Abbreviata hastaspicula, it is important

to determine the obligatory arthropod intermediate host. Therefore, in the first study, I

examined whether Coptotermes acinaciformis (Isoptera: Rhinotermitidae), the most

widely distributed termite species in Australia, is the intermediate host for the

nematodes A. hastaspicula and A. antarctica. The results showed that C. acinaciformis

is not a potential intermediate host for either Abbreviata spp. In the second study, I

elucidated the life-cycle of Abbreviata hastapicula in its final hosts. I dewormed and

experimentally infected the Varanus gouldii lizards that were captured from the wild.

Accordingly, adults of Abbreviata hastapicula were found in the stomachs of the

experimental V.gouldii. The infection rate of the experimental final host in this

experiment was 100% although no cysts or larva were found in the experimental

paratenic host Christinus marmoratus. This outcome has provided a considerable piece

of information on the biology of this species of nematode.

In the third study, I followed up the biology finding of A. hastapicula in terms of

phylogenetic background. I utilized molecular methods to investigate the genetic

knowledge of nematodes A. hastaspicula and A. antarctica. The present phylogenetic

understanding of this genus of nematodes is poor. The 18S ribosomal DNA was

sequenced based on the available Physalopterinae strains in NCBI genome database.

The novel result of this study contributed a considerable understanding to the evolution

of this group of nematodes.

Finally, in my last study, I developed a new insect-nematode model (Drosophila

melanogaster/ Drosophila simulan- Abbreviata hastapicula) to examine the effect of

resveratrol analogues and gastrodin on delaying aging. Capillary Feeder (CAFE) assay

and drug supplementation methods were used in three life stages (health span, transition

span and senescence span) of Drosophila spp. flies. Both Drosophia flies and adult A.

hastapicula showed an increase in life expectancy. These findings suggested that the

natural compounds I tested in this study slowed senescence.

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TABLE OF CONTENTS

SUMMARY i

TABLE OF CONTENTS ii

ACKNOWLEDGEMENTS iv

DECLARATION OF CONTRIBUTION v

CHAPTER ONE: GENERAL INTRODUCTION 1

1.1 Drosophila as a testing model 2

1.2 Abbreviata hastaspicula as a testing model 3

1.3 Thesis approach and aim 5

CHAPTER TWO: IF THE TERMITE COPTOTERMES ACINACIFORMIS

(BLATTODEA: ISOPTERA: RHINOTERMITIDAE) IS NOT A POTENTIAL

INTERMEDIATE HOST FOR THE NEMATODES ABBREVIATA

HASTASPICULA AND ABBREVIATA ANTARCTICA (SPIRURIDA:

PHYSALOPTERIDAE)? 9

2.1 Abstract 10

2.2 Introduction 11

2.3 Materials and Methods 12

2.4 Results 14

2.5 Discussion 15

CHAPTER THREE: THE LIFE CYCLE OF THE REPTILE-INHABITING

NEMATODE ABBREVIATA HASTASPICULA (SPIRURIDA:

PHYSALOPTERIDAE: PHYSALOPTERINAE) IN AUSTRALIA 22

3.1 Abstract 23

3.2 Introduction 24

3.3 Materials and Methods 25

3.4 Results 27

3.5 Discussion 29

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CHAPTER FOUR: MOLECULAR SEQUENCING OF THE NEMATODES

ABBREVIATA HASTASPICULA AND ABBREVIATA ANTARCTICA

(SPIRURIDA: PHYSALOPTERIDAE) FROM AUSTRALIA 40

4.1 Abstract 41

4.2 Introduction 42

4.3 Materials and Methods 43

4.4 Results and Discussion 46

CHAPTER FIVE: ANTI-AGING EFFECTS OF RESVERATROL ANALOGUES

AND GASTRODIN USING A NEW DROSOPHILA –ABBREVIATA

HASTASPICULA (NEMATODA) MODEL 58

5.1 Abstract 59

5.2 Introduction 60

5.3 Materials and Methods 66

5.4 Results 69

2.5 Discussion 72

CHAPTER SIX: GENERAL DISCUSSION 87

6.1 Abstract 88

6.2 Is the termite Coptotermes acinaciformis a potential host? 89

6.3 The life cycle of the reptile-inhabiting nematode Abbreviata hastaspicula 90

6.4 Molecular sequencing of Abbreviata hastaspicula and Abbreviata antarctica 91

6.5 Anti-aging effects of resveratrol analogues and gastrodin 94

6.6 Concluding Remarks 95

REFERENCES 97

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ACKNOWLEDGEMENTS

I would first like to thank my supervisors Jiake Xu, Hugh Jones and Alfred Tay. They

provided me with a plethora of advice on how to think, not just the science and

technical aspects but everything, their unstinting support has made a difference in my

life. Jiake, thank you for being my coordinating supervisor, this thesis was made

possible owing to your support. Hugh, thanks for your guidance throughout this

constant challenging PhD journey. Alfred, you really are Jack of all trades, thank you

for being there whenever I am in need. I am truly blessed to have three amazing

supervisors; I thank you all from the bottom of my heart.

I would also like to thank the School of Animal Biology, for providing housing for the

lizards, our collaborator, Jason Kennington for providing the Drosophila flies,

laboratory facility and equipment, Laura Travers and Robert Dugand for sharing

knowledge on caring for flies, Maxine Beveridge and Leigh Simmons who made the

crickets and laboratory facility available, Rick Roberts, Husnan Ziadi and Nicolas

Nagloo for catching the lizards from the wild. I would like to particularly thank Rick

Roberts for his technical support along the way, without his help; this research could not

have been carried out.

I am very grateful to Geoff Richardson who provided the laboratory termites and made

the laboratory facility available. Geoff, thank you for being so nice and helping me

unstintingly. I thank Cathy Lambert for showing me the termite harvesting methods,

Mao Zhu for assisting the collection of termites and caring for flies, Marty Firth for the

statistical analysis.

Last but not least, I would like to extend my acknowledgements to everyone who helped

make this thesis a success, whether their names are mentioned above or not.

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DECLARATION OF CONTRIBUTION

This thesis is submitted as a series of discrete manuscripts, it contains work under

review and work prepared for publication. This declaration is to clarify the contribution

of the student to the work.

I. King. C., Jones, H. and Tay, A. (2016).If termite Coptotermes acinaciformis

is not a potential intermediate host for the nematodes could it be a safe food

source for humans? Accepted by Journal of Insect as Food and Feed.

II. King. C., Jones, H. (2016).The life cycle of the reptile-inhabiting nematode

Abbreviata hastaspicula (Spirurida: Physalopteridae: Physalopterinae) in

Australia. International Journal for Parasitoogy: Parasites and Wildlife,

5(3), 258-262.

III. Molecular sequencing of the nematodes Abbreviata hastaspicula and

Abbreviata antarctica (Spirurida: Physalopteridae) from Australia. Under

review by Parasitology International.

IV. Anti-aging effects of resveratrol analogues and gastrodin using a new

Drosophila –Abbreviata hastapicula (Nematoda) model. Prepared for

publication.

All manuscripts were written in collaboration between Chloe King (CK) and her

supervisors Jiake Xu (JX), Hugh Jones (HJ) and Alfred Tay (AT). For each experiment,

Chloe King was the main contributor and responsible for experimental design,

laboratory work, data analysis and manuscript writing. Hugh Jones, Jiake Xu and Alfred

Tay contributed to experimental design, data analysis and manuscript revision. Author

contributions to manuscript I: CK= 80%, HJ= 10%, AT=10%, manuscript II: CK=

90%, HJ= 10%, manuscript III: CK=80%, AT=10%, HJ=10%, manuscript IV:

CK=90%, JX=10%.

All authors have given permission for all work to be included in this thesis.

------------------ ------------------- -------------------- -----------------

Chloe King Jiake Xu Hugh Jones Alfred Tay

(Candidate) (Supervisor) (Supervisor) (Supervisor)

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CHAPTER ONE

General Introduction

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1.1 DROSOPHILA AS A TESTING MODEL

The common fruit fly, Drosophila spp. is one of the most studied organisms in

biological research. This species possesses a number of attributes that make it an

attractive model for aging research. Drosophila is a convenient model organism in the

laboratory because it has a rapid lifecycle; it is easy to culture, economical to maintain

and reproduce in high numbers. They can be anesthetised by carbon dioxide readily

with simple equipment, providing a quick way for drug screening compared with

traditional mammal-based models. D. melanogaster was the first major complex

organism to have its genome completely sequenced and annotated after Nobel Prize

winner Ed Lewis’s pioneering research in defining its gene structure (Adams 2000).

Studies found that 77% of disease related genes in human have highly similar cognates

in D. melanogaster. Genetic analysis showed that 714 of the 929 distant human

pathologic genes matched the 548 unique D. melanogaster sequences, and of the 548

fruit fly genes associated to human diseases, 153 are allied with known mutant alletes

and an additional 56 are tagged by P-element insertions. This discovery suggests that

this gene appears to be an excellent candidate for study (Reiter et al. 2001). The species

of fruit fly we use in this thesis, D. simulans, is very closely related to D. melanogaster

except for a deviation of male external genitalia and the absence of segregating

chromosomal inversions. Unlike D. melanogaster, its complete genome has not yet

been fully sequenced (Ballard 2005; Garrigan et al. 2012; Palmieri et al. 2015).

Drosophila spp. fruit flies differ from many other insects; their aging process is the

same as in humans, gradually degenerating despite the fact that the post-reproductive

lifespan of Drosophila is a lot shorter than that of humans. The fly has four distinct

morphological developmental stages: eggs, larva, pupa and adult. Although each

developmental phase could be used as a model to investigate physiological process and

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behaviours, the sophisticated structure of an adult Drosophila and its manifestation of

aging changes are very useful in understanding the basic mechanism of human aging

(Conn 2006). Since there is no consensus on the biomarkers of physiological and

neurodegenerative aging, it is therefore unambiguous for me to measure the rate of

aging by analysing the age-at death pattern. Age-specific demographic studies on

mortality demand large population sizes, particularly when weare estimating the

maximum lifespan. Drosophila spp. flies are easy to culture and simple to monitor in a

large number, therefore it is an ideal model for my demographic aging analyses and

high throughput drug screening. The well-developed accessible genetic tools in

Drosophila for molecular demographic and biochemical studies (Paaby & Schmidt 2009)

allow the fly to be a versatile candidate for genetic expression (expressed, under-

expressed, overexpressed or deleted) in an inexpensive and timely manner compared

with mammalian testing system (Pandey & Nichols 2011).

1.2 ABBREVIATA HASTASPICULA AS A TESTING MODEL

Abbreviata hastaspicula (Spirurida: Physalopteridae: Physalopterinae) is the

predominant parasitic gastro-intestinal nematode in the Australian lizards in the genus

Varanus (Lacertilia: Varanidae). It is a zoonotic parasite and humans are the accidental

hosts. Morphologically, A. hastaspicula is a white cylindrical worm with a cervical

collarette, elongated in shape, tapered at each end, bilaterally symmetrical and

possessing a pseudocoel. Females are longer, stouter and slightly more numerous than

males (details refer to Chapter 3 of this thesis). It has a pharynx, a nerve ring, and

complete digestive and reproductive systems. Its body is covered with a non-cellular

cuticle which is secreted by an underlying hypodermis and is shed four times (four

larval stages) during ontogeny. The muscles of a nematode’s body wall are only one

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layer thick and they are all longitudinally arranged with no separate circular layer. It has

different cell types which have similar organization to that of vertebrates (Lee &

Ogilvie 2002). The life cycle of physalopterid nematodes consists of six stages

involving an egg, four larval stages (or juvenile stages) and lastly, adult. They are

infective from the beginning of the third larval stage (Anderson 2006). Ingestion of

arthropod intermediate hosts is essential for transmission. Its lifecycle can only be

completed by the final host (larger lizards) feeding on insects (intermediate host) or

paratentic hosts (smaller lizards) containing infective third stage larvae. However, in

paratenic hosts, the infective stage of a parasite persists without essential development

and usually lack of growth (Anderson 2006) (details of its life-cycle refer to Chapter 3).

Nematodes, like humans and all multicellular organisms and probably all living things,

mature, then grow old, and eventually die. I use A. hastaspicula as a model to test

whether the natural compounds resveratrol analogue and gastrodin can retard aging

because firstly, this nematode has a heteroxenous life-cycle. Only certain species of

arthropods have the potential to be its intermediate host (the full species list of potential

arthropods is not known yet) and until the ‘right’ arthropod ingests its eggs, A.

hastaspicula will not develop. I am intrigued about the developmental mechanism in the

nematode that allows it to switch between growing and not growing. If humans can

have the same ability to ‘press the button’ whenever we want to alter our developmental

mechanism, can we solve the problem of getting age- associated illnesses when we

grow old? Can we stop going along a pathological pathway when we age? A.

hastaspicula can persist in the abdominal tissues of tail-regenerating paratenic gecko

hosts, and it does not mature to adult until it is ingested by the final hosts, larger lizards.

Although the regenerated tails of the geckos are not exactly the same as the original one

(Grismer & Chan 2010), if A. hastaspicula can live inside the gecko as a third-stage

larva until or unless ingested by a final host then does A. hastaspicula share the gene

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that facilitates tissue regeneration in geckos? If it does, can A. hastaspicula regenerate

itself if the appropriate drug compounds are given to it? In addition, A. hastaspicula

does not mature after being encysted in the tissues the paratenic gecko (similar to the

facultative diapausal form of Caenorhabditis elegans third stage larvae). So what is the

secret of not maturing and delaying aging? What mechanism prevents A. hastaspicula

from growing old? What are the physiological determinants underlying this plasticity of

maturing? If it has the ability to accidentally develop in humans, is it possible that it can

harmonize with the human aging process? Therefore investigating how it ages may

throw light on the basic mechanism of human fundamental aging. I want to know

whether, with all these unusual characters that A. hastaspicula possesses, certain natural

compounds can postpone its ageing and increase its health span?

1.3 THESIS APPROACH AND AIM

Resveratrol and gastrodin are both natural phenols. The beneficial biological properties

of these two natural compounds have been extensively studied both in vitro and in vivo.

They may have the potential to make a positive impact on human health and life

expectancy. Studies using resveratrol to extend lifespan have been particularly fruitful

despite emerging evidences showing that its pro-aging result is in fact determined by

dietary composition. Not many studies have concentrated on the anti-aging effect of

gastrodin, while its pharmacological properties such as antioxidative, anticonvulsant,

neuroprotective, sedative, analgesic, and immunomodulatory etc. have received

widespread attention. The present thesis aimed to find out whether intervention of

resveratrol analogues and herbal gastrodin can control senescence in higher organisms

using our new insect-nematode, Drosophila-Abbreviata hastapicula model. It is hoped

that my research can help to prevent aging-associated degenerative diseases such as

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osteoporosis, osteoarthritis and Alzheimer’s disease etc. This research thesis is

presented in the form of a series of papers; the main focus of each paper is as follows:

1. In order to use Abbreviata hastaspicula as a testing model for anti-aging, it is

vitally important for us to better understand the biology of this nematode by

first of all, identifying its essential arthropod intermediate host. In Chapter

Two, I examine whether Coptotermes acinaciformis (Isoptera:

Rhinotermitidae), the most widely distributed termite species in Australia, is

the intermediate host for the nematodes A. hastaspicula and A. antarctica. I

exposed a total of 13,500 laboratory bred native C. acinaciformis to the

infected faeces of Varanus gouldii (Squamata: Lacertilia: Varanidae) that

contained embryonated eggs of the nematodes A. hastaspicula and A.

antarctica. I divided the termites into 9 groups to find out under what

environmental conditions the termites would ingest the dung and what kind of

dung would attract most termites. A total of 11,699 termites were dissected at

6 intervals and were observed under dissecting microscope to determine

whether they were the potential intermediate hosts. Statistical computing and

graphics were also utilized to estimate the statistical significance of our

findings.

