TABLE OF CONTENTS
CHAPTER ONE
1.0.
INTRODUCTION
AND LITERATURE REVIEW
1.1.
INTRODUCTION
1.2.
3-MERCAPTOPYRUVATE
SULFURTRANSFERASE
1.2.1.
DISTRIBUTION
OF 3-MST
1.2.2.
OCCURRENCE
1.2.3.
MECHANISMS
OF ACTION
1.2.4.
MOLECULAR
FORMULA AND MOLECULAR WEIGHT
1.2.5.
STRUCTURE
OF 3-MST
1.2.6.AMINO
ACID COMPOSITION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
1.2.7.CATALYTIC
ACTIVITY OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
1.2.8.
ENZYME
REGULATION OF 3-MERCAPTOPYRUVATE
1.2.9. STABILITY OF 3-MST
1.3.
PHYSICO-CHEMICAL
PROPERTIES OF 3-MST
1.3.1.
OPTIMAL
TEMPERATURE
1.3.2.
OPTIMUM
pH
1.3.3.
EFFECT
OF METALS/ IONS ON 3-MST
1.3.4.
SPECIFIC
ACTIVITY OF 3-MST
1.3.5. INHIBITORY STUDIES OF 3-MST
1.4.
CYANIDE
1.5.
ORYCTES
RHINOCEROS LARVAE
1.5.1.
TAXONOMY
OF ORYCTES RHINOCEROS
1.5.2.
NUTRITIONAL
QUALITIES OF RHINOCEROS LARVAE
1.5.3.
LIFE CYCLE OF ORYCTESRHINOCEROS LARVA
1.5.4.
DAMAGE
1.5.5. NATURAL ENEMIES
1.5.6.
MANAGEMENT
1.5.7. ECONOMIC IMPORTANCE
1.6.
THE GUT
1.7.
PURIFICATION OF 3-MST
1.8.
JUSTIFICATION
OF STUDY
1.9.
OBJECTIVES
OF STUDY
CHAPTER
TWO
MATERIALS
AND METHODS
2.1.
MATERIALS
2.1.1. REAGENTS
2.1.2. APPARATUS
USED
2.1.3. STUDY
SAMPLE
2.2.
METHOD
2.2.1.
PREPARATION
OF BUFFER AND REAGENTS
2.2.1.1.
Preparation
of 0.25M Potassium Cyanide
2.2.1.2.
Preparation
of 0.5M Potassium Cyanide
2.2.1.3.
Preparation
of 38% Formaldehyde
2.2.1.4.
Preparation
of 0.25M Ferric Nitrates (Sorbo Reagent)
2.2.1.5.
Preparation
of Bradford Reagent
2.2.1.6.
Preparation
of 0.38 M Tris-HCl Buffer
2.2.1.7.
Preparation
of 0.30M Mercaptoethanol
2.2.2.
PREPARATION
OF ENZYME CRUDE EXTRACT FROM THE RHINOCEROS
LARVA GUT
2.2.3.
PROTEIN CONCENTRATION DETERMINATION
2.2.4. ASSAY FOR 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
2.2.5. ENZYME
PURIFICATION
CHAPTER THREE
RESULTS
CHAPTER FOUR
DISCUSSION,
CONCLUSION AND RECOMMENDATION
4.1.
DISCUSSION
4.2.
CONCLUSION
4.3.
RECOMMENDATION
REFERENCES
CHAPTER
ONE
1.0
INTRODUCTION
AND LITERATURE REVIEW
1.1 INTRODUCTION
One of the major metabolic enzymes that have gained so
much interest of scientists is 3-Mercaptopyruvate sulfurtransferase (3-MST).
This enzyme occurs widely in nature (Bordo, 2002 and Jarabak, 1981).
It has been reported in several organisms ranging from
humans to rats, fishes and insects. It is a mitochondrial enzyme which has been
concerned in the detoxification of cyanide, a potent toxin of the mitochondrial
respiratory chain (Nelson et al.,
2000). Among the several metabolic enzymes that carry out xenobiotic
detoxification, 3-mercaptopyruvate sulfurtransferase is of utmost importance.
