The Effect Of Alkanes On The Physiology Biology Essay

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Abstract:

The influence of different alkanes on spore morphology, growth, cyt-P450 containing membrane fractions, enzymes of citric acid/glyoxlate cycle, total lipid contents and fatty acid composition by Isaria fumosorosea were investigated under laboratory conditions. Fungal spores grown on alkane sources showed higher germination and mycelium formation when compared to control. A strong induction of different enzyme systems (cytochrome P-450, glyoxylate and tricarboxylic acid enzymes) in cell free extracts or microsomal fractions was observed for cells grown on different alkanes when compared to glucose and control. Higher activities of glyoxylate and tricarboxylic acid cycle enzymes were observed for cells grown on alkanes when compared to other treatments. The even numbered fatty acids (oleic, linoleic and palamatic acids) accounted for almost majority of fatty acid production and there was a significant significant increase in relative amounts of linoleic acid and of palmatic acid for the conidia grown on alkanes. These results indicate that addition of alkane to culture media might be a tool to improve the metabolic functions which are directly related to virulence of entomopathogenic fungi.

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Key words: Entomopathogenic fungi; alkane growth; Cytochrome P-450; fatty acids, glyoxylate and tricarboxylic acid

1. Introduction

Insect-pathogenic fungi are widely distributed across the fungal kingdom. Some species are host specific, while others have a wide host range with individual isolates being more specific (Clarkson & Charnely, 1996). Unlike other insect pathogenic micro-organisms (viruses, bacteria, nematodes and protozoa) which require ingestion for disease intiation, entomopathogenic fungi enter into its host through the penetration of host cuticle. Insect cuticle acts as the first line of defence against different biochemical agents. Insect cuticle consists of three layers, epicuticle, procuticle and epidermis. Insect surface of the outermost layer or epidermis is composed of long chain hydrocarbons, fatty alcohols, fatty acids or wax esters (Pedrini et al., 2007; Figueria et al., 2009; Pedrini et al.,2010).

Entomopathogenic fungi can breakdown the insect cuticular lipids by using hydrocarbons which are supposed to support the fungal growth (Napolitano & Jaurez, 1997). Previously, Crespo et al., (2000) observed the catabolism of insect-like hydrocarbons by Beauveria bassiana and Metarhizium anisopliae.. B. basiana spores grown on alkane source showed higher virulence, by increased mortality and reduced time for host control (Crespo et al., 2002; Pedrini et al., 2009). Huang et al., (2012) also showed increased germination rate and higher virulence of alkane grown Lecanicillium muscarium conidia. Although much work has been carried out to observe the interaction between insect-pathogenic fungi and cuticular hydrocarbons of insect host (Pedrini et al., 2007), very few studies are available about the nature of proteins/enzymesinvolved in alkane degradation. A P450 enzymatic system is supposed to initiate the alkane dergradtion in most of the eukaryotic organisms. Tanaka et al., (1982) reported that cytochrome P-450 and NADPH reducatse was involved in alkane oxidation by. Apart from producing a fatty alcohol, P-450 also enhanced the production monooxiadtion products, finally yielding α-ω acids (Scheller et al., 1998). The fatty acids can extend across the membranes of peroxisomes, which will eventually be converted into fatty acyl-CoA by uninterrupted transformations of alcohol dehydrogenase, aldehyde dehydrogense and acyl-CoA synthetases, for the oxidation of substrates in peroxisomes (Tanaka & Ueda 1993; Crespo et al., 2000; Jaurez et al.,., 2004; Pedrini et al., 2007). The enzymes responsible in alkane degradation by entomopathogenic fungi have not been isolated, although the metabolic pathways are similar to yeast systems (Pedrini et al., 2007).

These studies were carried out to observe the changes in spore morphology, growth, cytochrome-P450 containing membrane fractions, enzymes of citric acid/glyoxlate cycle, total lipid contents and fatty acid composition of Isaria fumosorosea grown on n-alkane enriched media. These studies can be help to observe the process of alkane degradation by fungi and the possible role of alkane degradation in fungal infection process.