2. Next, I elucidated the life-cycle of the reptile inhabiting nematode Abbreviata

hastaspicula (Spirurida: Physalopteridae) in its final hosts in Australia. In

Chapter Three, eight Varanus gouldii (Lacertilia: Varanidae), and two

Christinus marmoratus (Reptilia: Gekkonidae) lizards were captured in the

wild. The six wild-caught V. gouldii and two C. marmoratus were dewormed.

After ascertaining that the V. gouldii were free from nematodes, I

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experimentally re-infected the four uninfected Varanus and two C.

marmoratus by feeding them with infected live Teleogryllus oceanicus

(Orthoptera: Ensifera: Gryllidae) crickets for 48 hours. Another two

uninfected V. gouldii were fed with the two C. marmoratus that had consumed

T. oceanicus. The remaining two untreated V. gouldii were used as controls

because I wanted to ensure that the nematodes in the experimental lizards

were the same as the species of nematodes that occurred in these lizards in the

wild. From the time I fed the infected crickets to the uninfected lizards to the

time of dissection, it took two months. Adult A. hastaspicula found in the

stomach of V. gouldii were examined under X4, X10 and X20 objectives.

Images of stained histological sections of stomach of V. gouldii with adult A.

hastaspicula and morphological images of both male and female A.

hastaspicula were obtained.

3. Having elucidated the postulated life-cycle of A. hastaspicula in its final hosts

and paratenic hosts, in Chapter Four, I used molecular methods to follow the

phylogenetic background of A. hastaspicula. After the nematodes had been

identified morphologically, I extracted and homogenized the DNA of A.

hastaspicula. Physalopterinae specific 18S rRNA primers were designed

based on the alignment of the published Physalopterinae 18S rRNA from the

National Center for Biotechnology Information (NCBI). The sequencing work

undertaken in the study was novel.

4. Finally, in Chapter Five, upon better understanding the biology and

phylogeny of A. hastaspicula, I developed the insect-nematode model

(Drosophila melanogaster/ Drosophila simulan- Abbreviata hastaspicula). In

this study, I investigated whether resveratrol analogues (Gu) and herbal

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gastrodin (GAS) have anti-aging capacities, and whether the aging process can

be experimentally retarded using our insect-nematode model. Wild type D.

melanogaster and D. simulans were kept at a density of 20 flies per vial. The

extract of gastrodin was supplemented to individual vial containing molten

media at a final concentration of 0.5% by weight/volume. The Capillary

Feeder (CAFE) assay was applied to 0.1M, 0.3M, 0.5M and 0.7M. While for

resveratrol analogues, the CAFE method was used for concentration 0.3M,

0.5M and 0.7M. To find out whether the natural compounds have age-specific

effects on health and lifespan, the flies were divided into three stages (3-30

days, 31-60 days and 61 days-die). I used in total 16,700 and 6,720 flies to test

gastrodin and resveratrol analogues respectively. I maintained the 75 adult

nematodes A. hastapicula (immediately removed from a dissected lizard) in

5mL hydrochloric acid and evenly divided them into three groups. Gu and

GAS powders were added to the hydrochloric acid solution to a final

concentration of 2% of weight/volume. Mantel-Cox log rank test and

likelihood ratio test were used to analyse the survivorship data. Maximum

lifespan was calculated using the 10% longest surviving flies and nematodes

of mean lifespan of a population. P-values were considered in relation to the

no drug controls.

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CHAPTER TWO

If the termite Coptotermes acinaciformis (Blattodea: Isoptera:

Rhinotermitidae) is not a potential intermediate host for the

nematodes Abbreviata hastaspicula and Abbreviata antarctica

(Spirurida: Physalopteridae)?

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2.1 ABSTRACT

This study examines whether Coptotermes acinaciformis (Isoptera: Rhinotermitidae) is

the intermediate host for the nematodes Abbreviata hastaspicula and Abbreviata

antarctica by exposing a total of 13,500 C. acinaciformis to the infected faeces of

Varanus gouldii (Squamata: Lacertilia: Varanidae) that contained embryonated eggs of

the nematodes A. hastaspicula and A.antarctica. The termites were dissected at 6

intervals (1 day, 2 day, 3 days, 4-23 days, 25-38 days and 48-53 days). Eggs of the

nematodes were recovered in the mid-guts of the termites during the first three intervals.

There was no evidence that eggs hatched within the termites. No eggs or larvae were

found in the last three intervals. We conclude therefore that C. acinaciformis is not a

potential intermediate host for either species of Abbreviata.

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2.2 INTRODUCTION

The termite Coptotermes acinaciformis is the most widely distributed species of termite

in Australia (Lee et al. 2015). Termites are closely related to cockroaches, occurring

mainly in tropical and subtropical regions. Recent research has indicated that termites

are most likely the sister group of the Cryptocercidae (woodroaches) (Rasplus &

Roques 2010). Not only are they notorious pests destroying buildings and structures (Su

& Scheffrahn 2000), but they are also keystone engineers in ecosystems as they are

active members of the soil microfauna (Abensperg 1994; Bignell 2006). They play a

critical functional role in affecting soil processes in deserts by increasing soil porosity

and infiltration, and their removal can result in a series of changes in the structural and

functional integrity of deserts (Anderson 2009). The African Hodotermes pretoriensis

termite is the intermediate host for the spirurid nematode Hartertia gallinarum (Theiler

1919). Termites may be involved in the transmission of third-stage physalopterid larvae

to the paratenic or final reptile hosts, principally the geckoes Diplodactylus

conspicillatus and Rhynchoedura ornata. Jones (1995a) showed a positive correlation

between termites in the diet of lizards and the prevalence of cysts containing larval

nematodes. This apparent relationship was strongest in smaller lizards that feed

exclusively on termites and hence he adduced that termites may act as an intermediate

hosts for the larvae of species of Abbreviata (Jones 1995a). Reptile-inhabiting

nematodes in the genus Abbreviata (Physalopteridae) require an arthropod intermediate

host to complete their life cycles. Abbreviata antarctica has been shown to develop in

Australian tropical crickets, Telelogryllus oceanicus (King et al. 2013). Since termites

are particularly abundant in arid and semi-arid Australia where lizards are the principal

termite eaters (Abensperg 1994) and the genus Abbreviata is predominant in terrestrial

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Varanus of Australia (Jones 2005), this study investigates whether C. acinaciformis

could be a potential intermediate host for the nematodes A. hastaspicula and A.

antarctica.

2.3 MATERIALS AND METHODS

Feeding termites the infected faeces

Termites were collected from tanks connected to the laboratory mound by gently

placing a lightweight dampened slat of wood on top of the soil and dirt. After the

termites congregated on the underside of the slat, they were tapped off directly into the

experimental containers (Lambert & Power, 1999).

Laboratory bred native termites Coptotermes acinaciformis (Rhinotermitidae) (provided

by TMA Corporation Pty. Ltd.) were divided into 9 groups: natural wet faeces (nw),

natural wet faeces with wood (nww), natural dry faeces (nd) , natural dry faeces with

wood (ndw), dry faeces (d), dry faeces with wood (dw), wet faeces (w), wet faeces with

wood (ww) and control (c). Natural dry faeces were faeces defecated by Varanus gouldii

(Squamata: Lacertilia: Varanidae) infected with the nematodes Abbreviata hastaspicula

and Abbreviata antarctica. Natural wet faeces were faeces removed from the colon of a

V. gouldii which was just dissected (UWA Animal Ethics Ref. RA/3/100/1248). Dry and

wet faeces were faeces of Varanus that contained the eggs removed from the uteri of

thirty female A. hastaspicula and A. antarctica manually, as shown in fig.1A & B, there

were large amount of eggs in the uterus Hummock grass (Triodia spp.) (Collected from

John Forrest National Park, Western Australia) was placed in the containers of the wood

groups to determine whether the termites would consume the faeces in the presence of

wood. Experimental termites were exposed to the faeces that contained viable larvated

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eggs of both Abbreviata spp. for 48 hrs. Each group was held in an individual container

(10cm Length x 3.5cm Width x 3cm Height). Approximately 11g of infected faeces was

placed in each container. The number of eggs in the natural faeces defecated by the

Varanus gouldii was about 2 eggs/ mg. The estimated total number of eggs in infected

faeces was 2000 eggs/g.

After each C. acinaciformis termite group was transferred to a new container (20cm

Length x 20cm width x 9cm height) with a 50mL test-tube filled with water and

plugged with cotton balls (water drinking device for termites). A thermo hydrometer

was placed in each container to monitor the humidity and temperature of the container.

Soil and a small jarrah wood piece (10cm length x 3.5cm width x 3cm height) were

given to each termite group to keep them alive as long as possible. They were reared at

60-80% RH and 30-33 °C laboratory temperature. About 1,000 C. acinaciformis (5 g)

were maintained in each container, approximately 9,000 termites in total.

This experiment was replicated on a smaller scale, at indoor room temperature, 24-

28 °C and 75-85% RH. Around 500 termites (2.5 g) were kept in each container (7.5cm

Length x 20cm Width x 9cm Height), 4,500 termites in total were tested.

Dissecting termites

A total of 11,699 Coptotermes acinaciformis were dissected at 6 intervals, 1day, 2 days,

3 days, 4-23 days, 25-38 days and 48-53days. Interval 1-5: The exoskeleton (outer

cuticle) of a termite was peeled off using two pointed forceps; the exposed alimentary

canal was mixed with normal saline solution and observed under X4, X10 and X20

objectives (King et al. 2013). At interval 6: all the termites were examined under a

dissecting microscope as described above and one in every ten termites were observed

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under compound microscope. A total of 11,699 C. acinaciformis were dissected in this

study.

Statistical significance testing

Software R version 3.2.0 was used to perform statistical computing and graphics. All p-

values from the termites were generated from Fisher's exact test, including Benjamini &

Hochberg multiple testing correction. P ≤ 0.05 was considered statistically significant.

Prevalence was defined as the total number of eggs that have been found in the nine

different experimental groups at the given time period (interval one to three). It showed

the likelihood of termites having eggs found in their guts.

2.4 RESULTS

Laboratory bred C. acinaciformis in all 9 groups had ingested faeces whether or not

Hummock grasses were present in the container (fig. 2A). The laboratory termites had a

very white and clean appearance, and once they had ingested the faeces, dark colour

could be observed in their abdomens. Termites had built firm nests and tunnels using

the soil provided in the containers as soon as two days after their exposure to the faeces.

Eggs found at different intervals

Fifty six eggs in total were found at intervals1-3. Twenty seven termites had eggs of A.

hastaspicula or A. antarctica recovered at interval 1, 13 termites at interval 2 and 2

termites at interval 3. Five termites had both species of Abbreviata eggs recovered (fig.

2B). Twenty one were soldier caste termites and 33 were worker termites had ingested

infective eggs. Three A. hastaspicula eggs were found in soldier termites and 13 were

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found in workers (fig. 3A). Nineteen and 21 eggs of A. antarctica eggs found in soldiers

and workers respectively. Thirty seven eggs were found at interval 1, 15 eggs at interval

2 and 2 eggs at interval 3. In total, 40 A. antarctica eggs and 16 A. hastaspicula eggs

were recovered from the hind-guts of the termites (fig. 3B). The proportion of eggs

found compared to termites numbers for interval 1-3 were 10.19%, 4.16% and 0.60%.

10,642 termites were dissected in total from interval 4 to interval 6 (604 termites at

interval 4, 754 termites at interval 5 and 9,284 termites at interval 6). Small numbers of

unidentified larvae of other species of nematode were recovered. No larvae of

Abbreviata spp. were found. TABLE 1 shows the prevalence of both Abbreviata spp.

eggs in different groups from interval 1 to 3. Except from ndw group, eggs were found

in the hindguts of termites in dw, nw and ww groups in spite of the presence of

Hummock grass in their containers.

The eggs of A. antarctica and A. hastaspicula are similar, A. antarctica eggs are oval in

shape, 60 -65 x 30µm, have smooth and think shells, embryonated. On the other hand,

the eggs of A. hastaspicula are slightly less elongated 53-40 x 32 µm, smaller than those

of A. antarctica, and have conspicuously thinner shells, embryonated.

2.5 DISCUSSION

Coptotermes acinaciformis as a potential intermediate host

The P-value of dw (P ≤ 0.005), nw (P ≤ 0.004), and ww (P ≤ 0.005) were the most

significant (Table 1). This suggests that the greater the number of eggs in the faeces, the

greater the chance that termites would ingest them. The faeces in these 3 groups

contained more eggs because of the manner in which the faeces were prepared (as

mentioned above). In addition, the unexpectedly high prevalence of eggs found in dw

16

and ww group indicated that termites would still feed on the infected faeces of V gouldii,

despite an alternative, Hummock grass, being available. This is probably because C.

acinaciformis is interested in the chemical elements in the faeces. According to

Freymann et al. (2008), although termites had no distinctive preference for mammalian

dung, and dung feeding was only of opportunistic importance to termites, the nitrogen

and phosphorus levels of cattle dung (0.95% nitrogen, 1.06 % phosphorus) was much

more favourable than Andropogon straw (0.32% nitrogen, 0.03 % phosphorus) and

maize straw (0.77% nitrogen, 0.18% phosphorus).

No egg was found in ndw group, possibly because the dry faeces of V. gouldii were

relatively old and therefore lacked moisture. Thus if presented with Hummock grasses,

the termites prefer this to the lizard faeces. Possibly because the number of eggs in the

natural dry faeces was sparse, the chance of termites ingesting eggs in the faeces was

lower. Unlike the nd group, C. acinaciformis had no choice of feeding material, and

thus fed on faeces. In this case, termites might forage in the faeces to seek water rather

than fibre (Ferrar & Watson 1970).

Under normal circumstances foraging activities are carried out by worker castes. Korb,

(2007) explained why worker caste termites have a higher prevalence of eggs found in

their hind-guts than that of soldier termites. Worker termites consumed the faeces, then

regurgitated them from their mouth or passed from their anal areas to the soldier

termites (Hadlington 1996).

The proportion of eggs found compared to termite numbers diminished noticeably from

interval 1 to interval 3, and no egg of Abbreviata spp. was observed from interval 4

17

onward, suggesting that C. acinaciformis is not a suitable intermediate host for either A.

hastaspicula or A. antarctica.

Literature on termites involved in the lifecycle of parasites is sparse. Hodotermes

pretoriensis worker termites are an intermediate host for nematode Hartertia gallinarum

in South Africa (Theiler 1919). Schedorhinotermes termites fed to domestic poultry can

act as an intermediate hosts for eleven different species of parasitic worms in poultry

(Alenyorege et al. 2011). Acquisition of nematode Physaloptera infection in the

endemic frog Proceratoprhys boiei depended on the presence of a high quantity of

infected termites in their stomach (Klaion et al. 2011). Jones (1995a) found a high

prevalence rates of physalopterid larvae within the tissues of the Australian geckoes

Diplodactylus conspicillatus (92.6%), Rhynchoedura ornata (79.4%), and in Gehyra

variegata (41.9%), which feed exclusively on termites, suggested that termites may be

involved in the complete development of species of Abbreviata . Hence, it appears that

other species of termites may still play a vital role in the transmission of reptile

nematode Abbreviata spp. regardless of the negative findings of C. acinaciformis in the

present study. Research on identifying species of termite which might be the potential

intermediate hosts would add considerably to the understanding of desert ecology.

18

TABLE 1. The prevalence of eggs of A. hastapicula and A. antarctica eggs varied in

treatment groups in interval 1-3. P value is compared to control group (c).

