ISOLATION AND PURIFICATION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE FROM THE GUT OF RHINOCEROS LARVA (Oryctes rhinoceros)
Cyanide is known to be one of the most toxic substances present in a
wide variety of food materials that are consumed by animals.
One of the cyanide detoxifying enzymes is 3-mercaptopyruvate
sulfurtransferase (3-MST). Indeed, recent studies have clearly shown that 3-
MST is involved in the detoxification of cyanide.
Rhinoceros (Oryctes rhinoceros) larva feeds on dead, decayed and living
plants, wood and palm. Plants are known to contain cyanide as a defence
mechanism for intruding/pesting organisms. Thus, for rhinoceros larva to be
able to live on plants, it must have possessed a cyanide-detoxifying enzyme.
3-MST, a cyanide-detoxifying enzyme was purified from Rhinoceros
(Oryctes rhinoceros) larva in this work.
The 3-MST enzyme was isolated from the gut of Oryctes rhinoceros larvae
and purified using Ammonium Sulphate Precipitation, Bio-Gel-P-100 Gel Filtration
Chromatography and Reactive Blue-2-Agarose Affinity chromatography.
The specific activity of the enzyme was 0.22U/mg.
The presence of this enzyme could be exploited by including it in the diet
of animals which would serve as a source of protein and 3-MST. Perhaps, these
rhinoceros larva could be introduced on farmland with contaminated soil whereby
they will process the dead roots and plants into soil thereby providing more space
and manure for plants to grow healthy.
TABLE OF CONTENTS
Title Page i
Table of Content v
List of Figures vi
List of Tables vii
1.0. Introduction and Literature Review 1
1.1. Introduction 1
1.2. 3-Mercaptopyruvate Sulfurtransferase 2
1.2.1. Distribution of 3-MST 5
1.2.2. Occurrence of 3-MST 5
1.2.3. Mechanisms of Action 6
1.2.4. Molecular Formula and Molecular Weight 7
1.2.5. Structure of 3-MST 8
1.2.6. Amino Acid Composition of 3-MST 8
1.2.7. Catalytic Activity of 3-MST 9
1.2.8. Enzyme Regulation of 3-Mercaptopyruvate sulfurtransferase 9
1.2.9. Stability of 3-MST 9
1.3. Physicochemical Properties of 3-MST 9 1.3.1. Optimal Temperature of 3-MST 9
1.3.2. Optimum pH of 3-MST 10
1.3.3. Effect of Metals/ions on 3-MST 10
1.3.4. Specific Activity of 3-MST 10
1.3.5. Inhibitory Studies of 3-MST 10
1.4. Cyanide 11
1.5. Oryctes rhinoceros Larvae 13
1.5.1. Taxonomy of Oryctes rhinoceros
1.5.2. Nutritional Qualities of Rhinoceros Larvae 13
1.5.3. Life Cycle of the Rhinoceros larva 15
1.5.4. Damage 16
1.5.5. Natural Enemies 16
1.5.6. Management 17
1.6. The Gut 18
1.7. Oryctes rhinoceros 19
1.7.1. Description of Development Stages 19
1.7.2. Distribution of Oryctes rhinoceros 21
1.7.3. Hosts/Species Affected 21
1.7.4. Economic Importance 23
1.8. Purification of 3-MST 27
1.9. Justification of Studies 28
1.10. Objectives of Research 28
2.0. Materials And Methods 29
2.1. Materials 29
2.1.1. Reagents 29 2.1.2. Apparatus Used 29
2.1.3. Study Sample 30
2.2. Method 30
2.2.1. Preparation of Buffer and Reagents 30
18.104.22.168. Preparation of 0.25M Potassium Cyanide 30
22.214.171.124. Preparation of 0.5M Potassium Cyanide 30
126.96.36.199. Preparation of 38% Formaldehyde 30
188.8.131.52. Preparation of 0.25M Ferric Nitrates (Sorbo Reagent) 31
184.108.40.206. Preparation of Bradford Reagent 31
220.127.116.11. Preparation of 0.38M Tris-HCl Buffer 31
18.104.22.168. Preparation of 0.30M Mercaptoethanol 31
2.2.2. Preparation of Crude Extract from the rhinoceros larva gut 32
2.2.3. Protein Concentration Determination 33
2.2.4. Assay for 3-Mercaptopyruvate Sulfurtransferase 34
2.2.5. Enzyme Purification 35
2.2.6. Substrate Specificity 37
4.0. Discussion, Conclusion and Recommendation 52
4.1. Discussion 52
4.2. Conclusion 52
4.3. Recommendation 52
1.0. INTRODUCTION AND LITERATURE REVIEW
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
3-mercaptopyruvate sulfurtransferase functions in the detoxifications of
cyanide; mediation of sulfur ion transfer to cyanide or to other thiol compounds.
