Tested insects

The almond moth (E. cautella) and the Indian meal moth (P. interpunctella) were reared on artificial diet consisting of ground wheat, ground sugar, dried yeast and glycerol pure (65:10:10:15 g by weight, respectively) under 27 ± 2 °C and 65 ± 5 % R.H. (Hussain 2008). Khapra beetle, T. granarium was reared on whole wheat grains at 30 ± 1 °C and 65 ± 5 % R.H. and the red flour beetle, T. castaneum was reared on diet of wheat flour mixed with yeast.

Production of ozone gas

Ozone gas was produced from air using an ozone generator Model OZO 6 VTTL (OZO Max Ltd, Shefford, Quebec, Canada) from purified extra dry oxygen feed gas at the laboratory of Food Toxicology & Contamination, National Research Centre. The amount of ozone output was controlled by a monitor-controller having a plug-in sensor on board which is changed for different ranges of ozone concentration and a belt pan in the monitor-controller allows controlling the concentration in a selected range.

Effect of ozone gas application on different test insects

The experiments were started using small jute bags (about 0.5 kg capacity each) and four replicates for each insect and concentrations used. Each bag contained 10 g of wheat artificially infested with 25 larvae (fourth instar) of T. granarium, and for T. castaneum the bag contained 10 g of wheat flour artificially infested with 25 larvae (fourth instar). In the case of P. interpunctella and E. cautella each bag contained 30 g of dates that was artificially infested with 25 larvae (fourth instar). The bags were closed and exposed to two concentrations of ozone (200 and 400 ppm) at different exposure periods (0.5, 1, 2, 4, 6 and 8 h). Four untreated bags were kept as control for each species. Jute bags for each replicate were observed after 24 h to count numbers of alive and dead larvae and calculate mortality percentage that was corrected according to Abbott's formula (1925).

Nitric oxide assay

Each 1 g of control larvae or LT₅₀ dose treated larvae was homogenised in 2 ml buffer (0.1 M phosphate buffer; pH 7.41; 0.015 M potassium chloride), followed by centrifugation for 10 min at 10000 rpm at 4 °C (n = 4). The supernatants were mixed in a proportion with Griess reagent (Sigma) and were incubated at room temperature for 15 min. The nitrite levels were estimated with an optical density measurement (595 nm), using a spectrophotometer (Nano Drop 2000c). Concentrations of nitrite were calculated against a silver nitrite-derived standard curve.

Superoxide dismutase (SOD)

For SOD assays, the procedure from Misra & Fridovich (1972) was used. The absorbance was measured at 44 nm with a UV/Vis Jcnway-7305 spectrophotometer (Bibby Scientific Limited, Staffordshire, U.K.). The SOD activity was expressed as OD 48 d µg proteins/minute.

Ultrastructrual studies

Target specimens (n = 4) were prepared in laboratories of the Faculty of Science, Al-Azhar University TEM unit. Simply control and treated larvae (fourth instars) (400 ppm, LT₅₀) were fixed in glutaraldehyde (2.5 %) in phosphate buffer (pH 7.2), and then they were post fixed in osmium tetroxide (1 %). This was followed by washing specimens in the buffer (three times) and then were dehydrated gradually in ethanol solutions of ascending concentrations till the absolute one. After that, specimens were embedded in resin and were sectioned at 1 µm thick using glass knives. Next, they were stained with methylene blue and examined. Ultra-thin sections were cut (90 nm thick) and were mounted on copper grids. Specimens were stained with uranyl acetate and lead citrate. Finally, they were examined using Joel JEM JE 1200EXII (Japan) TEM and photographed. (For more details see Ghazawy (2012) and Ghazawy et al. (2007)).

Statistical analysis

Data were analysed using the SPSS computing program using ANOVA, as described by Snedecor & and Cochran (1956). Data on the effect of exposure periods on the tested insects were subjected to Probit analysis, as described by Finney (1971). LT₅₀ and LT₉₉ values were calculated using the computer program developed by Noack & Reichmuth (1978). All values were expressed as means ± standard error. P-values less than 0.05 were considered statistically significant.