Group

Total no. of

eggs recovered

(both species)

Total no. of

termites

dissected

Proportion of eggs

found compared to

termites numbers p-value

c 0 120 0.00%

d 6 117 5.13% 0.047

dw 10 119 8.40% 0.005

nd 4 112 3.57% 0.076

ndw 0 118 0.00% 1.000

nw 12 123 9.76% 0.004

nww 3 115 2.61% 0.137

w 9 120 7.50% 0.007

ww 10 113 8.85% 0.005

Group

Total no. of A.

hastaspicula

eggs recovered

Total no. of

termites

dissected

Proportion of eggs

found compared to

termite number p-value

c 0 120 0.00%

d 3 117 2.56% 0.494

dw 1 119 0.84% 1.000

nd 1 112 0.89% 0.776

ndw 0 118 0.00% 1.000

nw 4 123 3.25% 0.490

nww 0 115 0.00% 1.000

w 3 120 2.50% 0.494

ww 4 113 3.54% 0.463

Group

Total no. of A.

antarctica eggs

recovered

Total no. of

termites

dissected

Proportion of eggs

found compared to

termites numbers p-value

c 0 120 0.00%

d 5 117 4.27% 0.096

dw 9 119 7.56% 0.028

nd 3 112 2.68% 0.137

ndw 0 118 0.00% 1.000

nw 8 123 6.50% 0.029

nww 3 115 2.61% 0.137

w 6 120 5.00% 0.059

ww 6 113 5.31% 0.038

19

Fig.1. (A) Uterus of a female A. antarctica that contained eggs (tiny white dots). (B)

Larvated eggs inside the uterus of a female A. hastaspicula.

20

Fig. 2. (A) C. acinaciformis consumed faeces (right) in the presence of Hummock grass

(left). (B) Eggs of both A. hastaspicula (right) A. antarctica(left) in the gut of a soldier

termite.

21

Fig. 3. (A) Proportion of A. hastaspicula eggs found in soldier and worker caste

compared to termite numbers; (n= 3,171). * P= 0.02. (B) Proportion of A. hastaspicula

and A. abbreviata eggs found compared to termite numbers;**P= 0.002.

22

CHAPTER THREE

The life cycle of the reptile-inhabiting nematode Abbreviata

hastaspicula (Spirurida: Physalopteridae: Physalopterinae)

in Australia

23

3.1 ABSTRACT

This study elucidated the life-cycle of the reptile inhabiting nematode Abbreviata

hastaspicula (Spirurida: Physalopteridae: Physalopterinae) in its final hosts in Australia.

Eight Varanus gouldii (Lacertilia: Varanidae), and two Christinus marmoratus (Reptilia:

Gekkonidae) lizards were captured in the wild. Two V. gouldii were used as controls

and no experimental procedures were carried out on them. Another six V. gouldii (final

host) and the two C. marmoratus (paratenic host) were treated with oral anthelmintics to

remove all parasitic worms and were fed with infected live arthropods containing third

stage larvae of Abbreviata spp. Faeces of V. gouldii were examined under the

microscope weekly to determine whether the third stage larvae had developed into

adults. Two months later, a total of 30 larvae and adults of A. hastaspicula were found

in the stomachs of four experimentally-infected V. gouldii lizards. No cysts or larva

were found in the C. marmoratus. Humans are usually accidental hosts to this subfamily

of nematodes and they may be underdiagnosed in patients. This is the first study to

demonstrate the life-cycle of this genus of nematode in their definitive reptile hosts.

24

3.2 INTRODUCTION

The nematode Abbreviata hastaspicula (Spirurida: Physalopteridae) occurs

predominantly in Varanus gouldii lizards, principally in the arid interior of Australia. It

requires an arthropod intermediate host to complete its life-cycle. Only one partial life

cycle of Abbreviata spp. is known from Australia (King et al. 2013). The life-cycle of

spirurid nematodes consists of six stages involving an egg, four larval stages (or

juvenile stages) and lastly, adult. For parasitic nematodes found in vertebrates, the

infective stage is always at the beginning of the third larval stage, L3 (Anderson 2006).

Species of Abbreviata exhibits a heteroxeous life cycle. However, in paratenic hosts, the

infective stage of a parasite persists without essential development and usually lack of

growth (Anderson 2006; Anderson et al. 2009). Its lifecycle can only be completed by

the final host (larger lizards) feeding on insects (intermediate host) or paratentic hosts

(smaller lizards) containing infective third stage larvae (Anderson 2006; Preston &

Johnson 2010). Larvae ingested by possible paratenic hosts generally encyst in the

abdominal tissues, where they can persist until they are eaten by a predaceous final host.

The ingested larvae then attach to the stomach wall and, depending on the amount of

food in the stomach, grow to adult (Lee 1955). Humans are the accidental hosts to

Abbreviata and there are records of Physalopteraine nematodes infecting humans in

different parts of the world (Ortlepp 1926; Morgan 1945; Singh & Rao 1954;

Vandepitte et al. 1964; Nicolaides et al. 1977), however, due to our poor understanding

of its biology, it may be underreported in human. Therefore, this study was undertaken

to elucidate the life-cycle of Abbreviata hastaspicula in in its final hosts in Australia by

infecting the wild caught Varanus gouldii (final host) and Christinus marmoratus

(paratenic host) with live arthropods that had been infected with the larvae of

Abbreviata species.

25

3.3 MATERIALS AND METHODS

Infecting the lizards

Eight Varanus gouldii and two Christinus marmoratus were caught from arid Paynes

Find (latitude:-29° 43' 21.5436", longitude: 117° 10' 24.3912") and Wooroloo (latitude:-

31° 48' 7.3218", longitude: 116° 18' 51.6384"), Western Australia respectively with the

permission of Department of Environment and Conservation Australia (Licence no.

SF009524). The average weight of the varanids was 4.15 ± 0.52 kg and that of the

geckkonidae was 17.1 ± 3.20 g.

The wild-caught V. gouldii and C. marmoratus were observed for two weeks. The faeces

of the lizards collected from the floor of their individual housing cage were checked

weekly for eggs of the nematodes Abbreviata spp. The faeces defecated by the lizards

were observed under compound microscope. For each slide used for microscopic

examination, the concentration of eggs in the faeces was 4 eggs/ 2mg. The estimated

total number of eggs was 2000 eggs/g. Two V. gouldii were used as controls and were

euthanized by sodium pentobarbital injection (dose: ≥ 100 mg/kg) as soon as

embryonated eggs were found in the faeces of V. gouldii. They were dissected to

ascertain that nematodes in the experimental lizards were the same as the species of

nematodes that occurred in these lizards in the wild.

The remaining lizards (six V. gouldii and two C. marmoratus) were treated with

fenbendazole (0.4mL/100g body weight PO once and repeat in 14 days). After

ascertaining that the V. gouldii were free from nematodes, four uninfected Varanus and

two C. marmoratus were fed with infected live Teleogryllus oceanicus (Orthoptera:

Ensifera: Gryllidae) crickets that ingested the embryonated eggs of A. hastaspicula 28

26

days earlier following the methodology previously described by King et al. (2013)

(feeding of arthropods was a one-off event). When T. oceanicus were examined under

microscope, cysts containing third stage larvae were found in their mid- and hindguts.

Each Varanus consumed 19 T. oceanicus crickets that had contained third stage larvae

of Abbreviata spp. and five T. oceanicus were given to each C. marmoratus. Faeces of

Varanus were examined microscopically every week to determine whether the third

stage larvae developed into adults.

Two months later, as only adult A. hasataspicula is able to pass its eggs in the faeces of

the lizards, in order to confirm that Abbreviata larvae can only develop into adults in a

final host, when the eggs of A. hastaspicula were found in the faeces of the four infected

V. gouldii, they and the two C. marmoratus (possible paratenic host), the V.gouldii and

C. marmoratus were dissected after euthanized by sodium pentobarbital injection and

carbon dioxide inhalation respectively. The stomachs and gastrointestinal tracts of the C.

marmoratus were opened to check whether they contained larval cysts of A.

hastaspicula.

After another month, the two V. gouldii that had consumed the two C. marmoratus were

euthanized by injection of sodium pentobarbital and were dissected (UWA Animal

Ethics Ref. RA/3/100/1248).

Adult nematodes of A. hastaspicula found in V. gouldii were observed under light

microscopy after clearing in chlorolactophenol, and lengths of the larvae and adult

nematodes (in mm) were measured with a map-measurer from drawings made with the

aid of a drawing tube. Their stage of development was assessed by the differentiation of

their sexual organs (Cawthorn & Anderson 1977).

27

Dissecting the lizards

The lizard was laid on its back, and a vertical ventral incision was made from the

sternum to the pubis. The connective tissue was peeled from skin and turned back so

that viscera were exposed. The lower oesophagus, stomach and intestine were released

from connective tissues and examined for adult nematodes and larvae. The stomach was

opened by vertical incision, food was noted and collected. Stomach, gastrointestinal

tracts, cysts and worms were removed with forceps, cleaned in normal saline, fixed in

formalin and preserved in 70% ethanol for morphological examination. Nematodes used

for sequencing were cleaned in sterile distilled water and stored in 70% ethanol.

Histology staining

Nematodes and small segments of stomach and gastrointestinal tracts of infected

Varanus were dehydrated, embedded in paraffin, and serially sectioned at 5 µm. Tissue

samples from animals were stained with hematoxylin and eosin (Gabe & Blackith

1976).

3.4 RESULTS

Prevalence and intensity of A. hastaspicula infection in V. gouldii before the

experiment.

Embryonated eggs of A. hastaspicula were present in the faeces of all wild-caught V.

gouldii, indicating that all the lizards were infected with this nematode in the wild.

Embryonated eggs of A. hastaspicula and A. antarctica were found in one of the

controls.

28

Feeding of infective Teleogryllus oceanicus

The V. gouldii had ingested all the T. oceanicus crickets as soon as they were given to

them. The two C. marmoratus had broken apart and ingested some shattered parts of the

crickets, but it was not certain whether they had ingested the infected cysts.

Infecting the experimental lizards

Two months after the lizards had ingested infected T. oceanicus, eggs of A. hastaspicula

were being passed in the faeces of all of the experimentally infected V. gouldii. The

infection rate was 100%. The numbers of A. hastaspicula found in the experimental V.

gouldii were 5, 3, 8 and 14 respectively. No larvae or adult nematodes of A. hastapicula

were found in C. marmoratus. Concurrent infection with A. antarctica occurred in one

of the two controls. Five hundred and eight and 834 nematodes were recovered

respectively from the two controls V. gouldii (Fig. 1).

Adult Abbreviata hastaspicula

Adult A. hastaspicula found in the stomach of V. gouldii were examined under X4, X10

and X20 objectives. Seventeen females and 13 males were recovered. Males were 8.24-

11.58mm long, and females 13.22-19.03mm long. Females were longer and stouter than

males; the average width of males (0.57mm) was about half of that of females

(1.10mm). Female A. hastaspicula contained eggs (Figs. 2A and 2B). The spearhead-

like tip of the male right spicule was diagnostic, figs. 3A and 3B (Jones 1979).

Diagnostic morphological features in the female include the thin-walled eggs compared

with those of potential concurrent species (A. antarctica and A. bancrofti) and the

tubular extension to the vulva (Figs. 2A and 4). Food residues were noted. The

nematodes were cleaned in normal sterile in which they were attached together in a

mass, fixed in formalin and preserved in 70% ethanol for morphological examination.

29

Histology staining

Histological sections showed little inflammatory cells and there was no evidence that A.

hastaspicula caused pathological changes that affect the healthof their final host, V.

gouldi (Fig. 5). No difference of host response to infection for A. hastaspicula and A.

antactica was noted in any lizards.

3.5 DISCUSSION

Physalopterinae nematodes in Australian lizards

Distribution of the nematode genus Abbreviata is worldwide (Bain et al. 2015). In

Australia, nematodes in this genus are widespread. They are most common in the lizard

fauna (Jones 2014), and physalopterid nematodes also occur in birds (Honisch & Krone

2008), and amphibians (Kelehear & Jones 2010). The arid Australian landscapes support

the richest and the most diverse lizard fauna in the world, due to the dry hot climate and

the dominant vegetation type, hummock grasslands (Triodia spp.), which provide niches

for many species of lizard (Pianka 1986). In addition, a range of shrubs and sparse trees

provide niches for a great variety of both diurnal and nocturnal lizards (Rich & Talent

2008). The Varanidae contain the world’s biggest lizards, with at least 25 endemic

described species in Australia (Bush et al. 2000). The species of lizard in this study,

Varanus gouldii is found in all areas of Western Australia except the coolest and wettest

parts.

Definitive host (large lizards) and paratenic host (smaller lizards)

Adult Abbreviata are widespread in the stomachs of large lizards in the genus Varanus

in Australia, with an infection prevalence close to 100% (Jones 1995a, 2005). In the

present study, adults or immature adults of A. hastaspicula were present in all the four

experimental Varanus gouldii that had ingested the infected tropical crickets, confirming

30

that A. hastaspicula recovered from V. gouldii did not result from natural infection but

experimental feeding (fig. 6). Previous studies by Jones (1983) have shown a positive

correlation between the numbers of Abbreviata sp. larvae and A.hastaspicula (P < 0.01).

The two C. marmoratus that had ingested the crickets and the two V. gouldii that had

consumed the C. marmoratus were not infected with adult Abbreviata species; however,

we were unable to ascertain that the geckoes had ingested nematodes from the offered

crickets, and thus we cannot confirm that C. marmoratus are or are not potential

paratenic hosts for A.hastaspicula. Many species of smaller lizards, mainly skinks and

geckoes, are paratenic hosts for physalopterid larvae (Jones 2010), in which there is a

lack of inflammatory response (Jones 1995b). No further development in these paratenic

hosts occurs unless they are consumed by a larger species of lizard (Jones 1995a). More

studies are required to ascertain the postulated life cycle in paratenic hosts (dashed

arrows in fig.6).

Geographic distribution

Abbreviata spp. are geographically widely distributed in lizards throughout Australia.

The morphologically primitive physalopteran nematode Kreisiella chrysocampa occurs

as adults in several species of smaller skinks lizards, in which cysts containing

physalopterid larvae occur but no adult Abbreviata. These observations suggest that

Abbreviata may have arisen in smaller lizards, and that their ancestor may have been

species related to Kreisiella (Jones 1995a). A. hastaspicula and A. antarctica coexist

over wide areas but the former replaces the latter in the drier inland of the continent

where an annual precipitation below 400 mm (Fig. 7) (Jones 1983). Distribution of

A.hastaspicula and A. antarctica indicate that climate and habitat may limit the

distribution of these two species. Since T. oceanicus does not occur in northern, central,

31

southern and southwestern Australia, other species of arthropod are implicated in the

development and transmission of these nematodes.

Host specificity

A. hastaspicula predominates in Varanidae, and A. antarctica was recovered at highest

prevalence and intensity in V. rosenbergi (Jones 2005, 2007, 2014). Factors affecting

the geographical pattern of these two Abbreviata spp. are probably firstly, the

distribution of the suitable arthropod intermediate hosts; secondly, the ability of the eggs

to survive and remain viable outside the final hosts (Jones 2014); thirdly, the availability

of prey for the hosts e.g. small lizards. A fuller understanding of the biology of species

of Physalopterinae would clarify the relative importance of these factors. Environmental

changes could theoretically expose lizards to different suites of parasites over time

(Poulin 2007; Poulin & Keeney 2008), and findings from the Australian lizard fauna

show that host-specificity in the subfamily Physalopterinae is at the family rather than

species level (Jones 2004; Jones 2005; Jones & Watharow 2010).

Histology

The absence of pathological changes produced by larval Abbreviata spp. infection is

probably the result of a long evolutionary association between this species of nematode

and their reptile final host. V. gouldii can live for at least seven years in captivity (King

& Green 1999). The lifespan of A. hastaspicula within the host is unknown, but the

reptile hosts outlive the third-stage physalopterid larvae (Jones 1995b).

