Induced oxidative stress by Metarhizium anisopliae spp . instigates changes in lipid peroxidation and ultra structure in Periplaneta Americana

Consequent to injection of conidia at LC30, LC50 and LC90 doses of the entomopathogenic fungal isolates of Metarhizium anisopliae (Metch.), alterations in lipid peroxidation and ultrastructural changes were observed in cockroaches (Periplaneta americana). A decrease in the level of lipid peroxidation was evident in the treated cockroaches compared to those of control. Present investigation reports the effects of fungal infection on the midgut ultrastructure of the adult cockroaches which was manifested in the form of damage to the microvilli, epithelial cell vacuolization, necrosis and disruption of the epithelial cell membrane which occurred with increase in the time interval post treatment with conidia from high virulent isolate (M20) at LC50 dosage. Our study reveals for the first time that fungal infection instigates oxidative stress in the cockroach and that the villi of the midgut are also the target organs for the oxidative damage.


Metarhizium
anisopliae (Metschnikoff) Sorokin (Hypocreales: clavicipitaceae), is a ubiquitous insect parasitic fungus (Rehner, 2005) and the causal organism of green muscardine disease.The entomopathogenic fungus, M. anisopliae (Metchnikoff) Sorokin has been reported to infect more than 200 species of insects belonging to different orders (Zimmermann, 1993).M. anisopliae produces destruxins both in culture and in vivo in the infected insects and were reported to be the candidates for mortality in insects.Kershaw et al. (1999) reported that, in the pathogenesis of M. anisopliae var anisopliae, there is a relationship between the titer of DTX production of isolates in vitro and the killing power.DTX A induces adverse ultrastructural changes in the epithelial cells and villi of midgut of the lepidopteran pest Galleria mellonella (Dumas et al., 1996).This toxin was found to possess uncompetitive inhibitory effect on the hydrolytic activity of vacuolar-type ATPase in the brush border membrane vesicles of the midgut of G. mellonella larvae (Bandani et al., 2001).The detection, characterization and analysis of the role of reactive oxygen species (ROS) is well established in both normal and pathological processes of cellular metabolism.
Lipid peroxidation refers to the oxidative degradation of lipids.It is the process in which free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage.This process proceeds by a free radical chain reaction mechanism and fatty acid radical is produced.The most notable initiators in living cells are ROS, such as Hydroxyl radical (OH • ) and HO 2 , which combines with a hydrogen atom to make water and a fatty acid radical.The peroxidation of lipids is basically damaging because the formation of lipid peroxidation products leads to spread of free radical reactions.Lipid peroxidation is one of the major outcomes of free radicalmediated injury to tissue.
Studies have revealed the susceptibility of the cellular components from the attack of ROS, but the major multifold effects are manifested in the form of loss of ions and protein cleavage.The generation of ROS like OH - and superoxide radical (O 2 -) radicals disintegrates biomembranes by lipid peroxidation which is a general mechanism of stress induced responses in living systems as was reported by Panda and Chaudhury (2003).ROS include oxygen ions, free radicals and peroxides, both inorganic and organic.These molecules are generally very small and highly reactive, because of the presence of unpaired electrons (Aslanturk et al., 2011).ROS are formed as a natural byproduct of the normal metabolism of oxygen.They play an important role in cell signaling and the induction of host defense genes (Dalton et al., 1999).Studies reported that organophosphate pesticides caused lipid peroxidation and the alterations in the antioxidant defence enzymes of insect (Gupta et al., 2010;Wu et al., 2011).
To minimize the potential threats of ROS, the cells are equipped with numerous antioxidant defense systems.Their function is to maintain low steady state levels of ROS and other radicals in the cell, a process involving precise regulation of their location and amount.The antioxidant enzymes, such as superoxide dismutase (SOD,E.C. 1.15.1.1),catalase (CAT,E.C. 1.11.1.6)and peroxidase (POX,E.C. 1.11.1.7)form a part of the defence system (Joanisse and Storey, 1996a).Catalases and peroxidases are the most important enzymes that degrade peroxide into water and oxygen.These enzymes and superoxide dismutase are the first lines of cell defence against ROS.
Antioxidant defence was measured by activities of SOD, CAT and ascorbate peroxidase (APOX).CAT and POX, more appropriately the specific APOX, act to remove these peroxides.The key step in oxidative stress is the production of ROS which initiate a variety of auto-oxidative chain reactions on membrane unsaturated fatty acids and proteins, producing lipid peroxides and protein carbonyls respectively resulting in a cascade of reactions ultimately leading to destruction of organelles and macromolecules (Jamieson, 1989).Hydroxyl radical (OH .), hydrogen peroxide (H 2 O 2 ) and superoxide radical (O 2 .-), the ubiquitous products of single electron reductions of dioxygen, are amongst the most reactive compounds known to be produced during oxidative stress (Dietz et al., 1999).Lipid peroxidation is a well-established mecha-nism of cellular injury in both plants and animals and is used as an indicator of oxidative stress in cells and tissues (Esterbauer et al., 1991).Lyakhovich et al. (2006) opined that various antioxidants that were present in the insect may decrease the level of lipid peroxidation.
Cockroaches are insects of the order Blattodea with about 4,500 species, is widely distributed throughout the world.30 species of cockroaches are associated with human habitations and about four species are well known as pests (Valles et al., 1999).Cockroaches are implicated as vectors of several human disease agents.Periplaneta americana (American cockroach) is the well known pest species and ubiquitous throughout the world.Considering cockroach diversity, it is necessary to evaluate virulence of the insect pathogenic fungal isolates of M. anisopliae on hand against the locally prevailing populations.There is paucity of information regarding pathogenecity of M. anisopliae against P. americana.Therefore, the present study is undertaken with the objective of exploring the mode of infection of M. anisopliae against P. americana.
The aim of the present study was to reveal the induced oxidative stress in terms of levels of lipid peroxidation in P. americana when infected with conidia of M. anisopliae through injection process with three different lethal concentrations, LC30, LC50 and LC90 at 1, 24 th and 48 th h post treatment.The dynamics in the levels of lipid peroxidation were studied among the insects that were subjected to infection with three isolates of M. anisopliae which differ in their virulence.Microscopic studies were made to observe changes in the ultrastructure of mid gut villi in treated cockroach to unravel the consequences of the oxidative stress induced through fungal infection.
The treated insects after death and mummification were transferred to humid chambers for promoting mycosis.The mycosed and sporulated insects were used to reisolate the fungus.The isolates at the first cycle after reisolation were used for evaluation.
Germination of the conidia tested on SDAY prior to experimentation revealed a range of 90 -95% values for the four isolates used in the investigation.All the solvents used as medium components of the culture media and artificial diets were from Merck (India) ltd.