2 Material and methods

2.1 Chemical and reagents:

Hexadecane, n-ocatcosane, n-tetracosane were obtained from Sigma. The other chemicals were purchased from Guangzhou Jinhuadu chemicals and reagents, Guangzhou, China and Sigma.

2.2 Fungi:

Isaria fumosorosea (isolate-IF 28.2), was for these studies. I. fumosorosea was cultured on PDA and incubated at 25 ± 2oC for 10 days. Conidia were harvested with distilled water having 0.03% Tween-80 and final concentrations were determined by direct counting using hemocytometer under compound microscope

2.3 Fungal cultivation on alkanes:

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For enzymatic studenzyme assays I. fumosorosea was cultured on complete liquid medium containing (g/l distilled water) KH2PO4,0.4 g; Na2HPO4 ,1.4g; MgSO4.7H2O, 0.6 g; KCl, 1.0g; NH4NO3.7H2O, 0.7g; glucose 10g and yeast extarct, 5g.I. fumosorosea was also cultured in minimal medium containing complete medium ingredients without glucose and yeast extract (1% w/v): hexadecane, n-tetracosane or n-octacosane. Five milliliters of 1Ã-107 spores/ml were added to the basal medium and flasks were incubated for 10 days at 150 rpm and 26°C. After 10 days, fungal mycelia were harvested by filtration through Whatman filter paper (no.1). For biomass determinations, the filtered mycelia were dried at 80°C until constant weight.

To perform total lipid and fatty acid analysis I. fumosorosea was cultured on minimal medium (MM) containing KH2PO4,0.4 g; Na2HPO4 ,1.4g; MgSO4.7H2O, 0.6 g; KCl, 1.0g; NH4NO3.7H2O, 0.7g. Culturing was performed in solid medium having 2% agar supplemented with different carbon sources (Hexadecane, n-octacosane, n-tetradecane). Each of the hydrocarbons, hexadecane, n-ocatcosane, n-tetracosane, [2.5ml of a 10% (w/v) hexane solution], were layered on the media and allowed to evaporate whereas the addition of 1% glucose served as a control. The conidia were collected by scraping the colony after 10 days of inoculation (Huang et al., 2012).

2.4 Alterations in microsomal membrane fractions of I. fumosorosea grown on alkanes

2.4.1 Preparation of spheroplasts :

Fungal mycelia obtained through filtration after 10 days of growth were processed to spheroplasts by following Wolska-Mitaszko et al., (1981). I. fumosorosea mycelia (50 mg) were washed and concentrated in 1M sorbitol. The conecentrated mycelia were suspended in 1.4M Sorbitol, 40mM HEPES (pH 7.5), 0.5mM MgCl2 and a trace of ß-mercaptoetahnol for 5 min. The suspension was centrifuged at 4°C for 15 min followed by the addition of lyticase (5 mg/ml). This suspension was shaken at 20°C for 45 min and samples were checked for spheroplast formation. The spheroplasts were separated from the suspension through centrifugation at 2,500g for 15 min at 4°C.

2.4.2 Cell free extracts preparation and differential centrifugation:

The spheroplasts obtained through the treatment of fungal mycelia grown on different carbon sources were suspended in fractionation medium pH 7.4 (200 ml) having 20mM Tris-base, 20mM KH2PO4, 0.33M sucrose,1 mM EDTA and 0.2% BSA and the whole mixture was homogenized to prevent the aggregation of subcellular particles and homogenate was centrifuged through differential centrifugation (Kovae et al., 1968; Mauersberger et al., 1984). Intact spheroplast, nuclei and large debris were separated from the suspension by centrifugation at 3,000g for 10 min and the pellets were divided into two parts. The pellet obtained after centrifugation was homogenized for 1 min, diluted and centrifuged by repeating the above mentioned procedure and the supernatant thus obtained was named as cell free extract. The second part of pellet was centrifuged at 6,000g for 30 min at 4°C and the supernatant was carefully separated from mitochondrial peroxisomal fraction. This fraction was suspended in fractionation medium followed by centrifugation at 15,000g for 20 min. The supernatant was discarded leaving the post-mitochondrial pellet. This pellet was centrifuged at 15,000g for 60 min after re-suspension in fractionation medium and the pellet obtained was named as microsomal pellet was was suspended again in the fractionation buffer.