To conclude, our study has elucidated the life-cycle of A. hastaspicula in its definite final

host. The findings of the present study may have relevance for human contact and hence

possible infection. As early as 1902, there are records of Physalopterinae nematodes

32

infecting humans in Caucasus, Central Africa, South America (Ortlepp 1926; Morgan

1945), India (Singh & Rao 1954) and Congo (Vandepitte et al. 1964). In Australia,

physalopteran larvae have caused life-threatening eosinophilic granulomata in an 11-

month-old male infant, but the species causing the infection could not be identified

(Nicolaides et al. 1977). This subfamily of nematodes may perhaps be underreported in

man because they are insufficiently known. Further studies of nematodes in the genus of

Abbreviata in Australian lizards should provide considerably more information for the

understanding of their biology, and thus the risk of humans acquiring physalopterid

infection.

33

Fig. 1. A large number of Abbreviata hastapicula were found in the stomach of a

control V. gouldii

34

Fig. 2. Female Abbreviata hastaspicula. (A) tubular vulva, laying eggs, dorsal view.

(B) Embryonated eggs in the uterus.

35

Fig. 3. Male Abbreviata hastaspicula. (A) Posterior end, dorsoventral view, where S is

the spicule. (B) T is the tip of right spicule, lateral view.

36

Fig

. 4

. A

dult

fem

ale

Ab

bre

viata

hast

asp

icula

. A

E, an

teri

or

end;

ME

, m

usc

ula

r oes

oph

agus;

GE

,

gla

ndula

r oes

ophag

us;

TV

, tu

bula

r vulv

a; U

B, eg

gs

in th

e 4 u

teri

ne

bra

nch

es;

A, an

us;

T, ta

il.

37

Fig. 5. Stain, H & E. Section of stomach of Varanus Gouldii with an adult Abbreviata

hastapicula. AT, apical tooth of A. hastaspicula

38

Fig. 6. The postulated complete life cycle of Abbreviata hastapicula

Thick arrows indicated the life cycle in the final host (life cycle elucidated in this paper):

Firstly, eggs passed from the faeces of the larger lizards. Secondly, eggs containing first

stage lava (e) are ingested by suitable arthropod intermediate hosts (e.g. Teleogryllus

oceanicus), and the 1st stage larva developed into 3rd stage larva/ larvae then encysted

on the guts of arthropod intermediate hosts. Lastly, arthropod intermediate hosts are

consumed by final/definitive host (larger lizard).

Or alternatively, as shown by the dashed arrows (this is not yet confirmed), if the life

cycle is achieved through paratenic hosts, the arthropod intermediate hosts would be

consumed by the paratenic host (smaller lizards), and the infective larva(e) persist

without essential development or growth until it is consumed by the final host (larger

lizards).

39

Fig. 7. Distribution of (A) Abbreviata antarctica (B) Abbreviata hastaspicula. ( )

distribution of Teleogryllus oceanicus in Western Australia. ( ) location of the 8

Varanus gouldii were captured. ( ) areas of the 2 Christinus marmoratus being

caught. Dashed line represents the 400-mm average annual precipitation. Scale bar

=800km. Amendment of this figure is made according Jones (2014).

40

CHAPTER FOUR

Molecular sequencing of the nematodes Abbreviata hastaspicula and Abbreviata

antarctica (Spirurida: Physalopteridae) from Australia

41

4.1 ABSTRACT

The nematodes Abbreviata antarctica von Linstow, 1899 and Abbreviata hastaspicula

Jones, 1979 are predominant spirurid nematodes in species of Varanus lizards in

Australia. However, genetic knowledge of these two species of nematode is lacking. In

this study, nematodes removed from Varanus gouldii were examined using integrated

morphological and molecular methods. DNA from both species of nematodes A.

hastaspicula and A. antarctica was extracted for PCR and sequencing. Specific 18S

ribosomal DNA primers were designed based on the existing Physalopterinae strains

from the NCBI genome database. Phylogenetic analysis revealed the genetic

relationship of the two species of Abbreviata within a limited component of the order

Spirurida. This is the first study reported the 18S sequences of these two Abbreviata

species. The findings of the present study contribute to the biological and genetic

knowledge of this group of nematodes.

42

4.2. INTRODUCTION

Abbreviata antarctica and Abbreviata hastaspicula (Spirurida: Physalopteridae) are

predominant gastro-intestinal nematodes parasitising in the Varanus lizards of Australia

(Jones 1983, 1985a, 1988, 2005). They are spirurid nematodes and exhibit a

heteroxenous life cycle (Anderson 2006). Their life-cycle can only be completed by the

final hosts feeding on arthropod intermediate hosts containing infective third stage

larvae (King et al. 2013; King & Jones 2016). Physalopterine nematodes infecting

human had been previously reported in different countries. Physaloptera caucasica was

the first species in this family isolated from infected human in Russia 1902. Physalotera

mordens Leiper, 1908 was reported in man in Central Africa and South America

(Ortlepp1926; Morgan 1945). An outbreak of Physalotera caucasica in Congo had

caused five cases of physalopterosis in outpatients aged from 16 to 40 years old, four

men and a woman (Vandepitte et al. 1964). An unidentified adult Physaloptera sp.

caused a purulent subcutaneous abscess in the neck of a female patient was reported in

India (Singh & Rao 1954). In Australia, an 11 month old Caucasian male baby was

reported suffering from gangrene of the distal portion of the small bowel as a result of

ingesting larval Physaloptera sp.. He might have eaten infected insects when he was

playing on the grass in front of his house in the country. It was assumed that following

ingestion, the larvae hatched and caused endarteritis and thrombosis of mesenteric

vessels when they attempted to migrate into tissue for encystment in what was for them

a final host. Unfortunately, due to the lack of diagnostic tools, the species of nematode

involved could not be identified further until studies in Australia find out more about

this subfamily of nematodes (Nicolaides et al. 1977).

43

Abbreviata antarctica was described as Physaloptera antarctica by von Linstow in

1899 and redescribed by Irwin-Smith (1922). Abbreviata hastaspicula was described

from specimens in Emerald, Central Queensland (Jones 1979). Travassos proposed the

genus Abbreviata in 1920 to accommodate certain species of physalopterid nematode

that possessed different spicules, uteri and papillae (Morgan 1945). Schulz added

another 23 species to Abbreviata in 1927, and subsequently many more species have

been described in this genus (Morgan 1945). The taxonomic criteria distinguishing the

nematodes A. hastaspicula and A. antarctica are the male copulatory spicules, egg shell

thickness,the form of the vulva in females, and variations of their cephalic morphology.

No phylogeny exists for these two physalopterid species to date.In this study we

collected live adult A. antarctica and A. hastaspicula from six dissected Varanus

gouldii. After identifying the nematodes morphologically, molecular methods were

applied to explore the phylogenetic framework of these two widespread Australian

nematodes.

4.3 MATERIALS AND METHODS

Specimen Collections

Viable nematodes A. antarctica and A. hastaspicula were collected from six freshly

killed and dissected Varanus gouldii which were captured in the wild from arid

Sandstone, Western Australia (Latitude: -32° 8' 2.7024", Longitude: 115° 55' 34.2114")

(Licence no.SF009524). The V. gouldii were euthanized by the injection of sodium

pentobarbital (dose: ≥ 100 mg/kg) before they were dissected (UWA Animal Ethics Ref.

RA/3/100/1248).

44

Morphological examination

The nematodes were firmly attached to the stomach wall of the V. gouldii. Live

nematodes for photographic imaging were removed from the stomach by forceps,

cleared in chlorolacophenol, placed on a clean microscope slide and examined by

microscopy under X10, X20 and X40 objectives under a compound microscope

(Specimen ID: UWA-HAC-A.antarctica and UWA-HAC- A.hastaspicula). Specimens

for further molecular analysis were cleared in glycerol instead of chlorolacophenol to

avoid DNA being damaged.

DNA extraction

After the nematodes were morphologically identified, single adult nematodes were

cleaned with distilled water and preserved in 70% ethanol for DNA extraction. Within

48h after collection each nematode was first submerged in 300 μl of distilled water, in a

2 mL centrifuge tube containing a 0.5 cm stainless steel ball, and homogenized via a

TissueLyserII (Qiagen, Cat#85300). Homogenized samples were then processed

according to Qiagen manufacturer protocol (Qiagen DNeasy® Animal Tissues Mouse

Tail, Spin-Column Protocol).

PCR amplification and sequencing

Physalopterinae specific 18S ribosomal RNA (rRNA) primers were designed based on

the alignment of five published Physalopterinae 18S rRNA from the National Center for

Biotechnology Information (NCBI) genome database: Physaloptera alata (AY702703),

P. apivori (EU004817), P. turgida (DQ503459), Turgida torresi (EF180069) and

Physaloptera sp. SAN-2007 (EF180065). The primers designed for this study for A.

45

hastaspicula were (Forward) 5’-GCGCGCAAATTAACCCAATCTC- 3’ and (Reverse)

5’-CGGGCGTCTCGCTACGG-3’. The primers used for A. antarctica, (Forward) 5’-

GTAACGGGTAACGGAGAG- 3’ and (Reverse) 5’- CACCGAATCAAGAAAGAG-3’

were previously described (King 2012). Primers were synthesized commercially

(Sigma-Aldrich). Each PCR reaction contained 2 μl (30 ng/μl) of DNA template, 5 μl

(10 pmol/μl) of each primers, 25 μl of GoTaq® Green Master Mix (Promega) and

nuclease-free water to a total volume of 50 μl. The PCR thermal cycle was as follow:

initial denaturation at 94°C for 1 min, 35 cycles of denaturation at 94 °C for 18 sec,

annealing at 45 °C for 30 sec, extension at 72 °C for 1 min and final extension at 72 °C

for 10 min. After analysing on 2% agarose gels, the successfully amplified PCR

products were purified using PCR clean up kit (Qiagen). Purified PCR products were

then sequenced using Sanger sequencing method via Australian Genomic Research

Facility (AGRF). All the molecular procedures were duplicated to confirm the

reliability and consistency of the findings.

Phylogenetic tree construction

A total of 60 18S rRNA reference nucleotide sequences from the order Spirurida were

downloaded from NCBI and aligned with the 18S rRNA sequence of A. antarctica and

A. hastaspicula via Multiple Sequence Comparison by Log Expectation (MUSCLE)

alignment. This alignment algorithm is known to be more efficient and accurate than the

conventional clustalW (Edgar 2004). Since the aforementioned reference nucleotide

sequences have different length, the final nucleotide alignment was trimmed to a final

size of 388 base pairs (bp). Two rooted Maximum Likelihood (ML) trees (Saitou & Nei

1987) were generated using Molecular Evolutionary Genetics Analysis (MEGA, version

6.0) with 1000 bootstraps. Two outgroup species, Priapulus caudatus (Priapulimorpha:

46

Priapulimorphida: Priapulidae) and Plectus aquatilis (Adenophorea: Araeolaimida:

Plectidae) were used to root the two ML trees respectively. These two outgroup choices

were supported by the previous phylogenetic analyses of Blaxter et al. (1998) and

Nadler et al. (2007). The ML trees were constructed using corrected pairwise distance

and the aligned sites with a bootstrap support lower than 50% were selectively filtered

(removed).

4.4 RESULTS AND DISCUSSION

Morphological analysis

A. hastaspicula was the only species of nematode that occurred in five out of the six

dissected V. gouldii, in the sixth V. gouldii there was concurrent infection of A.

antarctica and A. hastaspicula. Microscopic morphological examination confirmed that

the nematodes removed from the stomach of the varanid lizards were adult stage A.

antarctica and A. hastaspicula. The distinguishing morphological features between the

two species of nematode are as follows: Firstly, the mouthparts, mouth corner denticles

are present in A. antarctica but absent in A. hastaspicula (Fig. 1). Besides, the spicule of

male A. antarctica is thicker and shorter than that of A. hastaspicula, and there is no

spearhead-like tip at the anterior end of the right speculum (Fig. 2). Also, for A.

antarctica, vulva situated on a slightly elevated distance (between 1/5 and 1/4) from the

anterior end, and it is behind the esophago-intestinal junction. Coils of uterus extend

anterior to the vulva (Fig. 3). In addition, when observing the female uterus, the eggs of

A. antarctica are darker than those of A. hastaspicula under the microscope (Fig. 4).

The eggs of A. antarctica are slightly bigger, less elongated and have conspicuously

thicker shells than the eggs of A. hastaspicula (Fig 5).

47

Identification of the two species of nematode was confirmed morphologically and

provided guidelines for the molecular analyses. Given a lack of existing genomic data

on the nematodes A. antarctica and A. hastaspicula, accurate identification of the

species by morphological features is essential and fundamental for an accurate

phylogenetic analysis. Both A. antarctica and A. hastaspicula conform to the basic

pattern of Physalopterine nematodes; cephalic dentition is a valuable character in the

identification of these nematodes. They possess a large single external apical tooth and a

small bifid internal apical tooth, doubled submedian teeth at the dorsal and ventral lip

margin. Mouth corner denticles are present in A. antarctica but are inconstant in A.

hastaspicula. The amphids are situated at the base of the lateral pseudolabia (Anderson

2006). In males, the caudal bursa is ornamented and the caudal alae are on the ventral

surface of the body.

The phylogeny of Abbreviata

The genome sequencing work undertaken in this study was novel. A. hastaspicula and A.

antarctica were clustered together with the other seven physalopterids of the

superfamily Physalopteroidea with a high bootstrap value of > 80% (Fig 6 & 7). The

resulting topology of the two rooted ML trees generated by the phytogenic analysis

showed that within the Physalopteroidea, A. hastaspicula and A. antarctica (bootstrap >

80%), Physaloptera apivori and Physaloptera alata (bootstrap > 50%), as well as

Physaloptera torresi and Turgida torresi (bootstrap ≥ 60 %) were monophyletic.

According to Anderson et al. (1974), in the order Spirurida, there are two sub-orders,

the Camallanina and the Spirurina. The Camallanina were divided into two

superfamilies, the Camallanoidea and Dracunculoidea. The ten superfamilies in

Spirurina were Physalopteroidea, Filarioidea, Thelazioidea, Diplotriaenoidea,

48

Habronematoidea, Acuarioidea, Spiruroidea, Gnathostomatoidea, Rictularioidea and

Aproctoidea. The ML tree out-grouped by the marine nematode Priapulus caudatus

(Martín-Durán et al. 2012) excluded the Gnathostomatoidea (hosted by mammals) away

from the Spirurina and the Camallanina with a 55% bootstrap value, the three

Gnathostoma spp. were aligned at the bottom of the ML tree, sister to the outgroup P.

caudatus with a strong 99% bootstrap support (Fig 6). On the other hand, the ML tree

out-grouped by the freshwater nematode Plectus aquatilis (Abebe et al. 2006) has

grouped the Gnathostomatoidea with the Camallanina (bootstrap < 50%), the three

Gnathostoma spp. were aligned just below the Spirurina. Although the

Gnathostomatoidea was placed closer to the Dracunculoidea, it was not sister to

Anguillicola crassus as suggested by Nadler et al. (2007), instead, the outgroup Plectus

aquatilis was sister to A. crassus (bootstrap > 70%) (Fig. 7). Sequences were submitted

to GenBank under accession number KX255660 for A. antarctica and KX255661 for A.

hastaspicula.

Limited phylogenetic studies were available for the Physalopterinae despite several

researchers have reconstructed the phylogenetic relationship of the Spirurina within the

phylum Nematoda over the last two decades. Blaxter et al. (1998) first defined the

phylum Nematoda molecularly into five clades, and the order Spirurida was belonged to

clade III under the Scernentea plus Plectidae (S + P). De Ley (2006) classified the sub-

order Spirurina under the order Rhabditida and suggested the Chromadoria lineage was

subdivided in Spirurina, Rhabditina and Tylenchina. Holterman et al. (2006) put the

Spirurina into clade 8. Van Megen et al. (2009) revised this phylogenetic structure by

including the Ascaridormorpha, Rhigonernatomorpha, Oxyuridomorpha and

Gnathostomatomorpha in the Spirurina. Blaxter & Koutsovoulos, (2015) described the

branching order of clade III Spirurina was resolved and the Enoplia were arising basal

49

to Dorylaimia plus Chromadoria. Although Nadler et al. (2007) focused on the

molecular phylogeny of clade III nematodes, little is known about the Physalopterinae.