Experimental insects
P. americana (American cockroach) adults were initially collected from their natural habitats like godowns.The adult cockroaches were selected and were transferred to wooden framed boxes of 30.5 x 30.5 cm dimensions with iron mesh on one side for aeration.The cages were kept dry and the insects were fed with hardened bread.An absorbent cotton piece soaked in water was kept in a bowl to serve as a source of water and moisture to the roaches.The food was changed for every two days.Rearing conditions were standardized so as to ensure 100% survival in laboratory conditions.The healthy adult roaches were kept for observation for one week before starting the experiment.Insects measuring 3.25 cm (± 0.1) length and 1.5 gms (± 0.1) weight were selected for treatments.The experiment set up was maintained at a temperature of 29± 1°C with photo period conditions of 12 h darkness followed by 12 h light.

Mode of treatment
The lipid peroxidation assays were conducted on P. americana using three isolates (M20, M48 and M19) of M. anisopliae.Injection method as adopted by Gunnarsson and Lackie (1985) was followed with slight modifications for treating the cockroaches with fungal conidia.Twenty micro liters of conidial suspension at 5x10 7 (1x10 6 conidia in 20 µl), 5x10 6 (1x10 5 conidia in 20µl), 5x10 5 (1x10 4 conidia in 20 µl), 5x10 4 (1x10 3 conidia in 20 µl), 5x10 3 (1x10 2 conidia in 20µl) conidia/ml was injected in to the haemocoel of the cockroach.Injection was done using a 1 ml disposable syringe holding a 0.30 x 8.0 mm needle on the ventral side of the roach body piercing through inter segmental region of 5 th and 6 th segments as was done by Vilcinskas and Matha (1997).The suspension was released gently so as to ensure effective spread of conidial suspension into haemocoel of the insect.Mortality data was recorded at 6 h intervals starting at 6 h post treatment and continued for 5 days.The data obtained from the bioassays was subjected to probit analysis using SPSS 11.0 version software to obtain values of median lethal concentrations that caused 30% (LC30), 50% (LC50) and 90% (LC90) deaths at the 48 th h post treatment.
Three lethal concentrations LC30, LC50 and LC90 were selected to treat the cockroaches (Table 1) for conducting lipid peroxidation assay.LC50 was used for scanning electron microscopy (SEM) and transmission electron microscopic (TEM) studies.The changes in the lipid peroxidation levels were recorded at 1, 24, and 48 h post treatment.LC50 and LC90 for M19 isolate and LC90 for M48 isolate were too high to count with haemocytometer and hence were not included in the study.The control cockroaches were treated with 0.02% tween solution.

Lipid peroxidation
The lipid peroxidation level was measured using the thiobarbituric acid-reactive substances (TBARS) assay.Briefly, the treated and control samples were homogenized (1:10 w/v) in 3 ml of 0.5% thiobarbituric acid (TBA) in 20% trichloroacetic acid (TCA) (w/v).The homogenate was incubated at 95°C for 30 min, and the reaction was stopped in ice.The samples were centrifuged at 10,000 g for 10 min and absorbance of the resulting supernatant was recorded at 532 and 600 nm.The non specific absorbance at 600 nm was subtracted from that at 532 nm.The absorbance coefficient of malondialdehyde (MDA) of 155 /mM`/cm was used for estimating the degree of lipid peroxidation (Heath and Packer, 1968).
Ten (10) insects were used for each treatment and the experiment has been repeated thrice.In each treatment, triplicate readings for lipid peroxidation were taken and the data obtained from the experiments was subjected to analysis of variance (ANOVA) using SPSS version 11.0 statistical software to obtain standard error means (SEM).Since the trend in dynamics of lipidperoxidation was found to be similar in repeated experiments, the results of the final experiment were taken as concluding values.

Transmission electron microscopic (TEM) study
For TEM studies, the anterior thorax region of the live insects after 10,12 16, 20 and 24 h post treatment was cut and gut was transferred to vials and fixed in 2.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 h at 41°C.These samples were post-fixed in 0.5% aqueous osmium tetroxide in 0.05 M phosphate buffer for 2 h.After post-fixation, samples were dehydrated in a series of graded alcohol, infiltrated and embedded in spurr's resin.The ultra-thin sections were cut with a glass knife on an ultra microtome (Leica Ultra Cut UCT-GA-D/E-1/100).The sections of 50-70 nm thickness were mounted on grids, stained with saturated aqueous uranyl acetate and counter-stained with 4% lead citrate (Bozzola and Russell, 1999).The sections were observed under a TEM (Hitachi, H-7500).

Scanning electron microscopy (SEM)
Scanning electron microscopy was done by the method proposed by John Bozzolla (1999).The cockroaches were injected with conidia (LC50) from high virulent isolate of M. anisopliae (M20).Live insects after 24 th and 48 th h post treatment were dissected and their fat bodies were processed for SEM.The samples were fixed in 2.5% gluteraldehyde in 0.1 M Phosphate buffer (pH 7.2) for 24 h at 4°C and post fixed in 2% aqueous osmium tetroxide for 4 h in the same buffer.After the fixation, samples were dehydrated in series of graded alcohols and dried to critical point drying with Electron Microscopy Science CPD unit.The dried samples were mounted over the stubs with double-sided carbon tape and applied a thin layer of gold coat over the samples by using an automated sputter coater (JOEL JFC-1600) for 3 min.The samples were scanned under scanning electron microscope (Model: JOEL-JSM 5600) at various magnifications.