Effect of ozone gas application on different tested insects

Results concerning the evaluation efficacy of ozonation on larvae of E. cautella, P. interpunctella, T. castaneum and T. granarium are shown in Table 1. The larval mortality of the four species increased with the increase of both concentration and exposure periods. Complete mortality (100 %) was observed in caterpillars (E. cautella and P. interpunctella) after the exposure time of 8 h whether at 200 or 400 ppm. However, in the case of grubs of T. castaneum and T. granarium, complete mortality (100 %) was noticed only at 400 ppm and after the exposure time of 8 h. Findings showed that grubs were more tolerant to ozone treatment than caterpillars.

LT₅₀ and LT₉₅ (time required to kill 50 and 95 % of the population at a certain concentrations) values of ozone gas against larvae of E. cautella, P. interpunctella, T. castaneum and T. granarium are presented in Table 2 and Fig. 1. Results show that ozone was more effective for the four species when concentration and exposure periods were increased from 200 ppm to 400 ppm. LT95 for E. cautella larva was 24.03 and 10.67 h at 200 and 400 ppm, respectively. Moreover, LT95 was 26.48 and 8.55 h at 200 and 400 ppm for P. interpunctella, respectively. On the contrary, O3 gas was less effective against T. granarium and T. castaneum larvae as LT95 was 21.61 and 23.51 h at 200 ppm, respectively. At 400 ppm, LT95 was 17.68 and 19.99 h, respectively. The obtained data revealed that caterpillars were more sensitive to O3 gas than grubs. In addition, data indicated that not all insects have the same sensitivity to ozone gas.

Table 1.

Effect of ozone concentration and exposure time on mortality (mean ± S.E.) of different larvae.

Table 2.

LT₅₀ and LT₉₅ values with their confidence limits for larvae of different insects exposed to two concentrations of ozone.

Fig. 1.

Mortality curves due to ozone for the four species studied. A, 200 ppm; B, 400 ppm.

Several chemicals have been proposed as replacements for methyl bromide since its listing as an ozone-depleting chemical, among those that have shown some promise is ozone gas. In this study, the researchers examined the effects of ozone concentrations on four larvae species belonging to Lepidoptera and Coleoptera. Ozone decays more naturally and rapidly (within about 50 min) than diatomic oxygen, leaving no residue. Our results indicated a remarkable difference in susceptibility between the four tested species. It was noticed that ozone was more effective for the four tested species when concentration and exposure periods were increased from 200 ppm to 400 ppm. The results indicated that 100 % of the larvae died within 8 h when being exposed to 400 ppm ozone. Moreover, Sadeghi et al. (2011) found that larvae of P. interpunctella are generally much more susceptible to gaseous ozone and carbon dioxide mixture than T. castaneum and R. dominica. Clearly, ozone has a place in stored product protection from insects and further research would define its role as a fumigant.

Effect of ozone on superoxide dismutase and nitric oxide

Results showed that there were cellular and biochemical changes like synthase activity and oxidative stress of enzymes such as superoxide dismutase (SOD) and nitric oxide (NO) activity in larvae of E. cautella and T. castaneum after ozone exposure using LT₅₀ dose (400 ppm) (Fig. 2). It was also noticed that there was a significant increase in SOD level in E. cautella after exposure to ozone than in the control larvae. Additionally, T. castaneum larvae exposed to the LT₅₀ dose recorded SOD concentration (0.1 ± 0.05) compared with the control larvae (0.0769 ± 0.006). This increment was significant (P < 0.05) (Fig. 2A). It was also found that there was a significant increase in SOD data in control larvae of T. castaneum than E. cautella. However, after LT₅₀ ozone exposure dose, there were no significant differences between them (P > 0.05).

Fig. 2.

A, SOD concentration in Ephestia cautella larvae control and treated with ozone (column 1, 2), respectively; and in Tribolium castaneum larvae control and treated with ozone (column 3, 4), respectively. Note: The same letter means there is no significant change between them at P < 0.05, (n = 3); B, nitric oxide concentration in Ephestia cautella larvae control and treated with ozone (column 1, 2), respectively; and in Tribolium castaneum larvae control and treated with ozone (column 3, 4), respectively. Note: The same letter means there is no significant change between them P < 0.05, (n = 3).

The results obtained in this study (Fig. 2B) indicated that there was a significant decrease in nitric oxide concentration in both of E. cautella and T. castaneum larvae after LT₅₀ dose of ozone exposure than the control larvae which indicated the power of ozone effect on the two different larvae belonging to two different orders to control them.