Therefore, the present study classified and grouped the nematode species according to

the classification keys of Anderson et al. (1974).

Within the Physalopteroidea, P. turgida Travassos, 1920 was considered synonymous

with T. torresi Schulz, 1927 (Morgan 1945), yet, molecular studies have shown that

they are different species (Smythe et al. 2006; Nadler et al. 2007). It appeared that the

host species may be the determinants for the forming of the monophyletic

physalopterids: A. antarctica and A hastaspicula are nematodes of reptiles (King et al

2013; King & Jones 2016); P. alata and P. apivori are found in birds (Ali 1961;

Anderson 2006); Physaloptera turgida and Turgida torresi are nematodes of marsupials

and rodents respectively (Smythe et al. 2006; Nadler et al. 2007). While for P. sp. SAN-

2007 and P. thalacomys, they were also monophyletic despite the low bootstrap support

(< 50%).P. thalacomys are hosted by rabbit-eared bandicoot (Baker et al. 1996)and the

striped skunk Mephitis mephitis is the host of P. sp. SAN-2007 (Nadler et al. 2007) . For

the rest of the the Spirurina, beginning from the top of the ML trees, mammal is the host

of Loa loa, Onchocerca cervicalis, Acanthocheilonema viteae, Litomosoides

sigmodontis, Wuchereria bancrofti, Brugia malayi, Thelazia lacrymalis, Dirofilaria

immitis, Setaria digitata, Serratospiculum tendo, Spirocerca lupi and Spirocerca sp.;

Onchoceridae sp. is the only free living nematode in the ML trees; Tetrameres

fissispina, Cyrnea leptoptera, Cyrnea mansioni, Cyrnea seurati and Echinuria borealis

are avian nematodes; lastly, Spinitectus carolini, Rhabdochona denudate, Ascarophis

arctica and Neoascarophis macrouri are hosted by fish. In the suborder Camallanina,

except the reptile-inhabiting nematodes (Micropleura australiensis and Dracunculus

oesophageus) and the mammalian nematodes (Dracunculus insignis and Dracunculus

50

medinensis), all the species are hosted by fish. The different host species may contribute

to the different genetic composition of the nematodes and thus the distance in the

branching of the phylogenetic tree.

Nucleotide diversity

Nucleotide diversity is the number of differences per nucleotide site between two

randomly chosen sequences; it is a measure of polymorphism at genetic level (Nei & Li

1979). According to the nucleotide diversity calculation, the divergences between A.

antarctica and A. hastaspicula is 1.8%. The differences between A. antarctica and other

Physaloptera species in the ML tree are: P. alata (3.08%), Physaloptera sp. JSL-2010

(3.35%), P. turgida (3.35%), Turgida torresi (3.35%), Physaolptera sp. SAN-2007

(3.35%), P. apivori (3.86%), P. thalacomys (4.2%). Yet, for A. hastaspicula, its

differences between P. alata (2.83%), P. sp. JSL-2010 (3.09%) and Physaloptera

apivori (3.6%) are smaller despite it is more diverse with Physaloptera turgida (3.87%)

when compared with that of A. antarctica. The genetic difference of A. hastaspicula

against Turgida torresi (3.35%), P. sp. SAN-2007(3.35%) and Physaloptera thalacomys

(4.21%) are the same as that of A. antarctica. The percentage nucleotide between

Abbreviata and the Spirurina (excluding the superfamily Physalopteroidea) is 4.38-

20.48%, while that of Abbreviata and the Camallanina is 11.76- 22.79%. Abbreviata is

approximately 18% genetically different from the outgroup Plectus aquatilis and is 25%

genetically different from the outgroup Priapulus caudatus.

In conclusion, the present study provided the detailed morphological characters for

identifying the nematode A. antarctica and A. hastaspicula, our molecular phylogenetic

51

findings briefly described how likely is the Abbreviate related to the order Spirurida.

The 18S rRNA was chosen for the phytogenic analysis in this study because it is the

only gene available in the database, but this gene is so conserve that it may not have

enough resolution to differentiate the closely related species. This is an additional useful

piece of information to the identification and understanding of physalopterans in

Australia. Although the Physalopterinae is considered rare in human and humans are

usually reported as accidental hosts to physalopterid nematodes, they may be

underdiagnosed and under-reported (particularly if they are immature larvae stage) due

to the poor understanding and the lack of identification tool. The combined

morphological-molecular detection tool developed in this study can certainly provide a

sound basis for further investigation of other Abbreviata species in Australian reptiles,

and aid in diagnosing infections in humans.

52

Fig. 1. Cephalic features, anterior end, lateral view AT, apical tooth; MCD, mouth-

corner denticles; SP, sessile papilla. (A) Abbreviata antarctica (B) Abbreviata

hastaspicula

53

Fig. 2. Male, Abbreviata antarctica, where S indicates the spicules, lateral view

Fig. 3. Female vulva, Abbreviata antarctica, lateral view

54

Fig. 4. Section of a female uterus, ventral view (A) Abbreviata antarctica (B)

Abbreviata hastaspicula. The eggs of A. antarctica are darker than those of A.

hastaspicula under the microscope

55

Fig. 5. Eggs of (left) Abbreviata antarctica (right) Abbreviata hastaspicula

56

Fig. 6. Phylogenetic relationships of nematodes in the order Spirurida based on

molecular data (18S rRNA sequences) obtained using the maximum likelihood method

(MEGA version 6.0). Scale bar units are branch lengths estimated by MEGA. Numerals

indicate bootstrap percentages following 1000 replications. Outgroup Priapulus

caudatus was used to root the tree. Bootstrap supports smaller than 50% are shown as

unresolved.

57

Fig. 7. Phylogenetic relationships of nematodes in the order Spirurida based on

molecular data (18S rRNA sequences) obtained using the maximum likelihood method

(MEGA version 6.0). Scale bar units are branch lengths estimated by MEGA. Numerals

indicate bootstrap percentages following 1000 replications. Outgroup Plectus aquatilis

was used to root the tree. Bootstrap supports smaller than 50% are shown as unresolved.

58

CHAPTER FIVE

Anti-aging effects of resveratrol analogues and gastrodin using a

new Drosophila –Abbreviata hastaspicula (Nematoda) model

59

5.1 ABSTRACT

Many natural compounds and extracts have been shown to have healthspan-promoting

and lifespan-extending effects. Resveratrol and gastrodin are both natural phenols. In

this study, we develop a new insect-nematodes model using the fruit fly Drosophila

melanogaster/Drosophila simulans and the reptile inhabiting nematode Abbreviata

hastaspicula to study the anti-aging effects of resveratrol analogue and gastrodin. Our

findings have shown that resveratrol analogue and herbal gastrodin delayed aging and

increased the survivorship in adult A. hastaspicula and all life-stages of Drosophila

depending on the drug feeding method. The 0.5% drug supplementation method

prolonged the life expectancy and slowed the rate of aging in either sex of Drosophila

spp. during all three life-stages (3 -30 days, 31-60days, 61days-die) regardless of the

type of yeast in the food. However, the Capillary Feeder (CAFE) assay increased the

mean lifespan of stage two flies but decreased the mean and maximum lifespan of some

stage one and stage three flies, and it also shortened the lifespan of male flies fed brewer

and baker yeast diets. The optimum dose for gastrodin was 0.1 molar and for resveratrol

analogue it was 0.3 molar. Female Drosophila was the longer living sex in general. We

hope the anti-aging capacity of these two natural compounds can help to prevent aging-

related diseases and ill health in human.

60

5.2. INTRODUCTION

Aging and senescence

Why do we grow old? Why do we age before we die? Why don’t humans just die

without aging? Why do our bodies deteriorate when we age? Why do we suffer from

age-associated pathophysiology when we grow old? Aging is a progress that

accumulates diverse deleterious changes which lead to an increase in the chance of

diseases and death (Harman 2001). It is a time-dependent functional degeneration that

affects most living organisms (López-Otín et al. 2013). Yet, our understanding of the

phenomenon of how and why we get old is still very inadequate. According to the

Gerontology Society of American, aging-related changes usually manifest in the post-

reproductive period and the process of aging progressively raises the probability of

dying. During the progress of aging, some series of different biomarker values and/or

gene expression patterns transit from highly bodily maintenance and normal functioning

to a state of low bodily maintenance, and gradually function abnormally (Arking 2006).

Senescent or senescence is defined as any noticeable body functional decline at the later

years of our lifespan (Lamb 1977). Aging is a disease- susceptible condition, but aging

is not a disease itself. Older individuals show a greater vulnerability to aging-related

disease burdens compared with younger people because they becomes frail and weak

(Carnes et al. 2003). The process of getting older increases our vulnerability to

pathological change, especially when the inexorable loss of molecular fidelity occurs in

our vital organs. Biological aging represents changes in the molecular structure and

function of our body. These aging changes exist for just the same reason that aging

exists in man-made objects or machines, according to Nobel-laureate immunologist

Peter Medawar’s theory: it is a default (DNJ de Grey 2015; Park 2015). It is a result of

entropy in accordance with the reinterpretation of the Second Law of Thermodynamics

61

(Amin et al. 2012). Until reproductive maturation, the fidelity of the energy state of

most molecules in our bodies is maintained, after which those energy states disperse and

cause biological molecular inactivation or malfunctioning. In terms of physics, the

aging process occurs because of the changed energy states. This loss of uniformity is

worsened by the diminution of repair and replacement capability as we grow older, and

eventually, when the repair and replacement system can no longer cope with the

escalating loss of molecular fidelity, our body becomes frail and thus more vulnerable

to age-associated diseases. For people in developed countries, cells that compose the

vascular system and those that are more prone to cancer are the weakest links (Hayflick

2007).

The lifespan of different species on earth varies. Caenorhabditis elegans nematodes live

for 3 weeks only, while mice and rats die at two to three years old. Elephants can make

it to 50 to 60 years old. The Galapagos giant tortoises live over 100 years in the wild

and at least 170 years in captivity. Bowhead whales have the capacity to live about 200

years (Mueller et al. 2015). Arctica islandica, a deep-sea clam lived for 507 years when

it died in 2006 (Gruber et al. 2015). These observations have led people to think about

why aging and death are dissimilar in different species? And what mechanisms

constitute the differences in rates of aging between different species? Promislow (1993)

suggested that animals with larger in body size tend live longer than those with smaller

size and this is the strongest phenotypic correlate with interspecies longevity (which

obviously does not fit into the aforementioned example of deep-sea clam). Nonetheless,

this relationship proposed by Promislow can be opposed with several arguments: firstly,

smaller species are more likely to be hunted by predators in the wild, so that under the

rule of natural selection, they will reproduce early in life and therefore perhaps age

more quickly than big animals. Secondly, the metabolic rates of large animals are

slower when normalized for body size and thus their rate of biological decline is

62

relatively slower compare to small species (Pitt & Kaeberlein 2015). Thirdly, the

lifespan of naked mole rats is ten times longer than the closely related rodents of similar

size, and, naked mole rats may never get cancer (Triplett et al. 2015). Lastly, certain

species of turtles (ages over 70) and rockfish (lifespan exceeds 100 years) exhibit

negligible senescence- the lack of symptoms aging (Finch 2009).

Growing old and getting ill

The global life-span of both genders has increased unprecedentedly; since the Industrial

Revolution in the 19th century, the worldwide average life expectancy by birth has

increased from around 45 years to 71.5 years in 2013. However, the healthy average life

expectancy at birth in 2013 was 62.3 years old (Murray et al. 2015), which means that

in the later years of our lifespan, we may suffer from poor health for 9.2 years before we

die. Men live five years less than women on average (Rochelle et al. 2015). There is no

sign that life expectancy is going to diminish despite the fact that the United Nations

estimated that a plateau would be reached (Westendorp 2006). As a result, almost every

industrialized nation in the world is experiencing a growth of older populations who are

living longer with multiple aging-related illnesses (Perry 2010). Older people are at

higher risk of dying (irrespective of the cause) and are more susceptible to disease, and

the likelihood of their being diagnosed with aging related disorders progressively

increases throughout the rest of their lives.

Aging is the major risk factor for leading causes of death. The chance of acquiring

heart diseases and cancers are 10 times higher for ages over 65 than under 65, and the

risk of death from Alzheimer’s disease has increased more than 50% every five years

(National Center for Health Statistics 2011). For adults aged 55–64, the percentage of

them being diagnosed with serious psychological distress in 2012–2013 was 22% higher

63

than in 2002–2003 (3.6%). Chronic conditions such as heart disease, stroke, high blood

pressure, hypercholesterolemia, diabetes, kidney disease, certain cancers, dementia,

Alzheimer’s diseases and osteoarthritis are common among those aged 55–64 (National

Center for Health Statistics 2015).

Aging-related progressive physiological deterioration provides a substratum for the

aging-associated pathophysiology such as osteoporosis, osteoarthritis, dementia,

Alzheimer’s disease, diabetes, cardiovascular diseases, atherosclerosis and cancers

(Masoro 2010). Genetic and environmental regulations can postpone aging related

degenerative changes in lifespan and health span (Fontana & Partridge 2015).

Pharmaceutical and nutraceutical interventions have shown that modulation of oxidative

stress and inflammation promoted health and delayed senescence (Sun et al. 2014).

Resveratrol

Resveratrol can be found in grapes (Vitis vinifera), blueberries, raspberries, mulberries,

peanuts and some medicinal plants such as Fallopia japonica (Baur 2010; Ghanim et al.

2010). The most common dietary source of resveratrol is red wine; the concentration of

resveratrol in red wine is about 5mg/L (Valenzano et al. 2006). Studies have showed

that resveratrol is capable of extending organismal lifespan of Drosophila,

Caenorhabditis elegans (Wood et al. 2004), Saccharomyces cerevisiae (Howitz et al.

2003) and mice Microcebus murinus (Marchal et al. 2012). Resveratrol slows aging by

activating sirtuins in vitro (Howitz et al. 2003) and increases sirtuin expression in vivo

(Rogina & Helfand 2004). Sirtuin2 (SIRT2) is an enzyme induced by calorie restriction

(Wood et al. 2004), ethanol feeding and exercise activation (Gambini et al. 2011).

Resveratrol consumption activates SIRT2 and could regulate the mechanisms that

prolong life expectancy. However, Vitaglione et al. (2005) suggested that resveratrol

64

alone may not take the credit for the French Paradox, and it is more likely that a

combination of polyphenols contributed to the lower incidence of cardiovascular

diseases in the French population regardless of their diet of highly saturated fats.

Similarly, recent findings postulated that SIRT2 was not in fact activated by resveratrol,

and a low-calorie diet increased Drosophia lifespan independently of SIRT2 (Burnett et

al. 2011).