2.4.3 Enzyme activities:

Cytochrome P450 assay was performed by following Estabrook & Werringloer (1978) through CO differential spectra. Microsomes samples (1.5-2.0 mg/ml protein) were added to 1 ml 50mM Tris-HCl, pH 8.0. The mixture was gently sparged with CO for 2 min, followed by the addition of solid sodium dithonite. The sparging was further carried out for 2 min and the cuvette was closed. Absorbance was recorded between 400 to 500 nm for 5 to 10 min after the addition of sodium dithionite. Cytochrome aa3 concentrations were quantified from dithionite reduced-oxidized absorbance difference between 600 and 630 nm by using an extinction coefficient of 16.5mM/cm (William, 1964).

NADPH-cyt c reductase assay was performed through reduction of cytochrome c (Honeck et al.,1982). The reaction mixture (1ml) consisted of 810 µl stock reaction mixture (40 mM phosphate buffer, pH 7.5, 32mM nicotinamide and 50 µM cytochrome c), 225 µM sodium cyanide and 75 µM NADPH at 25°C. Protein samples (10μl) were added to the reaction mixture and absorbance was recorded at 550nm. The average change in absorbance for 1 min was recorded. The correction factor used for the calculation of nanomoles of cytochrome c reduced at 550 nm was 51.

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Cytochrome b5 concentration was quantified by observing the differential spectra between NADH-reduced and oxidized microsomes (Estabrook & werringloer, 1978; Pearce et al 1996). Dilution of Samples was carried out by adding 1 mg/ml protein sample to 100 mM KH2PO4, pH 7.4. After obtaining baseline of equal light absorbance between 400 and 500 nm, 5 μl of 20 mM β-NADH was added to the sample cuvette and the change in absorbance was observed between 410 -425 nm. The concentration of cytochrome b5 was detrmined fro the absorbance difference, using an extinction coefficient of 185mM/cm.

The cytochrome c oxidase activity was determined by using cytochrome c oxidase assay kit by Sigma. 0.95 ml of assay buffer (10mM tris-HCl, pH 7.0, 120mM KCl diluted with Buffer 5-fold with water) was added to cuvette and spectrophotometer was calibrated to zero reading. After this 0.2 ml enzyme sample was added to the cuvette and the solution was mixed by inversion. The reaction was started by the addition 50 µl ferrocytochrome c substrate solution. The change in absorbance per minute at 550 nm was observed and one unit of enzyme activity was defined as the enzyme that oxidize 1.0 mM of ferrocytochromec ph 7.0 per minute at 25°C.

Catalase activity was assayed by the method described by Beers and Sizer (1952). The H2O2 decomposition was observed at 240 nm. Reagent grade water (1,9ml) and 0.059M H2O2 (1.0 ml) were pipetted into the cuvette. The cuvette was incubated in spectrophotometer for temperature equilibration and to establish blank rate if any. After 5 min, 0.1 ml sample was added and change inabsorbance was recorded for 2-3 min. One unit of enzyme activity was defined as the enzyme that can decompose 1mM hydrogen peroxide per minute at an initial concentration of 30mM at pH 7.0 and 25°C.

Protohaem contents were determined by observing the changes in dithionite reduced-oxidized absorbance spectar between 541-557 nm with extinction coefficient 0f 20.7 mM/cm (Mauresberger et al 1984). Long chain ADH and ALDDH were quantified by the method of Yamada et al., (1980). The reaction mixture (1.5 ml) consisted of 50 mM Tris-HCl pH 8.9, 3.3 MM sodium azide, 3.3 mM NAD+ and 0.27 mM decanol. The alcohol dehydrogenase measurement were performed using ethanol (130mM) as substrate.