Ozone is considered as a strong oxidant, reactive to biomolecules. Ozone also has a toxic effect on insect pests (Ahmadi et al. 2009). Sousa et al. (2008) stated that T. castaneum were susceptible to ozone. Insecticide toxicity was associated with biochemical defence mechanisms in insect populations (Sousa et al. 2008; Oliveira et al. 2007).

Analysis of variance of the data obtained by SOD and NO activity showed that the LT₅₀ of the ozone dose exposure for E. cautella and T. castaneum larvae had an obvious significant effect on SOD and the NO activity compared to normal insects. Larvae exposed to ozone for 1 h and 30 min at LT₅₀ dose in E. cautella and 2 h and 33 min in T. castaneum stimulated antioxidant activity, which led to an increase in the prooxidant building. LT₅₀ dose of more ozone-generated O^–2 radicals that may result in an increase in the SOD which were determined immediately after exposure to ozone. In the present study, the SOD levels were considered as a marker of oxidative stress and it was observed that there was a high significant relation between the ozone dose exposure and the SOD concentration level in both larvae (E. cautella and T. castaneum). On the other hand, it was found that the relationship between ozone dose and NO level was independent; NO levels decreased.

In addition, the nitric oxide system was considered as a marker of oxygen-free radicals and cellular oxidation process that could further generate nitrogen-centred free radicals. Therefore, NO can be considered as free radicals (Bianchini et al. 2017). The reactive nitrogen species (RNS) is produced by activity of nitric oxide synthase, an enzyme involved in cellular signalling and immunological responses. In oxidative metabolism, NO is associated with the production of the peroxy nitrite radical (ONOO^–) (Dusse et al. 2003). Consequently, this reason represented clarification of reduction or downgrading in the NO activity in both treated specimens. Moreover, the intra-mitochondrial reaction of NO with superoxide anion resulted in the production of peroxy nitrite which led to unrecoverable reactions such as oxidative stress (Ghafourifar & Codenas 2005). Furthermore, oxidative stress, when produced generates relative oxygen species (ROS), can have an indirect effect on a NO response. Besides, the previous reason or justification given for an action of reduction in the NO levels, there is an extra clarification by the arginase–mediate removal of L-arginine, which inhibits the expression of inducible NO. Moreover, arginase decreases the NO formation by several actions which can be generated as an effect of the combination of ozone dose and the duration of exposure.

Many studies considered the potentiality of ozone as a fumigant to control insect pests of stored products and a potential alternative control method to other dangerous insecticides (Rozado et al. 2008; Sousa et al. 2008).

Effects of ozone on ultrastructure

The brain histology of controlling larvae consists of an outer sheath called neural lamella which enclosed the glial cells and the less-packed neuron layer. In the middle part, there is a dense mass of fibrous tissue called neuropile. The neurosecretory cells are distributed in various regions of the brain which consisted of large glandular bodies filled with neurosecretory material. This could be observed in the axon of neurosecretory cells and terminates into the corpora cardiaca.

Neurosecretory cells in the central nervous system control many major physiological events in the post embryonic life of insects. Neuroblasts were observed during post-embryonic development which stopped dividing prior to adult eclosion (Ganeshina et al. 2000). Lately, the neuroblasts are found in the mushroom bodies which are a paired structure in the protocerebrum and contain densely packed cell bodies, Kenyon cells and the neuropile forming a calyx.

In E. cautella, the normal muscles (Fig. 3A) are seen with myofibrils that appeared homogenous with sarcomeres showing a pattern of striations with an opaque myosin and light actin filaments. A muscle fibre is regarded as multinucleated cell which is bounded by a delicate coat ‘the sarcolemma'. The sarcoplasm contains large and numerous nuclei. The muscles are innervated by large axons ending with axon terminals (Fig. 3A). On the other hand, in ozone-treated larvae, the muscles lack a homogenous pattern with degenerated nuclei and loose fibres; moreover, nervous supply is lost. Many vacuoles appeared. A big gap appeared between muscle fibres due to detachment in between (Fig. 3B).