RESULTS AND DISCUSSION
The assay of the lipid peroxidation in the cockroaches treated with fungal conidia revealed a decrease compared to those of the untreated samples.The insects treated with M20 isolate at LC30 dose displayed a decrease  of 54% over the control at 1 h post treatment.At LC50 treatment, an increase in lipid peroxidation was recorded compared to the corresponding values of LC30 at 1 st and 48 th h post treatment but showed a decrease at the 24 th h (Figure 1).On the other hand, at LC90 treatment, the lipid peroxidation levels increased over the control values by 28% at 1 h post treatment but as the time of treatment increased, at the 24 th and 48 th h, the values decreased.On the other hand, with M19 a high virulent isolate and M48 a low virulent isolate, decrement in lipid peroxidation levels with progression in the post treatment time was displayed.
Among the treated ones with high virulent isolate M20 at 24 th h post treatment, the insects infected with LC30 displayed an increase in lipid peroxidation by 11% with the advancement of post treatment time from 1 h and by the 48 th h, 11% decrease was revealed.At LC50, there is an initial decrement of lipidperoxidation of about 24% which increased by 50% by the 24 th h.For the same isolate, 28% increase in lipid peroxidation in insects infected with LC90 at 1 h post treatment and a gradual decrease by 60% by the 24 th h was recorded.With another high virulent isolate M19, at LC30, similar trend as shown by insects treated with LC30 of M20 has been revealed where an increase in lipid peroxidation from 1 st to 24 th h post treatment and a decrease by 48 th h was recorded.With the low virulent isolate M48, at LC30, lipid peroxidation decreased by 13% at the 24 th h post treatment and 5% increase from the 24 th h to 48 th h was recorded (Figure 1).
Decrease in the extent of lipid peroxidation, as was revealed by the assay of lipid peroxides, can be attributed to increase in the proportion of antioxidant enzymes like peroxidases and ascorbate peroxidases that the cockroaches secrete to fight the oxidative stress induced by the fungal infection.The antioxidant machinery contributes to decrease in the concentration of lipid peroxides that were the end products of lipid peroxidation.The increased activities of peroxidases and ascorbate peroxidases with increase in the post treatment time (Naren, 2013) could be correlated to decrease in the lipidperoxidation.MDA, a major oxidation product of peroxidized polyunsaturated fatty acids, has been used to determine the degree of lipid peroxidation and as a biological marker of oxidative stress (Rael et al., 2004).Besides, under environmental stress, ultraviolet irradiation, bacterial infections, antibiotics and pesticides exposure, the ROS level may increase remarkably and result in oxidative stress in insects (Lopez-martinez et al., 2008;Buyukguzel and Kalender, 2009;Durak et al., 2009).CAT and POX, more appropriately the specific APOX, act to remove ROS and cellular haemostasis in addition to the non-enzymatic antioxidants such as thiols, ascorbate and glutathione (Joanisse and Storey, 1996a).The decreased lipid peroxidation level is a result of antioxidants that neutralized the lipid peroxides.The increased lipid peroxidation indicates the elevated levels of lipid peroxides as a result of increased oxidative stress which is beyond the reach of insect's antioxidant defense.To neutralize the toxicity of ROS, insects have developed a suite of antioxidant enzymes like other eukaryotes to overcome oxidetive stress.Several antioxidant enzymes may decrease the level of lipid peroxidation in insects (Felton and Summers, 1995).This can be attributed to the scavenging activity of the antioxidant enzymes on the lipid peroxides which, in view of increase in the activity of peroxidases and ascorbate peroxidases, there has been a decrease in the concentration of the lipid peroxides.Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds.Polyunsaturated fatty acid peroxides generate MDA and 4-hydroxyalkenals upon decomposition.Measurement of malondialdehyde and 4-hydroxyalkenals has been used as an indicator of lipid peroxidation (Esterbauer  , 1991).Along with lipid peroxides, the APOX activity found in fat body tissues, suggests that ascorbate peroxidase may be important in removing lipid peroxides in insects (Mathews, 1997).With the increase in the fungal dose, the extent of lipid peroxidation increased among the treated insects in all the three fungal isolates.