Protein concentration was quantified by the Lowry's method using BSA as standard (Lowry et al., 1951)

2.4 Influence of tricarboxylic and glyoxylate cycle enzymes.

Citrate synthase activity was analysed by using citrate synthase assay kit provided by Sigma. Briefly, the mycelium (50 mg) were dissolved in 50mM Tris- HCl buffer pH 8.0 (3ml). The suspension was centrifuged at 15,000 g for 20 min and the cells free extract was resuspended in 125 µl cell lytic reagent. Reaction mixture (1ml) consisted of 50mM Tris-HCl buffer pH 8.0 (930µl), 30mM Acetyl CoA solution (10 µl), 10mM DTNB (10 µl) and cell free extract (50 µl). Absorbance of reaction mixture was observed at 412 nm for 1.5 min to calculate baseline reaction. After this 50 µl of 10mM oxaloacetic acid solution was added and mixed by inversion. The absorbance was read for 1.5 min and the net citrus synthase activity was calculated by using difference in absorbance and extinction coefficient of 13.6. One unit of enzyme activity was defined as nanomoles of substrate converted into product per minute per mg protein.

Aconitase activity assay was performed by following Morrison (1954). Briefly, 15 mg mycelium and 2.15 ml activation buffer (Prepared by combining 4 ml of 100 mM Tris buffer, pH 7.4, 0.10 ml 0f 1 MM ferrous ammonium sulphate solution and 0.2 ml of 50 mM L -Cysteine Solution, pH 7.4) were mixed and incubated at 0°C for 1 hr. In the mean time pipette out 1.45 ml of deionized water, 1.0 ml of 100 mM Tris buffer, pH 7.4, 0.1 ml of 2 mM citric acid solution, 0.1 ml of 5.4 mM ß -Nicotinamide Adenine Dinucleotide Phosphate solution, 0.20 ml of 20 mM Manganese Sulfate and 0.05 ml of Isocitric Dehydrogenase enzyme Solution in two different cuvettes marked as as test and blank. Mix the solution by inversion and equilibrate at 25°C. Monitor the A340nm until constant. Then Activated Aconitase Enzyme Solution 0.10 ml was added to the test cuvette while to the blank 0.10 ml activation buffer was added. The solution was immediately mixed by inversion and change in absorbance at 340nm was observed for 5 minutes. Of enzyme activity was defined as the enzyme that can convert 1.0 µM of citrate (via cis-aconitate) to isocitrate per minute at ph 7.4 and 25°C.

Isocitric dehydrogenase (NADP) assay was performed by following Bergmeyer (1974). Briefly, the mycelium (50 mg) were dissolved in 250 mM Glycylglycine Buffer, pH 7.4 (3ml). The suspension was centrifuged at 15,000 g for 20 min to obtain the cells free extract. The reaction mixture (2.9ml) contained 1.95 ml of deionized water, 0.50 ml of 250 mM Glycylglycine buffer, pH 7.4, 0.20 ml of 6.6 mM DL-isocitric acid solution, 0.15 ml of 20 mM ß-Nicotinamide adenine dinucleotide phosphate, and 0.10 ml of 18 mM manganese chloride solution. The blank (3ml) contained 1.95 ml of deionized water, 0.60 ml of 250 mM Glycylglycine Buffer, pH 7.4, 0.20 ml of 6.6 mM DL-isocitric acid solution, 0.15 ml of 20 mM β-Nicotinamide adenine dinucleotide phosphate, 0.10 ml of 18 mM manganese chloride solution. The solutions were mixed by inversion and equilibrated at 37°C and absorbance was recorded at 340 nm. Then 0.10 ml of cell free extract was added to the reaction mixture and change in absorbance was observed for 5 minutes. Change in absorbance per minute was calculated for test and blank. One unit of enzyme activity was defined as the enzyme which can convert 1.0 µM of isocitrate to α-Ketoglutarate per minute at pH 7.4 at 37°C.

Isocitric lyase assay was performed by following chell et al., (1978). Briefly, the mycelium (50 mg) were dissolved in 50 mM Imidazole Buffer, pH 6.8 (3ml). The suspension was centrifuged at 15,000 g for 20 min to obtain the cells free extract. The test as well blank mixtures (1.0 ml) contained 0.50 ml of 50 mM Imidazole buffer, pH 6.8, 0.10 ml of 50 mM magnesium chloride solution, 0.10 ml of 10mM EDTA solution, 0.10 ml of 40 mM Phenylhydrazine HCl solution, and 0.10 ml of 10 mM DL-Isocitric Acid Solution. The solutions were mixed by inversion and equilibrated at 30°C and absorbance was read at 324 nm until constant. Then 0.10 ml of cell free extract was added to the test mixture and 0.10 ml of 50 mM Imidazole buffer was added to the blank. The change in absorbance was observed for 5 minutes. The change in absorbance per minute was calculated for test and blank. One unit of enzyme activity was defined as the enzyme which can catalyze the formation of 1 µM glyoxylate per minute at pH 6.8 and 30°C.