The neurosecretory cells in the pars intercerebralis in the brain showing a lightened neuroblasts with normal appearance due to the liberation of neurosecretory material to their normal path through nervi corpori cardiaci to corpora cardiaca (Fig. 4A). In treated larvae, the neurosecretory cells showed a trapped neurosecretion inside the cells and accumulation of neurosecretory material without liberation (Fig. 4B). In addition, there was a darkened pattern due to their trapped material, the greater number of neurosecretory cells contained masses of inclusions while others were vacuolated.

The neuropile glia have their cell bodies (Fig. 5A) around the neuropile of the brain. They appeared to fill in the irregular spaces between neuronal elements; most of them have abundant microtubules. In the treated larvae (Fig. 5B), the neuropile glia appeared loosely with many vacuoles. Many irregular spaces in neuronal elements appeared hollow without glia filling as in control. Furthermore, it was a case known as filopodia in which organelles for restoring cell contiguity after separation, so it is a general property of insect cells that occurred as a response to the separation of cells from one another (Locke 1987).

Fig. 3.

A, Normal muscles with dark and light striations (D, L) and axons end with axon terminals (axt); B, striations appearing loosely without clear dark and light bands, many vacuoles (v) appeared, and a large muscle detachment was obvious.

Fig. 4.

A, Lightened neuroblasts (NB) and normal matrix; B, accumulated secretion in neuroblasts with dark matrix.

Fig. 5.

A, Wrapped glial cell (gl) around neuropile (ni) in a normal appearance; B, uniform appearance of glial cells and neuropile appeared loosely and degenerated also a filopodia case (fl) were obvious.

The larval antenna (Fig. 6A) appeared with an obvious large channel, narrowing to the antennal artery and a small inextensible and uncompressible trachea. In treated larvae, the antenna appeared degenerated with loose sinus, no artery nor trachea present (Fig. 6B).

In P. interpunctella larvae (Fig. 7A), the brain was homogenous mushroom calyces and Kenyon cells distributed on the periphery. In the middle, the ganglion mother cells are large and small neuroblasts around, matrix appeared uniform. In treated larvae (Fig. 7B) the mushroom body calyces appeared without normal distribution of Kenyon cells also ganglion mother cells and neuroblasts appeared degenerated with many vacuoles and loose manner.

The muscles (Fig. 8A) showed a definite layer with axon nerves and uniform muscle fibres but in treated larvae (Fig. 8B) the muscle fibres appeared clear without dark and light striations as in control ones. The researchers also noticed a splitting in fibres with vacuoles.

The normal fat body cells were homogenous with normal mitochondria and smooth endoplasmic reticulum (Fig. 9A). In the treated larvae (Fig. 9B), the cells appeared with abundant amount of lipid droplets as an indication of cell destroying or lysis.

The neuropile axon in the antennal nerve was filled with neuroblasts and surrounded by glial cells (Fig. 10A) but in the treated larvae the axon appeared with disintegrated neuroblasts and many vacuoles scattered in between (Fig. 10B).

The cuticular layer with normal chitin deposition and normally located epidermal cells (Fig. 11A). In treated larvae (Fig. 11B), the cuticular layer was degenerated and cells appeared loose in manner and irregularly distributed as in control, there were also many vacuoles and lysosomal bodies.

In T. granarium treated larvae, there were many alterations in the brain as degeneration in the synaptic neuropiles and appearance of many vacuoles in the glial cells area. The cuticle appeared normal with deposition of chitin (Fig. 12A) while in treated larvae the layers showed no chitin deposition (Fig 12B).

As shown in Fig. 13A, tracheae appeared with solid uncollapsible intima. In treated larvae, the tracheae collapsed and appeared plain without differentiation (Fig. 13B).

The neuropile appeared with neural lamina in a consistent manner (Fig. 14A); but in treated larvae, the neuropile appeared damaged and disorganised without distinct neural lamella and vacuolised tissues were observed (Fig. 14B).

In T. castaenum larvae, the tract glia wraps around the axons in the central nervous system, many glial processes form layers around giant axons. The glial cytoplasm is filled with variably staining round clusters of glycogen granules and uniform nuclei appeared as compared with nucleus (Fig. 15A). Treatment led to the appearance of less chromatin and no distinct glial wrapping around axon (Fig. 15B).

