developing an insect-nematode drosophila …...developing an insect-nematode drosophila- abbreviata...
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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
iv
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
8
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
12
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
13
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
14
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
15
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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