Gastrodin

Gastrodin is a phenolic glucoside and the main bioactive compound isolated from the

orchid Gastrodia eleta Blume (GEB). It is a pharmacologically active substance whose

tranquilisation, sedative and analgesic effects have been known since ancient times (Ha

et al. 2000; Qiu et al. 2014; Liu et al. 2015b; Woodbury et al. 2015). Gastrodin is a

potential anti-inflammatory drug in neurodegenerative diseases (Dai et al. 2011). It

played a significant role in protecting liver damage and hepatic fibrosis by attenuating

oxidative stress and inflammation (Zhao et al. 2015). It treats migraine through

regulating the neurotransmitters in the central nervous system (Wang et al. 2016). 4-

hydroxybenzaldehyde (a polyphenol) in GEB exhibited antiepileptic and anticonvulsive

activities (Ojemann et al. 2006). It lowered blood pressure in hypertensive patients (Liu

et al. 2015a) and showed neuroprotective effects in subchronic toxin-induced

Parkinson’s disease mouse model (Kumar et al. 2013). It may prevent osteoporosis, it

inhibits adipogenesis and osteoclastogenesis; it decreases serum oxidative and

osteoclast-specific markers (Huang et al. 2015). Gastrodin decreased the expression of

MAPKK4, Sortilin-1 and Rab6A in the hippocampus and prefrontal cortex of

senescence-accelerated mouse prone 8 (SAMP8), which suggested it has a positive

effect on vertebrate aging (Li et al. 2015).

65

Abbreviata hastapicula

Abbreviata hastapicula (Spirurida: Physalopteridae: Physalopterinae) is a parasitic

nematode found predominantly in the Australian lizard Varanus gouldii. It requires an

arthropod intermediate host to complete its life cycle (Anderson, 2006). Its life cycle

consists of six stages involving an egg, four larval stages and finally, adult. The life

cycle can only be completed by the final host (larger lizards) feeding on insects

(intermediate host) or paratentic hosts (smaller lizards) containing infective third stage

larvae. Only the beginning of the third larval stage is the infective stage (Anderson

2006). Humans are the accidental hosts. We used A. hastaspicula as a testing model

because only certain species of arthropods have the potential to be its intermediate host

(King et al. 2013) and until a suitable arthropod has ingested its eggs, A. hastaspicula

will not develop. In some paratenic hosts of the nematode family Physalopteridae, the

infective stage of a parasite persists within a cyst in the abdominal tissues of a tail-

regenerating paratenic gecko host (similar to the facultative diapausal form of

Caenorhabditis elegans third stage larvae) (Anderson 2006; Anderson et al. 2009;

Golden & Riddle 1984). So, what are the physiological determinants underline this

plasticity of maturing? Does A. hastaspicula share the particular gene that facilitates

tissue regeneration with the geckos? We want to know if this nematode has the ability to

accidentally develop in humans, and if so, is it possible that it can harmonize with the

human aging process. Therefore investigating how the nematode matures may throw

light on the basic mechanism of human aging.

In this study, we investigate whether resveratrol analogues (Gu) and herbal gastrodin

(GAS) exhibit any anti-aging capacity, and whether the aging process can be

experimentally retarded using our new insect-nematode, Drosophila -A. hastapicula

model. We applied drug supplementation method and Capillary Feeder assay to test the

66

action of Gu and GAS using various concentrations of the drugs. If Gu and GAS can

postpone senescence in our testing model, it may also have the potential to make a

positive impact on human health and life expectancy.

5.3 MATERIALS AND METHODS

Fly stocks and maintenance of flies

Two species of wild type Drosophlia population, D. melanogaster and D. simulans

were used. The two wild Drosophila species were collected from rotting fruits or baited

traps from 32 sites along a 3000-km transect on the east coast of Australia in 2000

(Kennington et al. 2003). Fly stocks have been cultured and maintained in respective

population cages ever since. Drosophila flies were reared in a humidified, temperature

controlled room at 25- 29°C and 40% or 50% RH on a 12-h light: 12-h dark cycle. All

flies were reared in standard density culture on standard laboratory sugar/yeast (SY)

medium (10% sugar/yeast: 2% agar, 10% sucrose, 10% autolysed yeast powder, 3%

Nipagin, 0.3% propionic acid)(Bass et al. 2007). Two types of autolyzed yeast powder,

baker yeast (Lesaffre Yeast Corporation, Milwaukee, USA) and brewer yeast

(Associated British Foods, London, UK) were used. Light CO2 was applied to

anaesthetise the flies before they were transferred to the fresh food vials for the first 40

days of their lifespan only; no CO2 gas was involved during the transferfor the rest of

their lifespan.

Testing Gastrodin (GAS) with Drosophila flies

For drug supplementation method, the extracted powder of gastrodin (purity ≥ 98%)

(Chengdu Must Bio-technology Co. Ltd, Product # MUST-13101011) was added to

individual vials containing molten media at 60 °C; the final concentration of gastrodin

67

in food was 0.5% by weight/volume and 0 %. For negative control, 100 μL of 100%

DMSO was added to the cooled media at 40 °C to a final concentration of 2%. Flies

were kept in 30 mL plastic vials containing 5mL of food/drug media. Newly enclosed

adult flies were allowed to mate 2 days before they were transferred to fresh 10%

sugar/yeast medium for the lifespan trials. Male and female flies were separated. Flies

were kept at a density of 20 flies per vial, four vials for each gender. The numbers of

dead flies were counted when the flies were transferred to fresh vials every 3 days until

all the flies had died.

The Capillary Feeder (CAFE) assay (William et al. 2007) was implemented to find out

the exact amount of drug the flies have ingested in a given period of time.

Concentrations 0 M, 0.1 M, 0.3 M, 0.5 M and 0.7 M were tested using a graduated glass

microcapillary pipette (Hirschmann Micropipette). Two μL of 5% (wt/vol) sucrose

solution and 0.5 μL of food colouring (Queen Fine Foods, Australia) were mixed with

each concentration of GAS powder and then fed to the flies using the CAFE for 3 hours

every three days. For no drug control, only the carrier, 5% sucrose solution with the 0.5

μL of food colouring was administered. For negative control, 2 μL of 100% DMSO was

mixed with 0.5 μL of food colouring fed to the flies using the CAFE without the 5%

sucrose solution. On average, individual Drosophila fruit flies consume 0.096 ± 0.008

uL of fluid each meal at a frequency of 0.65 ±0.08 meal/h (William et al. 2007). Flies

were transferred back to normal SY medium after 3 hours of feeding. Eighty males and

80 females were tested for each concentration.

All flies were divided into three stages (Sun et al. 2014). Stage one, from age day 3 to

30 (health span), stage two, from age day 31 to 60 (transition span) and stage three,

from age day 61 until the death of the flies (senescence span). Drugs were only given to

the flies at the specific testing stage; flies were fed with normal SY diet without

68

administering the drug in any way at the non-testing stages. There were approximately

160 flies in each concentration except 40 flies for final concentration 0.5% by

weight/volume. A total of 14,394 flies were used. Gastrodin was tested on both species

of Drosophila.

Testing resveratrol analogue (Gu) with Drosophila flies

For resveratrol analogue (Gu) powder (provided by Sun Yat-Sen University), in the

original trial, concentration 0 M, 0.3 M, 0.5 M and 0.7 M were tested by the CAFE

assay with Drosophila melanogaster on brewer yeast for their entire lifespan from two

days after the newly enclosed flies had mated. Duplication trials for supplementation

method were carried out on final concentration 0.5% by weight/volume at three

different stages with both Drosophila species on two types of yeast using 4 vials for

each sex (20 flies in a vial). Only concentration 0.3 M was replicated using the CAFE

method. There were approximately 220 and 960 flies in each concentration for the

original and duplicated trial respectively. A total of 6,720 flies were tested.

Nematode Abbreviata hastapicula as a novel testing model

A total of 75 viable Abbreviata hastapicula nematodes were immediately removed from

a dissected lizard, evenly divided into three groups and were maintained in 5mL of

hydrochloric acid. Hydrochloric acid (HCI) is a gastric acid in the stomach of human

and many animals. It was used in this experiment to mimic the acidic environment of

the lizard’s stomach, the concentration of the HCI/kg was 37% and it had a pH value of

1.Gu and GAS powders were added to the hydrochloric acid solution to a final

concentration of 2% by weight/volume. For no drug control, only the carrier, 5ml of

hydrochloric acid was administered. A small piece of lizard stomach (5mm length x

69

5mm width x 3mm height) was placed in each container for the nematodes to consume.

Drugs were given to them for the remainder of their lives. The hydrochloric acid

solution was changed every 24 hours and the numbers of dead nematodes were checked

every 12 h then every 3 h after the initial 72 h. This nematode testing model was

replicated to confirm the reliability and consistency of the findings.

Statistical analysis

Mantel-Cox log rank test and likelihood ratio test were used to analyse the survivorship

data. P-values were adjusted using the Benjamini & Hochberg method to account for

multiple testing (R version 3.0.2). Maximum lifespan was calculated using the 10%

longest surviving flies of mean lifespan of a population (Sun et al. 2014). Lifespans

were measured after the initiation of the interventions. P ≤ 0.05 was considered

statistically significant.

5.4 RESULTS

Effect of Gu and Gastrodin on Abbreviata hastaspicula

The mean lifespan of adult nematodes supplemented with Gu and GAS was 58.8 ± 4.80 h

(P ≤ 0.003) and 54.0 ± 4.89 h (P ≤ 0.026) respectively compared to 41.3 ± 3.54 h in the

no drug nematodes (fig. 1), which indicated an increase of 42.37% in Gu and 30.75% in

gastrodin. Median lifespan of Gu, GAS and no drug were 60 h, 48 h and 36 h

respectively. Drug supplementation considerably promoted the maximum lifespan of A.

hastaspicula by 25.64% relative to the untreated nematodes. The maximum lifespan of

nematodes that had consumed Gu and gastrodin was 98 h, and that of no drug nematodes

was 84 h (78 ± 6 h) (Fig. 2).

70

Effect of resveratrol analogue, Gu on flies

In the first Gu trail, all concentrations tested by the CAFE showed an increase in the

maximum lifespan of both genders, the percentage growth in males was 3.48%, 2.21%

and 1.40%; in femaleswas 5.88%, 1.72% and 4.52% for 0.3 M, 0.5 M and 0.7 M

respectively, relative to no drug control, however, their log-rank P-value analysis was

not significant. In the duplicated trial, CAFE 0.3 M and final concentration 0.5% by

weight/volume extended the life expectancy of flies fed either type of yeast (Table 1);

the differences were statistically significant. The mean lifespan of flies fed baker yeast

diet lived longer and females slightly outlived males. D. melanogaster showed a more

statistically significant increase in survival rate than D. simulans in both sexs (Fig. 3).

Residual Gu solution and powder left in the CAFE glass microcapillary pipettes ranged

from 0- 1.2 μL in the original trial, no solution but only powder was remained in the

duplicated trial (because the Gu powder did not dissolve fully in the sucrose solution, a

very small amount of powder stayed inside the microcapillaries after the flies had

sucked out all the Gu solution). DMSO decreased the maximum lifespan of flies by

about 20% or 14-16 days in all categories.

Gu has shown an increase in the lifespan of the flies at all three life-stages regardless of

the drug feeding method. Supplementation 0.5% Gu increased mean lifespan of stage

two (31-60 days, transition span) D. melanogaster the most, by approximately 12 days

(relative to the stage two no drug control). Concentration 0.3M using the CAFE assay

also showed a maximum increase in that of stage two D. melanogaster, by

approximately 12.5 days (relative to the stage two no drug control). For D. simulans, 0.5%

supplementation increased the mean and maximum lifespans of stage three (61days to

die, senescence span) flies the most, by approximately 5 days (relative to the stage three

no drug control), but the effect of CAFE 0.3M was most obvious during stage one (3-30

71

days, health span), approximately 5.5days increase in mean lifespan and 10 days

extension in maximum lifespan (relative to the stage one no drug control) (Table 4).

Effect of gastrodin, GAS on flies

Final concentration 0.5% by weight/volume in either yeast diet showed a statistically

significant increase in the mean lifespan of both male and female flies (table 2).The

CAFE method increased the lifespan of female fed with either yeast type but slightly

decreased that of the male (0.4 d) in brewer yeast and 0.2 d in baker yeast. Drosophila

fed baker yeast diet survived longer than those fed brewer yeast. In terms of

concentration, GAS extended the maximum lifespan of male CAFE 0.1M (86.90 ± 0.52

d) fed brewer yeast and female CAFE 0.7 M (95.13 ± 0.48 d) fed baker yeas the most

(Table 3). Effect of gastrodin supplement promoted the life-expectancy of female D.

simulans most statistical significantly (Fig. 4). Approximately 1 μL of residual

gastrodin solution stayed in the CAFE glass microcapillaries in every trial. DMSO

decreased the maximum lifespan of flies by about 20 days in all categories.

The lifespan of both Drosophila spp. was extended most significantly during the

transition span life-stage, 31-60 days. The 0.5% GAS supplementation increased the

mean lifespan of stage two D. melanogaster by approximately 9 days while the CAFE

method increased its mean lifespan by approximately 4 days but slightly decreased the

maximum lifespan by 0.21 days or 0.35% (relative to the stage two no drug control).

For stage two D. simulans, 0.5% supplementation method increased its mean and

maximum lifespans by approximately 8 days, the CAFE assay slightly increased its

mean lifespan by 0.24 day and maximum lifespan by approximately 4 days relative to

the stage two no drug control (Table 5).

72

5.5 DISCUSSION

Nematode testing model

Our nematode A. hastaspicula model has shown that resveratrol analogue and gastrodin

can successfully prolong both maximum and mean lifespans. The adult A. hastaspicula

supplemented with Gu and GAS survived 17.5 ± 1.3h and 12.7± 1.4h longer than no

drug control, their changes of mean lifespan are very significant, especially for Gu, over

40%. The Gu curve moved slightly more to the right than the GAS curve during 36 to

72 h while the proportion of surviving nematodes during 72 to 98 h were the same (Fig.

2). This suggested that at any specific hour between 36-72 h, a higher proportion of

nematodes with ingested Gu survived compared with nematodes administered with

GAS. Gu extended the lifespan of nematodes most effectively at approximately the

second stage of the adult lifespan. The lifespan of parasitic A. hastaspicula within the

host is not known (Jones 1995a) but in a free-living environment, we saw a statistically

significant anti-aging effect of Gu and GAS supplementations compared to the

untreated no-drug nematodes. The mortality of nematodes may be or may be not

associated with aging and differences in lifespan itself may not with certainty, reflect

the differences in the rate of aging albeit the patterns of age-at-death analysis are

unambiguous and as far as the measurement of the rate of aging is concerned. We

therefore analysed the maximum lifespan of the nematodes as it allowed us to estimate

the decreased aging rate of A. hastaspicula, the best measures existing at present (Conn

2006). It is now clear that the basic aging process and molecular or genetic pathways is

fundamental that it contains the same elements that affect longevity in all animal species

(Pitt & Kaeberlein 2015). Hence, our findings with A. hastaspicula are considered

applicable to humans.

73

Different life-stages

Aging processes in animals can be classified into three patterns. Firstly, rapid aging

which is represented by semeloparous (death after first reproduction) organisms such as

Atlantic salmon and mayflies. Secondly, negligible senescence, which is typified by

colonial invertebrates, some turtles and fish. Lastly, gradually getting old, which is

characterized by most birds and mammals including humans (Finch, 1990). As with

humans, the adult lifespan of gradually aging Drosophila can be divided into three life-

phases including health span, transition span and senescent span (Arking 2006). Each

life-stage is characterised by different gene expression patterns (McDonald et al. 2013)

and oxidative damage at molecular levels (Sun et al. 2014). In this study, Gu promoted

the longevity of both Drosophila spp. during all three different life stages. The increase

of mean lifespan and maximum lifespan in D. melanogaster was particularly robust at

stage two (31- 60 d); indicating Gu intervention was most beneficial to D. melanogaster

when implemented during the transition span. The last survivor died more or less

around the same time at all three stages, whether Gu was administered with the CAFE

method or 0.5% supplementation (Fig. 5 and Table 4). For D. simulans, the increase of

mean lifespan was most noticeable in stage three flies whatever the drug feeding

methods, which suggested that the prolongevity effects of Gu was best if it was

administered when the flies are entering the senescence span. Stage one D. simulans

using the CAFE assay extended its maximum lifespan the most, which suggested that

the earlier Gu was given to D. simulans, the slower the flies aged. However, because

stage one flies were the healthiest and the strongest, they may have been able to suck

harder on the CAFE microcapillary pipettes and thus ingest more drug solution, which

may explain the increase of maximum lifespan.