Malate synthase assay was performed by following chell et al., (1978). Briefly, the mycelium (50 mg) were dissolved in 50 mM Imidazole buffer, pH 8.0 (3ml). The suspension was centrifuged at 15,000 g for 20 min to obtain the cells free extract. The test as well blank mixtures (1.0 ml) contained 0.50 ml of 50 mM Imidazole buffer, pH 8.0, 0.10 ml 100 mM magnesium chloride solution, 0.10 ml of 2.5 mM Acetyl CoA solution, 0.10 ml of 10 mM Glyoxylic acid solution, and 0.10 ml of 2 mM 5,5-Dithio-bis(2-Nitrobenzoic acid) solution (DTNB). The solutions were mixed by inversion and equilibrated at 30°C and absorbance was read at 412 nm until constant. Then 0.10 ml of cell free extract was added to the test mixture and 0.10 ml of 50 mM Imidazole buffer was added to the blank. The change in absorbance was recorded for 5 minutes. The change in absorbance per minute was calculated for test and blank. One unit of enzyme activity was defined as the enzyme which can catalyze the cleavage of one micromoles of acetyl-CoA per minute at pH 8.0 and 30°C, in the presence of glyoxylate.

NADP- dependent Glutamate dehydrogenase assay was performed by following Shimizu et al., (1979). Briefly, the mycelium (50 mg) was dissolved in 0.1 M Tris-HCl buffer, pH 8.3 (3ml). The suspension was centrifuged at 15,000 g for 20 min to obtain the cells free extract. The reaction mixture (2.95 ml) contained 2.5 ml of 0.1M Tris-HCl buffer, pH 8.3, pH 8.0, 0.20 ml of 3.3M NH4Cl solution, 0.10 ml of 0.225M α-Ketoglutarate solution, 0.10 ml of 7.5 mM NADPH solution and 0.05 ml cell free extract. The solutions were mixed by gentle inversion and change absorbance at 340 nm against water was recorded for 2-3 minutes. The change in absorbance per minute was calculated for test and blank. One unit of enzyme activity caused the oxidation of 1.0µM of NADPH per minute at pH 8.3.

Fumarase assay was performed by following Bergmeyer (1974). Briefly, the mycelium (50 mg) were dissolved in 100mM phosphate buffer, pH 7.6 (3ml). The suspension was centrifuged at 15,000 g for 20 min to obtain the cells free extract.Then 2.90 ml of 50mM L-Malic Acid (Malate) prepared in 100mM Potassium Phosphate Buffer, pH 7.6 at 25°C was added to the test and blank cuvettes. Blank cuvette also contained 0.10 ml of 0.1% Bovine Serum Albumin (BSA). The solutions were mixed by gentle inversion and and equilibrate to 25°C and absorbance was read at 240 nm until constant. Then 0.10 ml of cell free extract was added to the test mixture The change in absorbance was recorded for 10 minutes. The change in absorbance per minute was calculated for test and blank. One unit of enzyme activity was defined as the enzyme which can convert 1.0 µM of L-Malate to Fumarate per minute at pH 7.6 and 25°C.