3-mercaptopyruvate sulfurtransferase functions in the
detoxifications of cyanide; mediation of sulfur ion transfer to cyanide or to
other thiol compounds.(Vandenet al., 1967).It
is also required for the biosynthesis of thiosulfate. In combination with
cysteine aminotransferase, it contributes to the catabolism of cysteine and it
is important in generating hydrogen sulphide in the brain, retina and vascular
endothelial cells (Shibuyaet al., 2009).
It also acquired different functions such as a redox regulation (maintenance of
cellular redox homeostasis) and defense against oxidative stress, in the
atmosphere under oxidizing conditionsNagaharaet al (2005).
Hydrogen sulphide (H2S) is an important
synaptic modulator, signalling molecule, smooth muscle contractor and
neuroprotectant (Hosokiet al., 1997).
Its production by the 3-mercaptopyruvate sulfurtransferase and cysteine
aminotransferase pathways is regulated by calcium ions (Hosokiet al., 1997).
Organisms that are exposed to cyanide poisoning
usually have this enzyme in them. This could be in food as in the
cyanogenicglucosides being consumed. It has been studied from variety of
sources, which include bacteria, yeasts, plants, and animals (Marcus Wischik,
1998).
Cyanide could be released into the bark of trees as a
defence mechanism. There are array of defensive compounds that make their parts
(leaves, flowers, stems, roots and fruits) distasteful or poisonous to
predators. In response, however, the animals that feed on them have evolved
over successive generations a range of measures to overcome these compounds and
can eat the plant safely. The tree trunk offers a clear example of the variety
of defences available to plants (Marcus Wischik, 1998).
Oryctes rhinoceros
larva is one of the organisms that are also exposed to cyanide toxicity because
of the environment they are found.
1.2 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
3-Mercaptopyruvate sulfurtransferase (EC. 2.8.1.2), is
a member of the group, Sulfurtransferases (EC 2.8.1.1 – 5), which are widely
distributed enzymes of prokaryotes and eukaryotes (Bordoand Bork, 2002).
3-Mercaptopyruvate Sulfurtransferase is an enzyme that
is part of the cysteine catabolic pathway. The enzyme catalyzes the conversion
3-mercaptopyruvate to pyruvate and H2S (Shibuya et al., 2009). The deficiency of this enzyme will result in
elevated urine concentrations of 3-mercaptopyruvate as well as of
3-mercaptolactate, both in the form of disulfides with cysteine(Crawhallet al.,
1969). It catalyzes the chemical reaction:
3-mercaptopyruvate
+ cyanide à
pyruvate + thiocyanate
3-mercaptopyruvate
+ thiolà
pyruvate + hydrogen sulphide (Sorbo 1957).
It transfers sulfur-containing groups and participates
in cysteine metabolism (Shibuya et al., 2013).
This enzyme catalyzes the transfer of sulfane sulphur from a donor molecule,
such as thiosulfate or 3- mercaptopyruvate, to a nucleophile acceptor, such as
cyanide or mercptoethanol.3-mercaptopyruvate is the known sulphur-donor
substrate for 3-mercaptopyruvate sulfurtransferase (Porter & Baskin, 1995).
3-mercaptopyruvate sulfurtransferase is believed to
function in the endogenous cyanide (CN) detoxification system because it is
capable of transferring sulphur from 3-mercaptopyruvate (3-MP) to cyanide (CN),
forming the less toxic thiocyanate (SCN) (Hylin and Wood, 1959). It is an
important enzyme for the synthesis of hydrogen sulphide (H2S) in the
brain (Shibuya et al., 2009).
The systematic name of this enzyme class is 3-mercaptopyruvate: cyanide
sulfurtransferase. It is also called beta-mercaptopyruvatesulfurtransferase(Vachek
and Wood, 1972).It is one of three
known H2S producing enzymes in the body (Hylin and Wood, 1959). It
is primarily localised in the mitochondria (Cipolloneet al., 2008).
The expression levels of
3-MST in the brain during the fetal and postnatal periods are higher than those
in the adult brain (unpublished data) although the promoter region shows
characteristics of a typical housekeeping gene (Nagaharaet al., 2004). The observation is supported by the finding
that3-MST expression in the cerebellum is decreased during the adult period
(Shibuya et al., 2013). On the other
hand, its expression level in the lung decreases from the perinatal period.
These facts suggest that 3-MST could function in the fetal and postnatal brain.
