ANALYSIS OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE (3-MST)
CHAPTERONE
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
Image
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.
ImageImage
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.
Image 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.
1.5.6. 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.
Image
Figure 1.4: Decaying palm trunk
1.5.7. 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.
1.6. 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).
1.7. 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.
1.8. 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.
1.9. 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- mercaptopyruvatesulfurtransferaseenzyme isolated from the gut of Oryctesrhinoceros larva.
.