Malate dehydrogenase assay was performed by following Glepi et al (1992). Briefly, the mycelium (50 mg) were dissolved in 0.1 phosphate buffer, pH 7.4 (3ml). The suspension was centrifuged at 15,000 g for 20 min to obtain the cells free extract.The test and blank cuvettes contained 2.6 ml of 0.1 M phosphate buffer, pH 7.4, 0.2 mlof 3.75mM NADH and 0.1 ml of 6mM Oxaloacetic acid. cuvettes were incubated in spectrophotometer for temperature and blank rate equilibration. O.1 ml cell free extract was added to the test cuvette and change in absorbance at 340 nm was observed for 5 minutes. The change in absorbance per minute was calculated for test and blank. One enzyme unit oxidized 1µM of NADH per minute at 25°C and ph 7.4

2.5 Analysis of fungal lipids and fatty acids

The spores grown on different carbon/alkane sources were extracted with chloroform:methanol (2:1, v/v) and diluted with distilled water (5:1, v/v) (Folch et al., 1957). Then, samples were reduced under vaccum or N2 stream and stored at -18°C. The chloroform layer was dried to constant dried weight to obtain the total lipids in the sample. Silica gel plates developed in petroleum ether: diethyl ether: acetic acid (80:20; v/v) were used to perform the total lipid assay. Plates were charred after immersion in 10% H2SO4. For individual fatty acid analysis, the chloroform extract was evaporated under N2 and resuspended in 1 ml toluene. Trans esterification was carried out with BF3-methanol for one hour at 100°C. The resulting fatty acid methyl esters (FAME) were extracted with chloroform and partitioned against distilled water (3:2, v/v).

2.6 Statistical analysis:

The studies were replicated three times on different dates and the values of the results were expressed as average of triplicate determinations. Enzymatic activities data were analyzed by ANOVA and treatment means were compared by using Tukey's HSD test at 5% level of significance. All the analysis was performed by using SAS 8.01 (SAS, 2000).

3 Results:

3.1 Effects of different carbon/alkane sources on growth morphology

The effect of different carbon/alkane sources on the morphology of I. fumosorosea cells was tested microscopically after 5 days of growth. Very low conidial germination or mycelium formation was observed when I. fumosorosea was grown on glucose. I. fumosorosea conidia grown on alkane sources (n-tetracosane, hexadecane and n-octacosane) showed higher rates of germantion as well as formation of pseudomycelium in various proportions (Fig. 1).

3.2 Characterization of I. fumosorosea cell free extracts (S3).

I. fumosorosea grown in liquid medium having 1% of different carbon sources produced a range of constituents of alkane hydroxylation system corresponding to different hydrocarbon sources. Contents of these constituents from cell free extracts of 5 day culture are shown in Table-1.

As shown in Table-1, Cytochrome-P450 activity was exhibited by I. fumosorosea under liquid culture conditions having different carbon sources; however, the amount os secreted enzyme varied significantly among treatments and control (F4,10 = 10.6, P<0.001). The highest level of Cytochrome-P450 (0.27 ± 0.02 nmol/mg ) was found in the cell free extracts of cultures produced on n- octacosane while the lowest Cytochrome-P450 activity was detected for contro having a mean value of 0.00 ± 0.00 U/mg.

The NADPH- cyt c reductase activity shown by cell free extracts of I. fumosorosea was significantly different among different treatments (F4,10 = 24.21,P<0.001). The lowest NADPH- cyt c reductase activity was observed for control having a mean value of 0.04 ± 0.00 µmol/min whereas the highest NADPH- cyt c reductase activity (0.38 ± 0.02 µmol/min) was found for cell free extracts grown on n- octacosane (Table-1).

The ADH production by I. fumosorosea grown on different carbon sources differed significantly among different treatments (F4,10 = 15.68; P< 0.001). Maximum ADH activity (0.34 ± 0.02 µmol/min) observed for cell free extracts of I. fumosorosea grown on n- octacosane whereas the lowest ADH activity was observed for control having a mean value of 0.03± 0.01 µmol/min.

ALDDH were produced in varying amounts by cell free extracts of I. fumosorosea grown on different carbon sources (F4,10 = 28.01, P<0.001). n-octacosane proved to be the most active inducer of ALDDH activity showing average activity of 0.41± 0.03 µmol/min while the lowest ALDDH activity (0.06± 0.01 µmol/min) was observed for control (Table-1).

Significant differences were also observed for cyt a-a3 and cytc oxidase production by cell free extracts of I. fumosorosea. Maximum cyt a-a3 and cytc oxidase contents were observed from cell free extracts of I. fumosorosea grown on n- octacosane having mean values of 0.25 ± 0.02 and 0.38 ± 0.03 nmol/mg, respectively whereas the lowest cyt a-a3 and cytc oxidase contents (0.09 ± 0.01 and 0.16 ± 0.01 nmol/mg) were observed for control.