It was reported that serotonin signaling via the 5-HT1A receptor in
the brain during the early developmental stage plays a critical role in the
establishment of innate anxiety during the early developmental stage
(Richardson-Jones et al., 2011).
In rat, 3-MST possesses 2
redox-sensing molecular switches (Nagahara and Katayama, 2005). A
catalytic-site cysteine and an intersubunitdisulfide bond serve as a
thioredoxin-specific molecular switch (Nagaharaet al., 2007). The intermolecular switch is not observed in
prokaryotes and plants, which emerged into the atmosphere under reducing
conditions (Nagahara, 2013). As a result, it acquired different functions such
as a redox regulation (maintenance of cellular redox homeostasis) and defense
against oxidative stress, in the atmosphere under oxidizing conditions
(Nagaharaet al., 2005).
Moreover, 3-MST can
produce H2S (or HS−) as a biofactor (Shibuya et al., 2009), which cystathionine
β-synthase and cystathionine γ-lyase also can generate (Abe and Kimura, 1996).
Interestingly 3-MST can uniquely produce SOx in the redox cycle of
persulfide formed at the low-redox catalytic-site cysteine (Nagaharaet al., 2012). As an alternate
hypothesis on the pathogenesis of the symptoms, H2S (or HS−)
and/or SOxcould suppress anxiety-like behavior, and therefore,
defects in these molecules could increase anxiety-like behavior. However, no
microanalysis method has been established to quantify H2S (or HS−)
and SOxat the physiological level (Ampolaet al., 1969).
MCDU was first recognized and reported in 1968 as an
inherited metabolic disorder caused by congenital 3-MST insufficiency or
deficiency. Most cases were associated with mental retardation (Ampolaet al, 1969) while the pathogenesis
remains unknown.
Human MCDU was reported to
be associated with behavioral abnormalities, mental retardation (Crawhall,
1985), hypokinetic behaviour, and grand mal seizures and anomalies (flattened
nasal bridge and excessively arched palate) (Ampolaet al, 1969); however, the pathogenesis has not been clarified
since MCDU was recognized more than 40 years ago. Macroscopic anomalies were
associated in 1 case (Ampolaet al,
1969); however, this could be an accidental combination. 3-MST deficiency also
induced higher brain dysfunction in mice without macroscopic and microscopic
abnormalities in the brain. 3-MST seems to play a critical role in the central
nervous system, i.e., to establish normal anxiety (Richardson et al., 2011)
1.2.1
DISTRIBUTION
OF 3-MST
3-MST is widely distributed in prokaryotes and
eukaryotes (Jarabak, 1981). It is
localized in the cytoplasm and mitochondria, but not all cells contain 3-MST
(Nagaharaet al., 1998).
1.2.2
OCCURRENCE
Human mercaptopyruvatesulfurtransferase (MPST; EC.
2.8.1.2) belongs to the family of sulfurtransferases (Vandenet al., 1967). These enzymes catalyze
the transfer of sulfur to a thiophilic acceptor (Sorbo 1957), where MPST has a
preference for 3-mercapto sulfurtransferase as the sulfur-donor. MPST plays a
central role in both cysteine degradation and cyanide detoxification. In
addition, deficiency in MPST activity has been proposed to be responsible for a
rare inheritable disease known as mercaptolactate-cysteine disulfiduria (MCDU)
(Hannestadet al, 2006).
1.2.3
MECHANISMS
OF ACTION
3-Mercaptopyruvate sulfurtransferasecatalyzes the
reaction from mercaptopyruvate (SHCH2C (= O)COOH)) to pyruvate (CH3C(=
O)COOH) in cysteine catabolism (Vackek and Wood, 1972). The enzyme is widely
distributed in prokaryotes and eukaryotes (Jarabak, 1981).
This disulfide bond serves as a thioredoxin-specific
molecular switch. On the other hand, a catalytic-site cysteine is easily
oxidized to form a low-redox potential sulfenate which results in loss of
activity (Nahagaraet al., 2005).
Then, thioredoxin can uniquely restore the activity (Nagahara, 2013).