74

Unlike Gu, not all flies administered with the GAS during three life-stages had an

increased lifespan. Flies fed with 0.5 % GAS supplementation lived slightly longer than

those implemented with the CAFE assay in stage one. For 0.5% supplementation

method, D. melanogaster from stage three and D. simulans of stage two supplemented

with GAS aged at the slowest rate (Fig. 6 and Table 5). However, some stage one and

stage three flies implemented with the CAFE assay showed a decrease in mean and

maximum lifespans. Possibly GAS may be detrimental when implemented during stage

one and three of their lifespan. Alternatively the herbal GAS powder may have been

bitter and even when mixed with 5% (wt/vol) sucrose solution, was disliked by flies,

consequently, and large amount of residual gastrodin solution was left in the CAFE

glass microcapillary pipettes in every trial. For 0.5% GAS supplementation, the taste

and smell of the yeast may have suppressed that of gastrodin, and flies ingested the food

media that contained GAS powder. The flies might have been stressed by being forced

to ingest and to smell the drug every three days, and thus their lifespan shortened. Due

to the very large number of flies that were used in the experiments, all differences in

this study were statistically significant (Jafari et al. 2007). We noted that some flies died

presumably from old age at stage three before we started the implementation of Gu and

GAS.

Concentration of drug

In order to find out the optimum dose of Gu and GAS for health and life span extension

in Drosophila spp. flies, various concentrations of natural compounds were tested. To

save cost, in the Gu duplicated trial, we only re-tested 0.3 M concentration because this

concentration increased the mean-lifespan of D. melanogaster the most in the original

trial. Our result from the replicated trial confirmed that 0.3 M Gu was the most effective

dose for both sexes. For GAS, the effect of doses varied (Table 3). Except for 0.1 M, a

75

decrease in either mean or maximum lifespans was observed in all dosages. This could

be explained with the same reasons that affected the GAS screening results at three life-

stages. When different doses were tested using the supplementation method, special

attention should be paid to the variability in dosage produced by the adaptive

mechanism of the flies (Conn 2006). When applying the CAFE assay, flies were not

starved to make certain that the prolongevity effect was not owing to dietary restriction;

drugs were given to them immediately after they were transferred to the vials contained

the drug microcapillaries. Even though the flies did not finish all the drugs powders and

solutions in the micocapillaries, there was no statistical correlation between the change

of lifespan and the amount of Gu remained in the CAFE device. The drug powders

could not fully dissolve in the 5% (wt/vol) sucrose solution suggesting that the amount

of drug the flies ingested might not be the actual concentrations we tested. Our findings

indicated that the drug feeding method caused a different effect of Gu and GAS on the

lifespan and rate of aging in Drosophila flies. It is noteworthy that our results were

achieved using a relatively small dosage of resveratrol analogues and gastrodin

compared with other studies (Bass et al. 2007; Jafari et al.2007; Schriner et al. 2013;

Sun et al. 2014).

The global life-span of both genders increased markedly. Since the industrial revolution

in the 19th century, the worldwide average life expectancy in humans by birth has

increased from around 45 years to 71.5 years in 2013. Men live five years less than

women on average (Rochelle et al. 2015). However, the healthy average life

expectancy at birth in 2013 was 62.3 years old (Murray et al. 2015), which means at the

later years of our lifespan, we may suffer from poor health for 9.2 years before we die.

Almost every industrialized nation in the world is experiencing a growth in older

populations who are living longer with multiple aging-related illnesses (Perry 2010).

Aging is a disease susceptibility condition despite the fact that aging is not a disease

76

itself. The chance of acquiring heart diseases and cancers are 10 times higher for those

aged over 65 than under 65, and their risk of death from Alzheimer’s disease has

increased more than 50% every five years (National Center for Health Statistics 2011).

Chronic conditions such as heart disease, stroke, high blood pressure,

hypercholesterolemia, diabetes, kidney disease, certain cancers, dementia, Alzheimer’s

diseases and osteoarthritis are common among those aged 55–64 (National Center for

Health Statistics 2015).

In conclusion, we are not trying to find a panacea for immortality, but we hope to

prevent and alleviate aging-related degenerative diseases such as osteoporosis,

osteoarthritis and Alzheimer’s diseases. The causal connections of aging and diseases

are probably interconnected in complex circuits, instead of curing the individual illness;

interventions that target the fundamental aging process can simultaneously delay the

onset and progression of most age-associated health problems (Kaeberlein 2013).

Laboratory model organisms such as yeast, nematodes, fruit flies, mice and rat are used

to investigate human ageing, and the findings of these studies have identified potential

interventions that can retard aging in taxa spanning very broad evolutionary distances. If

these interventions can indeed slow human senescence, good health in old age may be

guaranteed (Pitt & Kaeberlein 2015). In the present study, although we have not

revealed the underlying mechanisms modulating lifespan, our findings from the

Drosophila- A. hastaspicula model suggested that resveratrol analogues and herbal

gastrodin possess anti-aging capacity and that they are worthy of further investigation.

77

Die

t S

ex

Dru

g C

on

c.M

ean

Lif

esp

an

Sta

nd

ard

Err

or

of

mean

life

span

(±

SE

)

Ch

an

ge

of

mean

life

span

(%)

Bre

wer

Mal

eN

o d

rug

86.5

0.6

9

GU

0.5

%9

10.3

75.2

0*

**

0.3

M C

AF

E

89.4

0.3

53.3

5*

**

DM

SO

65.2

0.6

3

Fem

ale

No d

rug

82

1.0

1

GU

0.5

%92.3

0.3

212.5

6***

0.3

M C

AF

E

90.3

0.4

10.1

2***

DM

SO

65.7

0.6

1

Bak

erM

ale

No d

rug

95

0.5

8

GU

0.5

%99.6

0.3

4.8

4*

**

0.3

M C

AF

E

100

.40.2

55.6

8*

**

DM

SO

73.4

0.8

3

Fem

ale

No d

rug

92.1

0.8

3

GU

0.5

%1

01

0.1

49.6

6*

**

0.3

M C

AF

E

100

.30.3

28.9

0*

**

DM

SO

71.7

0.7

3

Tab

le 1

. E

ffec

t of

Gu o

n t

he

surv

ivin

g t

ime

of

the

10%

longes

t su

rviv

ing D

. m

elanogast

er a

nd D

. si

mula

ns

. ***P

≤ 0

.001 b

y l

og

-ran

k a

nal

ysi

s.

78

Die

t S

ex

Dru

g C

on

c.M

ean

Lif

esp

an

Sta

nd

ar

d E

rror

of

mean

life

span

(± S

E)

Ch

an

ge

of

mean

life

span

(%)

Ch

an

ge

of

mean

life

span

(%)

Bre

wer

Mal

eC

AF

E

86

0.3

-0.4

6%

No d

rug

87

0.6

9

GA

S 0

.5%

91

0.1

95.3

2%

5.3

2*

**

DM

SO

65

0.6

3

Fem

ale

CA

FE

8

40.3

72.4

4%

No d

rug

82

1.0

1

GA

S 0

.5%

90

0.5

19.5

1%

9.5

1*

**

DM

SO

66

0.6

1

Bak

erM

ale

CA

FE

9

50.2

7-0

.21%

No d

rug

95

0.5

8

GA

S 0

.5%

99

0.3

94.2

1%

4.2

1*

**

DM

SO

73

0.8

3

Fem

ale

CA

FE

9

40.3

31.7

4%

No d

rug

92

0.8

3

GA

S 0

.5%

10

00.3

8.9

0%

8.9

0*

**

DM

SO

72

0.7

3

Tab

le 2

. E

ffec

t o

f G

AS

on t

he

surv

ivin

g t

ime

of

the

10%

longes

t su

rviv

ing D

. m

ela

no

ga

ster

an

d D

. si

mu

lan

s. T

he

CA

FE

incl

uded

all

the

conce

ntr

atio

ns

(0.1

M,

0.3

M,

0.5

M,

0.7

M).

Posi

tive

num

ber

= l

ifes

pan

incr

ease

, N

egat

ive

num

ber

= l

ifes

pan

dec

reas

e.

***P

≤ 0

.00

1 b

y

log

-ran

k a

nal

ysi

s.

79

Die

tS

ex

Dru

g C

onc.

# o

f fl

ies

(n)

Mea

n

Lif

esp

an

Sta

nda

r

d E

rro

r

for

mea

n

life

spa

n

(± S

E)

Ma

xim

um

life

spa

n

Sta

nda

r

d E

rro

r

for

ma

x.

life

spa

n

(± S

E)

P-v

alu

e

Bre

wer

Mal

eN

o d

rug

339

50.4

30.9

86.5

20.6

9

0.1

M351

52.3

40.9

186.9

0.5

20.1

4

0.3

M337

51.0

40.8

985.8

50.6

10.6

3

0.5

M338

51.0

10.8

785.4

80.6

80.6

4

0.7

M364

51.9

50.8

685.5

40.6

20.2

2

Fem

ale

No

dru

g336

47.1

90.8

181.9

61.0

1

0.1

M350

50.3

70.8

184.2

70.7

10.0

05**

0.3

M358

51.7

50.8

485.7

10.7

30.0

001

****

0.5

M343

50.0

40.8

883.7

30.7

40.0

17*

0.7

M333

48.3

60.8

381.8

50.8

0.3

1

Bak

erM

ale

No

dru

g381

59.4

60.8

181.9

61.0

1

0.1

M388

59.5

10.8

184.2

70.7

10.9

7

0.3

M374

55.3

30.8

485.7

10.7

30.0

004***

0.5

M379

55.8

10.8

883.7

30.7

40.0

02**

0.7

M384

56.7

20.8

381.8

50.8

0.0

2*

Fem

ale

No

dru

g351

54.9

50.9

792.0

80.8

3

0.1

M364

56.3

30.9

392.9

20.6

50.3

0.3

M362

55.7

30.9

292.4

40.7

30.5

6

0.5

M362

55.9

0.9

794.0

40.7

30.4

9

0.7

M358

57.3

51.0

395.1

30.4

80.0

9

Tab

le 3

. E

ffec

t o

f G

AS

on t

he

surv

ivin

g t

ime

of

the

10%

longes

t su

rviv

ing D

. mel

an

og

ast

er a

nd

D. s

imu

lan

s. * P

≤ 0

.05, ** P

≤ 0

.01,

***P

≤ 0

.001, ****

P ≤

0.0

001 b

y l

ikel

ihood r

atio

tes

t.

80

Sp

ecie

sD

rug

Co

nc.

Inte

rve

nti

on

pe

rio

d

(sta

ge

)

# o

f fl

ies

(n)

Me

an

Lif

esp

an

a

Sta

nd

ard

Err

or

for

me

an

life

sp

an

a

Ch

an

ge

of

me

an

life

sp

an

(%)

P v

alu

e

by

Lo

gR

an

k

Me

dia

n

Lif

esp

an

a

Max

.

Lif

esp

an

a

Max

life

sp

an

usin

g

the

10

%

lon

ge

st

su

rvin

vin

g

flie

s o

f

me

an

life

sp

an

a

Sta

nd

ard

Err

or

for

the

10

%

lon

ge

st

su

rvin

vin

g

flie

s o

f

me

an

life

sp

an

a

Ch

an

ge

of

max

.

life

sp

an

usin

g t

he

10

%

lon

ge

st

su

rviv

ing

flie

s (

%)

D.

mela

no

ga

ster

1319

53.1

91.2

153

100

90.0

60.8

3

2295

26.3

60.9

424

71

60.8

31.1

7

398

15.6

41.0

614

40

35.4

1.1

1

1159

64.0

31.9

720.3

6%

<0.0

001

69

100

97.1

30.6

57.8

4%

2147

38.3

31.4

545.4

1%

<0.0

001

36

71

68.2

70.8

912.2

2%

368

17.2

81.4

510.4

6%

0.1

683

14

41

39.7

10.4

212.1

9%

1160

63.3

31.6

619.0

6%

<0.0

001

65

100

93.8

11.1

34.1

6%

2139

38.8

31.5

47.3

1%

<0.0

001

36

71

68.1

40.6

512.0

2%

393

18.4

41.2

817.8

9%

0.0

502

14

41

39.4

40.7

511.4

2%

D.

sim

ula

ns

1320

53.7

41.1

453

100

87.4

11.0

4

2283

26.1

11.1

124

72

60.6

81.0

6

392

19.3

81.1

518

41

36.8

90.7

3

1160

55.8

81.8

83.9

9%

0.0

24

53

101

94.8

80.6

98.5

4%

2148

30.3

31.8

216.1

7%

0.0

074

24

72

68.5

30.5

912.9

4%

374

24.1

11.5

124.3

9%

0.0

011

26

42

41.8

60.1

413.4

7%

1160

59.1

91.6

210.1

4%

0.0

021

53

101

97.5

0.8

911.5

5%

2145

28.6

81.6

99.8

6%

0.0

624

72

67.6

41.0

811.4

8%

357

22.1

11.6

214.0

6%

0.0

455

22

41

40.3

30.3

39.3

4%

No d

rug

Gu 0

.5%

GU

0.3

M C

AF

E

No d

rug

Gu 0

.5%

Gu 0

.3M

CA

FE

Tab

le 4

. T

he

spec

ific

lif

e-st

ages

eff

ect

of

Gu o

n t

he

surv

ivin

g t

ime

of

D. m

elanogast

er a

nd D

. si

mula

ns.

a M

ean l

ifes

pan

, m

edia

n

life

span

and m

axim

um

lif

espan

of

the

spec

ifie

d s

tage

afte

r th

e in

itia

tion o

f in

terv

enti

on.

81

Specie

sD

rug C

onc.

Inte

rventi

on

peri

od

(sta

ge)

# o

f fl

ies

(n)

Mean

Lif

esp

an

a

Sta

ndar

d E

rror

for

mean

life

span

a

Change

of

mean

life

span

(%)

P v

alu

e

by

LogR

ank

Media

n

Lif

esp

an

a

Max.

Lif

esp

an

a

Max

life

span

usi

ng th

e

10%

longest

surv

invin

g

flie

s of

mean

life

span

a

Sta

ndard

Err

or

for

the 1

0%

longest

surv

invin

g

flie

s of

mean

life

span

a

Change

of

max.

life

span

usi

ng t

he

10%

longest

surv

ivin

g

flie

s (%

)

D.

mela

no

ga

ster

1319

53.1

91.2

153

100

90.0

60.8

3

2295

26.3

60.9

424

71

60.8

31.1

7

398

15.6

41.0

614

40

35.4

1.1

1

180

59.1

92.6

911.2

7%

0.0

224

61

100

96.8

81.2

77.5

6%

272

35.1

52.0

733.3

4%

0.0

039

32

70

64.4

31.6

55.9

1%

340

15.6

82.0

20.2

1%

0.9

245

14

40

39

0.5

810.1

7%

11276

51.7

40.5

9-2

.73%

0.3

838

53

100

88.3

10.5

1-1

.94%

21143

30.7

70.4

616.7

3%

0.0

039

28

71

60.6

20.4

4-0

.35%

3434

14.8

40.5

5-5

.14%

0.9

245

14

41

36.0

20.5

41.7

6%

D.

sim

ula

ns

1320

53.7

41.1

453

100

87.4

11.0

4

2283

26.1

11.1

124

72

60.6

81.0

6

392

19.3

81.1

518

41

36.8

90.7

3

180

61.2

62.6

614.0

0%

0.0

019

61

101

96.8

80.9

910.8

3%

279

33.8

52.4

429.6

4%

0.0

036

28

72

68.2

50.8

212.4

8%

339

18.1

52.0

9-6

.33%

0.9

348

14

41

39.5

0.6

57.0

8%

11275

53.3

60.5

7-0

.70%

0.9

739

49

100

88.6

60.4

61.4

3%

21173

26.3

50.5

70.9

3%

0.6

976

20

72

64.2

30.3

75.8

5%

3444

17.3

30.5

1-1

0.5

6%

0.1

911

14

42

35.8

0.3

3-2

.96%

No d

rug

Gas

0.5

%

Gas

CA

FE

(all

concentr

ation)

No d

rug

Gas

0.5

%

Gas

CA

FE

(a

ll

concentr

ation)

Tab

le 5. T

he

spec

ific

lif

e-st

ages

eff

ect

of

GA

S o

n t

he

surv

ivin

g t

ime

of

D. m

elanogast

er a

nd D

. si

mula

ns.

a M

ean l

ifes

pan

, m

edia

n

life

span

and m

axim

um

lif

espan

of

the

spec

ifie

d s

tage

afte

r th

e in

itia

tion o

f in

terv

enti

on.