The basal lamina (BL) (Fig. 16A) appeared regular and corpora allata cells with intercellular spaces (is) and smooth endoplasmic reticulum (SER); on the other hand the basal lamina had less consistence and neurosecretory fibres appeared vacuolated with lysosomal bodies which is a sign of cell lysis and many lipid droplets distributed beneath (Fig. 16B). More obvious in Fig. 16C, there are many lysosomal bodies with disorganisation of tissues, vacuoles appeared and many droplet cells.

The apical regions of receptor cells were connected by cellular junctions beneath the rhabdomeres structure (Fig. 17A). Few organelles appeared in cytoplasm while many slender mitochondria and granules, electron dense content occur as compared with rhabdomeres. By treatment, they took no fenestrae-like shape and not in clusters; as well as many lysosomal bodies distributed in cytoplasm (Fig. 17B).

The part played by the neurosecretory cells in the brain during moulting and metamorphosis is well-known (Wigglesworth 1972). While a few reviewers deal with ultra-structural NSC changes in the brain (Ghazawy 2012; Awad & Ghazawy 2016). It is the first study concerned with this point under the effect of ozone fumigation.

The present results showed the damage in the histology of the brain tissues, cells and muscles around after ozone fumigation exposure.

Ozone proved to be a good killer as fumigated to some insects (Jian et al. 2013). Most work done using ozone has hardly expressed its effect on ultrastructure. Perhaps, changes taken place in the present work could be attributed to the destruction of proteins that led to insect death (Holmstrup et al. 2011). Ultrastructural examination for caterpillars and grubs exposed to ozone showed that the brain tissues and neurosecretory cells, muscles and fat body were affected.

Fig. 6.

Obvious antenna (an) with small trachea (tr) (A), in treated larva degenerated antenna appeared without any details (B).

Fig. 7.

Normal mother cell (mc) and neuroblasts (NB), Kenyon cells were distributed on the periphery (Kc) (A), but in treated larva mother cell and neuroblasts were degenerated (B).

Fig. 8.

Light and opaque muscle striations with nuclei (n) (A) but in treated one the striations appeared cleared with many vacuoles (v) and splitting in between (B).

Fig. 9.

Normal larva appeared with homogenous fatty tissues (ft), normal mitochondria (mt) and smooth endoplasmic reticulum (ser) (A), many lipid droplets appeared in treated larva (ld) and mitochondria without cristae (mt) (B).

Fig. 10.

Neuropile (ni) surrounded with glial cell (gl) and neuroblasts (NB) inside (A) but in case of treated larva the neuropile appeared disintegrated and many vacuoles (v) appeared between neuroblasts (B).

Fig. 11.

Deposition of chitin (ct) in normal larva (A) but in treated one cuticle appeared degenerated with many vacuoles (v) (B).

Fig. 12.

Normal chitin deposition (ct) in controlled larvae (A) but in treated ones the cuticle appeared cleared without dense area of deposition (B), (v) vacuole.

Fig. 13.

Tracheae appeared with solid un-collapsible folded intima (f) in normal larva (A) but in treated larva tracheae appeared cleared without detected striations also many vacuoles (v) appeared due to splitting between layers (B).

Fig. 14.

Normal homogenous matrix with neural lamella (nl) on the border (A) and the matrix in treated larva appeared loosely organized with vacuoles (v), neural lamella was not distinct (B).

Fig. 15.

Normal glial cell with normally packed chromatin nucleus (N)and mitochondria (mt) with cristae (A) but in treated larva nucleus appeared with less dense chromatin with degenerated nuclear membrane and mitochondria appeared without cristae (B).

Fig. 16.

Brain tissues appeared with normal distinct neural lamella (nl) (A) but in treated larva vacuoles (v) appeared with disintegrated tissues (B) and with lysosomes (ly) and lipid droplets (ld) (C).

Fig. 17.

Normal rhabdomeres (rhd) with uniform fenestrae like structure (A), in treated larva the structure appeared lacking fenestrae in the above region and take a different direction in the rest (B).

Due to the activity of ozone, lipids, proteins and nucleic acids are oxidised and altered at molecular and cellular level (Iriti & Faori 2007). Oxygen released from ozone degradation might induce ‘oxidative stress’ (Khadre et al. 2001) that led to destruction of such molecules and finally cellular lesions and death (Holmstrup et al. 2011). The principle is that the released O2 reacted with the double bond in the unsaturated fatty acids. The sulfhydryl groups found in amino acids, peptides and proteins were vital molecules.