Thus, a catalytic site cysteine contributes to
redox-dependent regulation of 3-MST activity serving as a redox-sensing
molecular switch (Nahagara, 2013). These findings suggest that 3-MST serves as
an antioxidant protein and partly maintain cellular redox homeostasis. Further,
it was proposed that 3-MST can produce hydrogen sulphide (H2S) by
using a persulfurated acceptor substrate (Shibuya et al, 2009).
As an alternative functional diversity of 3-MST, it
has been recently demonstrated in-vitro that 3-MST can produce sulfur oxides
(SOx) in the redox cycle of persulfide (S-S-) formed at the
catalytic site of the reaction intermediate (Nagaharaet al, 2012).
1.2.4
MOLECULAR
FORMULA AND MOLECULAR WEIGHT
The molecular formula of 3-MST is C3H4O3S
(Vachek and Wood, 1972).
3-MST
has a molecular weight of 120.127g/mol or 23800 Daltons (as summarized by
PubChem compound).
1.2.5
STRUCTURE
OF 3-MST
Figure1.1: Structure of
3-mercaptopyruvate sulfurtransferase
Source:
www.ebi.ac.uk/thornton-srv/databases/cgi
bin/enzymes/GetPage.pl?ec_nnumber=2.8.1.2
1.2.6 AMINO
ACID COMPOSITION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
3-mercaptopyruvate sulfurtransferase is a
crescent-shaped molecule which comprises of three domains (Vachek and Wood,
1972). The N-terminal and central domains are similar to the thiosulfate
sulfurtransferaserhodanase and create the active site containing a persulfurated
catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74
and Arg-185 (Nahagara and Nishino 1996). A serine protease-like triad,
comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a
conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from
thiosulfate sulfurtransferases (Nahagaraet
al 1995).
1.2.7
CATALYTIC
ACTIVITY OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
3-mercaptopyruvate
+ cyanide = pyruvate + thiocyanate (Fiedler and Wood, 1956).
1.2.8
ENZYME
REGULATION OF 3-MERCAPTOPYRUVATE
Regulation is by oxidative stress and thioredoxin. Under
oxidative stress conditions, the catalytic cysteine site is converted to a
sulfenate which inhibits the mercaptopyruvate enzyme activity. Reduced
thioredoxin cleaves an inter-subunit disulfide bond to turn on the redox switch
and reactivate the enzyme (Nagahara, 2013).
1.2.9
STABILITY
OF 3-MST
3-MST is remarkably stabilized during purification and
storage by the presence of monovalent cations.
Maximal stability is obtained if purification and
storage are carried out at pH 6.5-7.5 in the presence of KCN and
2-mercaptoethanol (Vachek and Wood, 1972).
3-MST was stored at 4oC and recorded no
loss of activity after 10 days (Vachek and Wood, 1972).
1.3
PHYSICO-CHEMICAL
PROPERTIES OF 3-MST
1.3.1
OPTIMAL
TEMPERATURE
Minimum temperature is at 45oC, the optimum
temperature is at 45oC – 50oC, and maximum temperature is
at 60oC after which there is no more activity (Vachek and Wood,
1972).
1.3.2
OPTIMUM
pH
The
minimum pH is at 9.3, optimum pH is between 9.4 and 9.5. The maximum pH is at
9.6 (Vachek and Wood, 1972).
1.3.3
EFFECT
OF METALS/ IONS ON 3-MST
KCl:
0.02M causes 70% activation of 3-MST.
Na2SO4:
0.02M causes 70% activation.
K2SO4:
0.02M causes 70% activation.
Furthermore,
0.5mM arsenite and 0.01mM copper acetate has no effect on 3-MST activity
(Vachek and Wood, 1972).
1.3.4
SPECIFIC
ACTIVITY OF 3-MST
The specific activity of
3-MST is 540mM/min/mgVanchek and Wood, 1972).
1.3.5
INHIBITORY
STUDIES OF 3-MST
The inhibitors of
3-mercaptopyruvate sulfurtransferase include:
2-mercaptoethanol:
high concentration of it inhibits the activity of 3-MST.
Cyanide:
it inhibits at a short-time intervals and slightly enhancement at longer
periods.
Cysteamine:
it inhibits 3-MST slightly.
Mercaptosuccinamic
acid: it inhibits 3-MST slightly.
Pyruvate:
17% inhibition when present in 10mM and gives 45% inihibition in 20mM.