The catalse activity shown by cell free extracts of I. fumosorosea was significantly different among different treatments (F4,10 = 19.77, P<0.001). The lowest catalse activity was observed for control having a mean value of 84 ± 2.37 µmol/min whereas the highest catalase activity (864 ± 17.49 µmol/min) was found for cell free extracts grown on n- octacosane (Table-1).

3.3 Characterization microsomal farctions (P100) of I. fumosorosea grown on different alkane sources.

As shown in Table-2, Cytochrome-P450 activity was exhibited by I. fumosorosea under liquid culture conditions; however, the amount of screted enzyme varied significantly among different treatments and control (F4,10 = 18.47, P<0.001). The highest level of Cytochrome-P450 (0.57 ± 0.03 nmol/mg ) was found in the microsomal fractions of cultures produced on P.xylostella cuticle extract while the lowest Cytochrome-P450 activity was detected for control having a mean value of 0.00 ± 0.00 U/mg.

The NADPH- cyt c reductase activity shown by microsomal fractions of I. fumosorosea was significantly different among different treatments (F4,10 = 23.47, P<0.001). The lowest NADPH- cyt c reductase activity was observed for control having a mean value of 0.20 ± 0.01 µmol/min whereas the highest NADPH- cyt c reductase activity (0.82 ± 0.04 µmol/min) was found for microsomal fractions grown on n- octacosane (Table-2).

The cyt b3 production by I. fumosorosea differed significantly among different treatments (F4,10 = 19.71, P<0.001). Maximum cyt b3 activity (0.73 ± 0.03 nmol/mg) was shown by microsomal fractions of I. fumosorosea grown on n- octacosane whereas the lowest cyt b3 activity was observed for control having a mean value of 0.17± 0.01 nmol/mg.

Significant differences were also observed for NADH cyt c reducatse production by cell free extracts of I. fumosorosea grown on different carbon sources. Maximum NADH cyt c reducatse contents were observed from microsomal fractions of I. fumosorosea grown on n- octacosane having mean value of 0.37 ± 0.03 µmol/min whereas the lowest NADH cyt c reducatse contents (0.07 ± 0.01 µmol/min) were observed for control.

Protohaem were produced in varying amounts by microsomal fractions of I. fumosorosea grown on different carbon sources (F4,10 = 23.67, P<0.001). n- octacosane was proved to be the most active inducer of protohaem showing average activity of 1.63± 0.05 µmol/min while the lowest protohaem activity (0.61± 0.01 µmol/min) was observed for control (Table-2).

3.4 Effect of different carbon/alkane sources on tricarboxylic acid and glyoxylic acid cycle enzymes.

The production of different TCC and glyoxylate cycle enzymes by I. fumosorosea cells grown on different carbon/alkane sources were significantly different among different treatments and control. Higher levels of enzyme production were observed for I. fumosorosea cells grown on different alkane sources when compared to the glucose grown cells (Fig- 2). Highest levels of enzyme production were observed for I. fumosorosea cells produced on n- octacosane whereas the lowest enzyme activities were observed in control. The activities of iso-citrate dehydrogense, fumarase, malate dehydrogense were inhibited in glucose treatment when compared to other treatments (Fig.2).

3.5 Total lipids and fatty acid production by entomopathogenic fungi

The total fungal lipid production by I. fumosorosea cells grown on different substrates was significantly different among different treatments and control. The highest total lipid production (14.67 mg/g spores) was observed for fungal cells grown on n-octacosane where as the lowest lipid production was observed for control having a mean value of 9.7 mg/g spores (Table-3). The fatty acid production profile of I. fumosorosea spores grown on glucose consisted of three major fatty acids: oleic acid (50.99%), linoleic acid (16.55%) and palmatic acid (14.59%). This pattern of fatty acid production by I. fumosorosea spores grown on different alkanes (n-tetracosane, hexadecane and n-octacosane) was different from glucose and control treatments. Oleic acid was the major component of fatty acids but a significant increase in relative amounts of linoleic acid and of palmatic acid production. Very low production of fatty acids with chain lengths over 18:3 were also observed in different treatments (Table-3).