(Vanden et 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 (Shibuya et 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 conditions Nagahara et al (2005).
Hydrogen sulphide (H2S) is an important synaptic modulator, signalling
molecule, smooth muscle contractor and neuroprotectant (Hosoki et al., 1997).
Its production by the 3-mercaptopyruvate sulfurtransferase and cysteine
aminotransferase pathways is regulated by calcium ions (Hosoki et al., 1997). Organisms that are exposed to cyanide poisoning usually have this
enzyme in them. This could be in food as in the cyanogenic glucosides 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. 22.214.171.124), is a member of the
group, Sulfurtransferases (EC 126.96.36.199 – 5), which are widely distributed
enzymes of prokaryotes and eukaryotes (Bordo and 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
(Crawhall et al., 1969). It catalyzes the chemical reaction:
3-mercaptopyruvate + cyanide à pyruvate + thiocyanate
3-mercaptopyruvate + thiol à 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
The systematic name of this enzyme class is 3-mercaptopyruvate:
cyanide sulfurtransferase. It is also called beta-mercaptopyruvate
sulfurtransferase (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 (Cipollone et 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
(Nagahara et al., 2004). The observation is supported by the finding that 3-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 intersubunit disulfide
bond serve as a thioredoxin-specific molecular switch (Nagahara et 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 (Nagahara et 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 (Nagahara et al., 2012). As an alternate hypothesis on the pathogenesis
of the symptoms, H2S (or HS−) and/or SOx could 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 SOx at the physiological level (Ampola et 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 (Ampola et 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) (Ampola et 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 (Ampola et 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)
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 (Nagahara et al., 1998).
Human mercaptopyruvate sulfurtransferase (MPST; EC. 188.8.131.52) belongs
to the family of sulfurtransferases (Vanden et 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) (Hannestad et al, 2006).
1.2.3. MECHANISMS OF ACTION
3-Mercaptopyruvate sulfurtransferase catalyzes 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 (Nahagara et 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 (Nagahara et 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
Figure 1.1: Structure of 3-mercaptopyruvate sulfurtransferase
1.2.6. AMINO ACID COMPOSITION OF 3-MERCAPTOPYRUVATE
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 sulfurtransferase rhodanase
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
(Nahagara et al 1995).
1.2.7. CATALYTIC ACTIVITY OF 3-MERCAPTOPYRUVATE
3-mercaptopyruvate + cyanide = pyruvate + thiocyanate (Fiedler and Wood,
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
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,
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/mg Vanchek 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
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
Thioglycolic acid: it slightly inhibits 3-MST. (Vachek and Wood, 1972).
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
The body has several mechanisms to effectively detoxify cyanide. The
majority of cyanide reacts with thiosulfate to produce thiocyanate in reactions
catalyzed by sulfur transferase 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;
Azcon et 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 osori by the Ijaws, tam by the
Ogonis and utukuru by the Ibos, all of Southern Nigeria.
ImageFigure 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
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 ORYCTES RHINOCEROS 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
Image Figure 1.3: Life Cycle of Oryctes Rhinoceros Larva
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
Rhinoceros begtetle 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 Metarhizium anisopliae and the
Oryctes virus disease.
Rhinoceros beetles .