Thioglycolic
acid: it slightly inhibits 3-MST. (Vachek and
Wood, 1972).
1.4
CYANIDE
Cyanide
is a chemical compound that contains monovalent combining group cyanide (CN).
This group, known as the cyano-group, consists of a carbon atom triple-bonded
to a nitrogen atom.
Cyanide
is a potent cytotoxic agent that kills the cell by inhibiting cytochrome
oxidase of the mitochondrial electron transport chain. When ingested, cyanide
activates the body own mechanisms of detoxification, resulting in the
transformation of cyanide into a less toxic compound called thiocyanate (Biller
and Jose, 2007).
The
cyanide anion is an inhibitor of the enzyme cytochrome-c oxidase (also known as
aa3) in the fourth complex of the electron transport chain (found in
the membrane of the mitochondria of eukaryotic cells).It attaches to the iron
with this protein. The binding of cyanide to this enzyme prevents transport of
electrons from cytochrome C to oxygen. As a result, the electron transport
chain is disrupted, meaning that the cell can no longer produce ATP aerobically
for energy (Nelson et al, 2000).
Tissues that depend highly on aerobic respiration, such as the central nervous
system and the heart, are particularly affected. This is an example of
histotoxic hypoxia (Biller and Jose, 2007).
Many
plants and plant products used as food in tropical countries contain cyanogenic
glycosides (Vetter, 2000). These plants include cassava, linseed, beans and peas,
which are known to contain linamarin coexisting with lotaustralin. Millet,
sorghum, tropical grass and maize are sources of dhurin. Amygladin is found in
plums, cherries, pears, apple and apricots. These compounds are also present in
plants such as rice, unripe sugar cane, several species of nuts and certain
species of yam (Osuntokun, 1981; Oke, 1979).
In
plants, cyanides are bound to sugar molecules in the form of cyanogenic
glycosides and defend plants against herbivores. Upon hydrolysis, these compounds
yield cyanide, a sugar and a ketone or aldehyde (Jones, 1998).
Initial
symptoms of cyanide poisoning can occur from exposure to 20 to 40 ppm of
gaseous hydrogen cyanide, and may include headache, drowsiness, dizziness, weak
and rapid impulse, deep and rapid breathing, a bright-red colour in the face,
nausea and vomiting. Convulsion, dilated pupils, clammy skin, weaker and more
rapid pulse and slower, shallower breathing can follow these symptoms. Finally,
the heartbeat becomes slow and irregular, body temperature falls, the lips,
face and extremities take on a blue colour, the individual falls into a coma,
and death occurs. These symptoms can occur from sub lethal exposure to cyanide,
but will diminish as the body detoxifies the poison and excretes it primarily
as thiocyanate and 2-aminothiazoline-4-caarboxylic acid, with other minor
metabolites.
The
body has several mechanisms to effectively detoxify cyanide. The majority of
cyanide reacts with thiosulfate to produce thiocyanate in reactions catalyzed by
sulfurtransferase enzymes such as rhodanase. The thiocyanate is then excreted
in the urine over a period of days. Although thiocyanate is approximately seven
times less toxic than cyanide, increased thiocyanate concentrations in the body
resulting from chronic cyanide exposure can adversely affect the thyroid.
Cyanide
has a greater affinity for methemoglobin than for cytochrome oxidase, and will
preferentially form cyanomethemoglobin. If this and other detoxification
mechanisms are not overwhelmed by the concentration and duration of cyanide
exposure, they can prevent acute cyanide-poisoning incident from being fatal.
Other adverse effects include delayed mortality, pathology, susceptibility to
predation, disrupted respiration, osmoregulatory disturbances and altered
growth patterns. Concentrations of 20 to 76 micrograms per litre free cyanide
cause the death of many species, and concentrations in excess of 200 micrograms
per litre are rapidly toxic to most species of fish. Invertebrates experience
adverse non-lethal effects at 18 to 43 micrograms per litre free cyanide, and
lethal effects at 30 to 100 micrograms per litre. (Clark, 1974;Azconet al., 1987).
1.5
ORYCTES
RHINOCEROS LARVAE
The rhinoceros
larvae are popular in oil palm growing areas of the rainforest and coastal
areas of Nigeria. The larvae are white and soft in texture.