But with the advancement in the post treatment time, for each dose, there are dynamics in the lipid peroxidation levels indicating the response of innate immunity of the insect in the form of antioxidant enzymes viz., POX and APOX (Naren, 2013).Lyakhovich et al. (2006) opined that various antioxidants that were released in the insect as a response to the oxidative stress may decrease the level of lipid peroxidation.
The cockroaches treated with entomopathogenic fungal spores displayed growth of the fungal mycelia in the fat body of the insect at the 24 th h post treatment and proliferated all over the insect hemocoel by the 48 th h (Figure 2a, b).The TEM observations on the mid gut in the treated cockroaches revealed ultrastructural changes in the form of deformed villi due to lipid peroxidation and structural change.Peroxidation of lipids can greatly alter the physicochemical properties of membrane lipid bilayers, resulting in severe cellular dysfunction.Lipid peroxidation in biomembranes is mediated by free radical reactions.It leads to membrane damage and has been proposed to be associated with the pathogenesis to tissue injuries (Tampo, 2000).The extent of damage increased with increase in the post treatment time in the cockroaches treated with fungal conidia.In contrast to the control (Figure 3), at 10 th h post treatment, the mid gut of the cockroaches injected with M20 displayed loss of the columnar cell architecture (Figure 4).As the post treatment time incremented at the 12 th h post treatment, necrosis of the tissue was observed in the site where villi attach to the gut (Figure 5).At the 16 th h post treatment and the extent of damage to the villi increased and the villi were found to be getting separated from the intact columnar epithelial wall (Figure 6).The TEM at the 20 th h post treatment displayed an extensively damaged, broken and detached microvillus (Figure 7).The highly reactive properties of ROS make them a potential threat to cellular macromolecules, and if the initiated oxidation processes are not inhibited by the enzymatic and nonenzymatic components of the antioxidant defense system, damage to the DNA, lipid peroxidation and dysfunction of enzymes can result in necrotic or apoptotic cells (Smith et al., 2008).By the 24 th h, the villi got totally deformed due to fragmentation of the cells (Figure 8) and the electron dense areas as a consequence of the fat body depositions were observed in transmission electron microscopic observations of midgut wall (Figure 9).
The ultrastructural localization of lipid peroxides was reported by Kayatz et al. (1999) in the glutaraldehyde fixed tissue of rat retina, which was reacted with tetramethylbenzidine (TMB) and then postfixed in osmium tetroxide to visualize the lipid peroxides as electron-dense structures.Joanisse and Storey (1996b) also reported that fatty acids are particularly susceptible to ROS attack and their metabo-lism can in turn lead to ROS formation.To defend the tissue from the harmful effects of these hiked free radical levels, the activity of antioxidant enzymes and the level of antioxidants increases.In the current investi-gation, the deleterious effects of such high levels of free radicals has been ultrastructurally depicted in the villi of the midgut epithelial cell of the adult cockroaches treated with LC 50 of the high virulent isolate (M20).Increase in the post treatment time beyond a certain limit lead to the unbalanced generation of free radicals which suppresses the innate defense mechanism leading to consequent death of the insect.