82

Fig. 1. The effect of (A) gastordin, GAS and (B) resveratrol analogue, GU on the mean

lifespan of nematodes ± Standard deviation, (n= 75). *P ≤ 0.05, ** P ≤ 0.01 , P value

based on likelihood ratio test.

Fig. 2. The effect of gastordin (GAS) and resveratrol analogue (Gu) on the adult

lifespan of A. hastaspicula. P-value compared with nematodes not administered GAS

and Gu.

83

Fig. 3. The effect of resveratrol analogue, Gu on (A) male, (B) female D. melanogaster

and D. simulans ± Standard deviation (n= 6,720). ***P ≤ 0.001, ****P ≤ 0.0001 by

likelihood ratio test. The CAFE included concentration 0.3 M, 0.5 M and 0.7 M.

84

Fig. 4. The effect of gastrodin, GAS on (A) male, (B) female Drosophila species ±

Standard deviation (n= 14,394). * P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001

by likelihood ratio test. The CAFE included concentration 0.1 M, 0.3 M, 0.5 M and 0.7

M.

85

Fig. 5. Surviving time of flies at different life stages. (A) stage one, (B) stage two, (C)

stage three. The effect of resveratrol analogue, Gu on both species of Drosophila. The

CAFE only tested on concentration 0.3 M. P-value based on log-rank analysis. The

duration of the Gu treatment in each life stage is marked with a dash line bordered with

arrows.

86

Fig. 6. Surviving time of flies at different life stages. (A) stage one, (B) stage two, (C)

stage three. The effect of gastrodin, GAS on both species of Drosophila., The CAFE

included concentration 0.1 M, 0.3 M, 0.5 M and 0.7 M. P-value based on log-rank

analysis. The duration of the GAS treatment in each life stage is marked with a dash line

bordered with arrows.

87

CHAPTER SIX

General Discussion

88

6.1 ABSTRACT

In this review, I provide a general summary and discussion on the four articles that

comprise this thesis. The research described in this thesis is designed to establish an

insect-nematode model using fruitfly Drosophila melanogaster/D. simulans and the

reptile inhabiting nematode Abbreviata hastaspicula to study the effects of natural

compounds resveratrol analogue and gastrodin on anti-aging and longevity. I elucidated

the life cycle of A. hastaspicula in its final hosts, and investigate the role of one termite

species as its possible arthropod intermediate host. I have provided useful evidence in

each paper leading to invaluable insights towards better understanding of our stated

objectives, and thus point the way for future research.

89

6.2 IS THE TERMITE COPTOTERMES ACINACIFORMIS A POTENTIAL

HOST?

In Chapter two, I have shown that Coptotermes acinacifomis is not a potential

intermediate host for either Abbreviata hastaspicula or Abbreviata antarctica. We

found that all laboratory bred experimental C. acinaciformis had ingested the faeces

whether or not Hummock grasses were present in the container. Although eggs were

found in the hind-guts of soldier and worker termites at intervals1-3, no larvae of

Abbreviata spp. were found in the end of interval 6. Even though our result is not

consistent with Theiler’s (1919) findings about the African worker termite Hodotermes

pretorensis hosting the chicken inhabiting nematode Hartertia gallinarum, recent

studies support the proposal that termites could be a potential intermediate host;

Alenyorege et al. (2011) demonstrated that Schedorhinotermes termites fed to domestic

poultry can act as intermediate hosts for eleven different species of parasitic worms in

poultry. Acquisition of the nematode Physaloptera infection in the Brazilian endemic

frog Proceratoprhys boiei depended on the presence of a high quantity of infected

termites in their stomach (Klaion et al. 2011). Jones (1995a) showed a positive

correlation between termites in the diet of lizards and the prevalence of cysts containing

larval nematodes. This apparent relationship was strongest in smaller geckoes

Diplodactylus conspicillatus, and Rhynchoedura ornata that feed exclusively on

termites and hence he adduced that termites may act as an intermediate hosts for the

larvae of species of Abbreviata. Since termites are particularly abundant in arid and

semi-arid Australia where lizards are the principal termite eaters (Abensperg 1994) and

the genus Abbreviata is predominant in terrestrial Varanus of Australia (Jones 2005), it

suggests that other species of termites may still play a vital role in the transmission of

reptile nematode Abbreviata spp. regardless of the negative findings of C. acinaciformis

in the present study. Research on identifying species of termite which might be the

90

potential intermediate hosts would add considerably to the understanding of desert

ecology.

6.3 THE LIFE CYCLE OF THE REPTILE-INHABITING NEMATODE

ABBREVIATA HASTASPICULA

In Chapter three, we elucidated the life-cycle of the Australian reptile inhabiting

nematode Abbreviata hastaspicula in its final host. Varanus gouldii (Lacertilia:

Varanidae), and Christinus marmoratus (Reptilia: Gekkonidae) lizards were captured in

the wild for the purposes of this study. Two months after we fed the infected crickets to

the dewormed experimental lizards, a total of 30 larvae and adults of A. hastaspicula

were found in the stomachs of the experimentally-infected V. gouldii final hosts. The

infection rate was 100%. This result conforms to the previous studies of Jones (1995a,

2005); nematodes in the genus Abbreviata are the predominant gastric nematodes in

larger lizards and snakes in Australia, often attaining a prevalence of 100% and

intensities of several hundred worms. Our findings have confirmed that this lizard is a

definitive host of A. hastaspicula.

No cysts or larva were found in the paratenic host C. marmoratus, thus they were not

infected with Abbreviata species. Since I was unable to ascertain that the geckoes had

ingested nematodes from the offered crickets, this result suggested that either C.

marmoratus had ingested the nematodes but are not potential paratenic hosts for A.

hastaspicula, or they had not in fact consumed the larvae in the crickets. According to

Jones (2010), many species of smaller lizards, mainly skinks and geckoes, are paratenic

hosts for physalopterid larvae, in which there is a lack of inflammatory response (Jones

1995b). No further development in these paratenic hosts occurs unless they are

consumed by a larger species of lizard (Jones, 1995a). Our results show adult

91

Abbreviata spp. caused no pathological changes on the health of V. gouldi ; this is

probably the result of a long evolutionary association between this species of nematode

and their reptile final host. Humans are usually accidental hosts to this subfamily of

nematodes and there are early records of Physalopterine nematodes infecting man in

different countries. However, current understanding about the biology of these

nematodes, particularly in Australia, is sparse. . Further studies of nematodes in the

genus Abbreviata in Australian lizards should provide more information for the

understanding of their biology, and thus the risk of humans acquiring physalopterid

infection.

6.4 MOLECULAR SEQUENCING OF THE NEMATODES ABBREVIATA

HASTASPICULA AND ABBREVIATA ANTARCTICA

In Chapter four, our morphological findings on identifying the nematodes A. antarctica

and A. hastaspicula provided guidelines for our molecular analyses. Given a lack of

existing genomic data on these two species of nematode, accurate identification of the

species by morphological features is essential and fundamental for an accurate

phylogenetic analysis.

The resulting topology of the two rooted ML trees joined A. hastaspicula and A.

antarctica together with the other seven physalopterids of the superfamily

Physalopteroidea with a high bootstrap support (Fig. 6 & 7). Within the

Physalopteroidea, A. hastaspicula and A. antarctica (bootstrap > 80%), Physaloptera

apivori and Physaloptera alata (bootstrap > 50%), as well as Physaloptera torresi and

Turgida torresi (bootstrap ≥ 60 %) were monophyletic. The nucleotide diversity

92

calculation has shown a 1.8% divergence between A. antarctica and A. hastaspicula.

This outcome together with the findings in Chapter 3, are consistent with the

suggestions of Jones (1995a) about the ecology and the evolutionary history of A.

antarctica and A. hastaspicula in Australia. In Chapter 3, our findings showed that

concurrent infection of A. hastaspicula and A. antarctica were found in one of the V.

gouldii we caught in arid Paynes Find only. This suggested A. antarctica in the arid

interior of Australia is replaced by A. hastaspicula. Jones (1995a) suggested that

Abbreviata may have arisen in smaller lizards, and that their ancestor may have been

Kreisiella (Jones 1995a) because he observed that the morphologically primitive

nematode Kreisiella chrysocampa occurs as adults in several species of smaller skinks,

in which physalopterid cysts occur but no adult Abbreviata (Jones, 1985).

A. antarctica and A. hastaspicula are widespread in Australia, with large areas of

sympatry (Jones 1983). In Western Australia, the morphologically primitive nematode

Kreisiella chrysocampa Jones 1985 (Jones 1985b) occurs in several species of skinks

only supports Abbreviata encysted as larvae in or around the host visceral tissues. This

suggested Abbreviata arose in smaller lizards, and that their ancestor may have been

Kreisiella (Jones 1995a). A. antarctica is more common in areas with higher rainfall,

they are replaced by A. hastaspicula in tropical north as well as in the arid interior of

Australia. The factors influencing the population dynamics and interspecific relations of

these two species of Abbreviata are not yet understood, but the availability of arthropod

intermediate hosts and survival of eggs in the external environment are probably factors.

The concurrent infection of both species and the replacement of one species over the

other suggest the coevolution of A.hastaspicula and A. antarctica with their

93

hosts.Throughout the course of evolution, the Australian continent has offered suitable

niches for the diverse lizard fauna to thrive and flourish. At the same time, it also

provided a good opportunity for potential parasites to develop and evolve inside the

lizards. Changes in sea levels during the glacial periods of the Quarternary resulted in

continental joining of Australia with New Guinea and Southeast Asia. The merging of

lands has created convenient conditions for the immigration and isolation of lizards. The

consequent geographical range extension of lizard species meant more vacant host

niches were available for new parasites to exploit. The extent to which this natural

competition happens is determined by the ecology of the new host species and the

ability of the parasites to adapt (Pianka 1986; Pianka 1989; Jones 1995a, James & Shine

2000). In fact, the infection density of the lizard is ecologically rather than

physiologically determined (Jones, 1988).

Future research of the biology of Abbreviata species should give a fuller understanding

of Physalopterinae and thus would clarify the relative importance of the aforesaid

factors. Environmental changes could theoretically expose lizards to different suites of

parasites over time (Poulin 2007, Poulin and Keeney 2008), and findings from the

Australian lizard fauna show that host-specificity in the subfamily Physalopterinae is at

the family rather than species level (Jones 2004, Jones 2005, Jones and Watharow

2010). The extent to which natural competition happens is determined by the ecology of

the new host species and the ability of the parasites to adapt (Pianka 1986; Pianka 1989;

Jones 1995a, James & Shine 2000). In fact, the infection density of the lizard is

ecologically rather than physiologically determined (Jones, 1988). ‘Infection density’ is

a measure for the degree of infection in relation to physiological effects. The PCR

detection tool developed in this study can provide a sound basis for further investigation

of other Abbreviata species in Australian reptiles.

94

6.5 ANTI-AGING EFFECTS OF RESVERATROL ANALOGUES AND

GASTRODIN

Upon better understanding the biology and phylogeny of A. hastaspicula, in Chapter

five, I have developed a new insect-nematodes model using the fruit fly Drosophila

melanogaster/Drosophial simulans and the reptile inhabiting nematode Abbreviata

hastaspicula to study the anti-aging effects of resveratrol analogue (Gu) and gastrodin

(GAS). Our nematode A. hastaspicula model has shown that Gu and GAS can

successfully prolong both maximum and mean lifespans. Gu extended the lifespan of

nematodes most effectively at approximately the second stage of the adult lifespan.

Although the lifespan of parasitic A. hastaspicula within the host is not known (Jones

1995a), in a free-living environment, we demonstrated a statistically significant anti-

aging effect of Gu and GAS supplementations compared to the untreated no-drug

nematodes. It is now clear that the basic ageing process and pathways at its molecular or

genetic level is fundamental and that it contains the same elements that affect longevity

in all species (Pitt & Kaeberlein 2015). Hence, our findings with A. hastaspicula are

believed to be applicable to humans.

In our Drosophila model, Gu promoted the longevity of both Drosophila spp. in all of

the three different life stages. The increase of mean lifespan and maximum lifespan was

particularly robust in stage two D. melanogaster, in D. simulans stage three flies have

the most obvious increase in mean lifespan, and stage one the flies showed most growth

in maximum lifespan. Yet, because stage one flies were the healthiest and the strongest,

they may be able to suck harder on the Capillary Feeder (CAFE) microcapillaries and

thus ingested more drug solution, which may explain the increase of maximum lifespan.

Unlike Gu, not all the flies administered with the GAS during the three different life-

95

stages had an increase in their lifespan. Flies fed with 0.5 % GAS supplementation lived

slightly longer than those implemented with the CAFE in stage one. For 0.5%

supplementation method, D. melanogaster from stage three and D. simulans of stage

two supplemented with GAS aged at the slowest rate. However, some stage one and

stage three flies implemented with the CAFE assay showed a decrease in mean and

maximum lifespans. This finding suggested that either GAS may in fact be detrimental

when implemented during stage one and three of their lifespan. Or, this may be because

the flies have been stressed by being forced to ingest and to smell the drug they disliked

and thus their lifespan was shortened. The optimum dose for GAS was 0.1 molar and

for Gu it was 0.3 molar. Female Drosophila was the longer living sex.

Our study has shown the anti-aging capacity of GAS and Gu using our new insect-

nematode model, and our finding that female Drosophila flies are the more long lived

sex corresponds to human life expectancy, with men living on average five years less

than women (Rochelle 2015). Our results were achieved using a relatively small dosage

of resveratrol analogues and gastrodin compared with other studies (Bass et al. 2007;

Jafari et al. 2007; Schriner et al. 2013; Sun et al. 2014). Future work can focus on the

underlying mechanisms modulating lifespan, the factors influencing the life-specific

survivorship and the elements inducing the optimal concentration of drugs. Further

conclusion on aging and its correlative relationship to the pathophysiological states of

senescence cannot be drawn until all these unknown aspects are understood.

6.6 CONCLUDING REMARKS

We accept the fact that everything has an inclination to become old, wear out and break

down: old toys, old cars, old machines—and old people (DNJ de Grey, 2015). However,

can we get old in the absence of a disease burden? When life is diminishing, can we

96

live a healthy productive elderly life? To date, the number of aged people is increasing

and such demographic change highlights the view that aging is interrelated with every

aspect of human life from biological, social, psychological, and environmental to

spiritual components (Ankri & Cassou, 2013). In considering our understanding of the

biological mechanism of aging, it is reassuring that we can find out the underling root-

cause of each related disease. Recent evidences point out that what was true for the

laboratory organism might also be true for humans (Pitt & Kaeberlein 2015). Hence, our

insect-nematode model has shown that Gu and GAS may have the potential to make a

positive impact on human health and life expectancy. We hope the anti-aging capacity

of these two natural compounds can help to prevent aging- related diseases and ill health

in humans.

97

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