The
larva, also called grub, is called osoriby the Ijaws, tam by the Ogonis and utukuru
by the Ibos, all of Southern Nigeria.
Figure 1.2:Rhinoceros Larva
It is either eaten raw, boiled, smoked or fried. It
may be consumed as part of a meal or as a complete meal.
1.5.1
TAXONOMY
OF ORYCTES RHINOCEROS
Domain: Eukaryota
Kingdom: Metazoa
Phylum: Arthropoda
Subphylum:
Urinamia
Class: Insecta
Order: Coleoptera
Family:
Scarabaeidae
Genius: Oryctes
Species: Oryctes rhinoceros
1.5.2
NUTRITIONAL
QUALITIES OF RHINOCEROS LARVAE
In
spite of the effects of the rhinoceros
larvae on palm trunk, these insects (Oryctes
rhinoceros larvae) possess delectable and nutritional qualities that are
appealing to humans. In Nigeria, rhinoceros
larvae are among the edible insect commonly eaten (Banjo et al, 2006). They are well eaten in the rainforest, riverine and
coastal states where the oil palm is grown. The larvae are roasted or fried to
taste.
The
nutritional qualities shows the percentage of Crude Protein which was 36.45%, and the Lipid,
Nitrogen-free extract and Crude fibre are 34%, 15.05% and 10.50% respectively (Banjo et
al.,2006).
It is rich in essential
Amino acids which include:
|
Leucine
|
Phenylalanine
|
Methionine
|
|
6.30g/100g
|
4.65g/100g
|
2.085g/100g
|
Table 1.1: Essential
amino acids present in rhinoceros larva
These rich amino acid
values meet the minimum daily requirements for humans as recommended by the
WHO. It is also rich in minerals as shown in the table below (Banjo et al.,2006).
|
Iron
|
Sodium
|
Potassium
|
Magnessium
|
Zinc
|
|
8.5mg/100g
|
440mg/100g
|
38.4mg/100g
|
175mg/100g
|
7.0mg/100g
|
Table
1.2: Essential Minerals in rhinoceros larva
The
high iron content of the larvae of the rhinoceros
beetle is of advantage to women in developing economies including Nigeria and
more so far pregnant women who are reported to suffer from iron deficiency
during pregnancy (Banjo et al.,
2006).
Magnesium
is useful to maintain normal muscle and nerve function. It steadies heart
rhythm, supports immune blood and regulates blood sugar levels. Magnesium is
needed for more than 300 biochemical reactions in the human body (Saris et al., 2000).
1.5.3
LIFE CYCLE
OF ORYCTESRHINOCEROS LARVA
Eggs are laid and larvae
develop in decaying logs or stumps, piles of decomposing vegetation or
sawdust, or other organic matter. Eggs hatch into larvae 8 days to 12
days, while the larvae feed and grow for another 82 days to 207 days
before entering an 8 to 13 day non-feeding pre-pupa stage.
Pupae are formed in a cell made in the wood or in the soil
beneath where the larvae feed. The pupa stage lasts 17 to 28 days.
Adults remain in the pupa cell 17 - 22
days before emerging and flying to palm crowns to feed. The beetles are
active at night and hide in feeding or breeding sites during the day. Most
mating takes place at the breeding sites. Adults may live 4-9 months and
each female lays 50-100 eggs during her lifetime.
Figure 1.3: Life Cycle of OryctesRhinoceros Larva
1.5.4 DAMAGE
Coconut rhinoceros beetle adults damage palms by boring into the
centre of the crown, where they injure the young, growing tissues and feed
on the exuded sap. As they bore into the crown, they cut through the developing
leaves. When the leaves grow out and unfold, the damage appears as
V-shaped cuts in the fronds or holes through the midrib.
1.5.5. NATURAL
ENEMIES
Rhinocerosbegtetle eggs, larvae, pupae, and adults may be attacked by
various predators, including pigs, rats, ants, and some beetles. They may
also be killed by two important diseases: the fungus Metarhiziumanisopliaeand
the Oryctesvirus disease.
1.9.6. MANAGEMENT
Rhinoceros beetles
can be controlled by eliminating the places where they breed and by
manually destroying adults and immature.