Figur
Figur e 1. Grap h showi ng lipid perox idatio n at 1, 24 and 48 h post treat ment in the cockr oach es treate d with conidi a from M. anisopliae isolates.

Figure 2 .
Figure 2. Scanning electron micrograph of the fatbody of treated cockroach.a, 24 th h post treatment; b, 48 th h post treatment

Figure 2 .
Figure 2. Scanning electron micrograph of the fatbody of treated cockroach.a, 24 th h post treatment; b, 48 th h post treatment

Figure 3 .
Figure 3. Transmission electron micrograph depicting the ultrastructure of the epithelial architecture of the midgut of P. americana.Arrows indicate the intact villi and epithelial cell wall.

Figure 4 .
Figure 4. Transmission electron micrograph depicting the ultrastructure of the midgut of P. americana injected with LC50 of conidia from M20 isolate at 10 th h post treatment.Arrows indicate the deformed structure of epithelia.

Figure 5 .Figure 6 .Figure 7 .Figure 8 .
Figure 5. Transmission electron micrograph depicting the ultrastructure of the midgut of P. americana injected with LC50 of conidia from M20 isolate at 12 th h post treatment.Arrows indicate the necrosis at the base of the villi after the deformation of epithelial structure.

Figure 9 .
Figure 9. Transmission electron micrograph of mid gut of P. americana showing electron dense areas and fatbody in the treated cockroach at 24 th h from the time of treatment.

Table 1 .
Values of lethal concentrations (conidia/ml) for the three isolates of Metarhizium species at 48 th h after the treatment through injection mode.