Discussion:

Very low rates of germination as well as mycelium formation were observed for I. fumosorosea conidia grown on and control treatments whereas higher germination as and mycelium differentiation was shown by fungal cells produced on different alkane sources. The possible explanation of the reason controlling these morphological changes is not very clear but higher production of lipids by the cells grown on alkane sources can be one of the reasons influencing this phenomenon (Omer & Rehm, 1980).

The growth of I. fumosorosea cells through alkane utilization was linked with significant changes in enzyme profiles of cell free extracts as well microsomal fractions. The growth of fungal cells on different alkane sources can produce microsomal cyt P-450 system and dehydrogenase enzymes (ADH, ALDDH), which play an important role in hydrocarbon degradation into fatty acids. It can also induce the action of catalase during ß-oxidation of fatty acids in peroxisomes (Fukui & Tanaka, 1979). Similar works have also been carried out in different alkane assimilating fungi at cellular as well as subcellular levels with special focus on these enzyme systems (Gilewicz et al., 1979; Takagi et al., 1980, Yamada et al., 1980; Marchal et al., 1982). These changes in enzymes composition should be seen in relation to ultrastructural changes in fungal cells during alkane utilization. Fukui & Tanaka (1979) gave a possible model explaining the role of different subcellular components during alkane utilization by observing the occurrence and changes in concentration of cyt P-450 and alkane hydroxylase in microsomal fractions. However, the subcellular existence of these enzymes has not been explained in detail due to the unavailability of reliable enzyme markers (Delaisse et al., 1981). Because of the heterogeneous production of cyt P-450 containing microsomal fractions and the varied production of ADH andALDDH at subcellualr distribution of level, it can be postulated that dehydrogenase activities in the microsomes was because of the contamination of mitochondrial outer membranes containing ADH or peroxisomal membrane fragments, which are sensitive to mechanical stress (Gallo et al., 1974; Depierre & Dallner 1976).

The higher production citrate synthase and isocitrate lyase in I. fumosoroesa cells produced on alkane sources can explain the role of glyoxylate cycle in alkane adaptation by this fungus. Omer & Rehm (1980b) also reported higher production of isocitrate lyase by Candida parapsilosis cells grown on tetradecane when compared to other treatments. Similar results have also been reported by Lozinov et al., (1976) and Nabeshina et al., (1977) with different fungal species. The high malate synthase and malate dehydrogenase activities on alkane grown medium is also another explanation of the role glyoxlate cycle in alkane adaptation. The lower production of malate dehydrogense in control and glucose treatments was also reported by Polaski and Bartley (1965) using Sacchromyces cereviasie. These results are in accordance with witt et al., (1966) and Ferguson et al., (1967) who explained the role of glyoxlate cycle in alkane adaptation of S. cereviasie.

The insect-pathogenic fungi need an external an external carbon source for their growth, which is usually supplied by glucose or yeast extract present in the culture media (Smith & Grula, 1981). In these studies, the growth of I. fumosorosea on different alkanes resulted in an increase in lipid contents of fungal conidia, which can lead to higher fat metabolism in these cells. Crespo et al., (2002) also showed change in total lipid contents of B. bassiana grown on n-hexadecane. The fatty acid profile of I. fumosorosea cells grown on different alkanes predominantly consisting of C14, C16 and C18 fatty acids which are in line with previous studies. The major change in fatty acid composition between alkane grown and glucose grown conidia was differential production of C16:0 and C16:1 fatty acids. The higher production of C18 fatty acids in glucose grown cells can be due to the de novo synthesis via malonyl pathway. The higher production of C16 fatty acids in alkane grown cells also explain that monoterminally the oxidized substrate can be directly incorporated into their cell lipids (Omar & Rehm 1980b).

The variation in proteins as well as lipid synthesis showed the intricacy of induction process by alkanes. However the regulation of the protein and lipids at different levels during alkane degradation as well assimilation require further investigation for the possible development and improvement of high virulent fungal strains.