In many countries, the fungus Metarhiziumanisopliaeor
the Oryctes virus are used to control the rhinoceros beetle. More recently a chemical attractant,
ethyl-4-methyloctanoate, has been used in traps to attract and kill the
beetles. Both Metarhiziumanisopliaeand the Oryctesvirus are
present and helping to reduce rhinoceros
beetle populations in American Samoa; however, these pathogens and
the attractant have not yet received approval from the United
States Environmental Protection Agency for use as pesticides to
control the rhinoceros beetle.
Figure 1.4:
Decaying palm trunk
1.9.7. ECONOMIC IMPORTANCE
On
oil palms, O. rhinoceros bores into
the cluster of spears, causing wedge-shaped cuts in the unfolded fronds or
spears. In young palms where the spears are narrower and penetration may occur
lower down, the effects of damage can be much more severe than in older palms
(Wood, 1968a). The young palms affected by the beetle damage are believed to
have a delayed immaturity period (Liau and Ahmad, 1991). Thus, early oil palm
yields could be considerably reduced after a prolonged and serious rhinoceros beetle attack. Although Wood et al. (1973) suggested that the damage
to the immature palms results in relatively small crop losses, field
experiments conducted by Liau and Ahmad (1991) revealed an average of 25% yield
loss over the first 2 years of production. This was possibly caused by the
reduction in the canopy size of more than 15% for moderately serious to higher
damage levels (Samsudinet al., 1993).
In India, the infestation in oil palm was more prevalent in mature plantations
(10-15 year old) compared to immature or younger plantings (Dhileepan, 1988).
Similarly, on coconut the
reduction in leaf area of the palms influences nut production (Zelazny and
Young, 1979) but the attack was more towards the tall, mature trees, from about
5 years of age onwards (Bedford, 1976b). Considerably serious attacks on
coconut were also observed in areas adjacent to a breeding site with a high
beetle population, especially in the coastal region of Peninsular Malaysia.
Zelazny (1979) reported 5-10% damage resulting in 4-9% yield reduction;
similarly 30% damage resulted in 13% yield reduction.
1.10.
THE
GUT
In zoology, the gut, also known as the alimentary
canal or gastrointestinal tract, is a tube by which bilaterian animals
(including humans) transfer food to the digestion organs (Ruppertet al., 2004). In large bilaterians, the
gut generally also has an exit, the anus by the animal disposes off solid
wastes. Some small bilaterians have no anus and dispose of solid wastes by
other means (e.g. through the mouth) (Barnes et al, 2004).
Animals that have guts are classified as either
protostomes or deuterostomes, as the gut evolved twice, an example of
convergent evolution. They are distinguished based on their embryonic
development. Prostotomes develop their mouths first, while deuterostomes
develop their mouths second.
Prostostomes
include arthropods, molluscs, annelids, while deuterostomes include echinoderms
and chordates.The gut contains thousands of different bacteria, but humans can
be divided into three main groups based on those most prominent (Zimmer, 2011).
1.11.
PURIFICATION OF 3-MST
The purification
of the 3-Mercarptopyruvate Sulphur Transferase enzyme from the Oryctes rhinoceros larva involves the
combination of several methods such as:
i.
Ammonium Sulphate Precipitation
and Dialysis.
ii.
Bio-Gel P-100 and Affinity
Chromatography.
iii.
Protein concentration
determination which is carried out by
using Bradford Method of Protein Determination
iv.
The use of Nelson and
Somogyi method of assay to determine the activity of the enzyme in the
fractions.
1.12.
JUSTIFICATION
OF STUDY
Rhinoceros
larva feeds on woods and plants, especially the decayed palm trees. Plants are
known to possess defensive but toxic chemical called cyanide (Marcus Wischik,
1998). Therefore, rhinoceroslarva
should possess a cyanide detoxifying enzyme of which 3-MST is one.
1.13.
OBJECTIVES
OF STUDY
The
aim and objective of this study is to:
i.
Isolate 3-MST from the
gut of Oryctes rhinoceros larvae.
ii. Purify the 3- mercaptopyruvatesulfur transfer as eenzyme
isolated from the gut of Oryctesrhinoceros
larva.
Click “DOWNLOAD NOW” below to get the complete Projects
FOR QUICK HELP CHAT WITH US NOW!
+(234) 0814 780 1594
Login To Comment