Direct Effects of Temperature Changes on Biochemical and Enzymatic for Cotton Leafworm, Spodoptera littoralis (Boisd.)

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 11(1)pp: 121136(2018) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Insects face adverse stressors on a daily basis, for instance, fluctuations in temperatures and thermal stresses which may effect on their fitness and metabolism (Rinehart et al., 2000).Temperature is profound biotic environmental factors that induce physiological changes in insects resulting in rapid metabolic variation, which can lead to disorder affecting on their lives (Parmesan&Yohe 2003).On other hands, some studies on the effects of heat on insect metabolism demonstrated some adaptability to thermally challenging environments (Neven, 2000).
Insects are relatively easily affected by thermal stress, which can quickly elevate their body temperatures to lethal levels (Denlinger &Yocum, 1998).Many authors concluded that, as a result of global warming, the frequency and degree of appearance of high temperatures are predicted to increase substantially (Easterling et al., 2000;Diffenbaugh et al., 2005).In addition, Parmesan (2006)) noted that the change of climate may cause insect species to spread at the mean rate of 6.1 km per decade.It can influence insect pests and the size of damage caused by them: directly through their development, reproduction and distribution and indirectly by altering host physiology and defence mechanisms.Moreover, temperatures tend to enhance insect survival because it accelerated metabolism which may lead to higher consumption, growth, and development rates.Faster development, in turn, may lead to population increases via reduced generation time and decreased warmer late winter and early-spring (Bale et al., 2002).Metabolism, growth and reproduction increase exponentially with warming.Global warming is producing increases in both average temperatures and in the frequency and severity of heat waves (Stone et al., 2010).Moreover, because insects can be killed by short exposure to an extremely high temperature, heat treatments can be applied to control horticultural and stored-product pests, with few insecticidal applications, decreasing the environmental threat, (Cui et al., 2008 andHansen et al., 2011).Recent studies by many authors suggested that the conditions in the upper elevation and also in higher latitude would become more suitable for organisms, because global warming will exceed threshold of tolerance,in almost all insect species, which, in turn, accelerates cellular energetic demands, (Englund et al., 2011;Lemoine & Burkepile, 2012).Insect metabolic rates are highly sensitive to temperature, roughly doubling with an increase of 10°C across the full range of regularly experienced temperatures (Berggren et al., 2009).Temperature affects diffusion, membrane fluidity, nucleic acid stability, salt and gas solubility, and, significantly, the behavior of enzymes (Lee et al., 2007).
The Egyptian cotton leafworm, Spodoptera littoralis is distributed throughout the world.It is a serious or major pest of cultivated crops primarily in tropical and subtropical regions, in Africa, Southern Europe, Middle East and Asia (Pineda et al., 2004).S. littoralis is one of the major economic pests of cotton in Egypt that causes considerable damage to many other vegetables and crops (Dahi et al., 2009).S. littoralis.attacks all major crops in Egypt, including cotton, clover, corn, cabbage, cowpea, castor bean, sweet potato, lettuce, tomato, pepper, okra, mulberry, soybeans etc. (El-Aswad et al., 2003).Enzymes are proteins that are critical to catalyzing reactions (Brooker et al, 2011).One of the properties of enzymes that makes them as important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze (Kumar et al., 2005).Previous studies by (Hochachka & Sommero,2002) found that temperature changes affected on the binding of a substrate to the enzyme, causing a shift in the Michaelis constant (Km), thereby affecting immediate metabolic compensation.Moreover, An and Choi (2010),suggested that thermal stress may decrease the antioxidant state and cause oxidative stress.
The aim of this work is trying to understand the effects of increasing temperature due to global warming on S. littoralis.Using different temperatures in order to examine their effects on the biochemical parameters such as total proteins, lipids, carbohydrates and free amino acids as well as the activity levels of enzymes groups (metabolic, digestive, neural and detoxification enzymes) of fourth larval instars of S. littoralis .

Maintanence of Spodoptera littoralis Culture:-
The original colony of the cotton leaf worm S.littoralis was obtained from a well-established culture at the Department of Cotton Leaf worm; Plant Protection Research Institute.The insects were maintained under constant conditions of 15, 20, 25, 30 and 35± 2 0 C, 70 ± 5% R.H. and 12:12 (L: D) photoperiod, separately.Larvae were reared on fresh castor oil leaves, (Ricinus communis L.) supplied daily in sufficient amounts.Maintenance of the different developmental stages was conducted according to method described by Gamil, (2004).Biochemical Bioassay: 1.Sample Preparation: Twenty-five of 4 th larval instars of S. littoralis from each rearing temperature degrees were collected in a chilled glass.These larvae were then homogenized in phosphate buffer (PH.7) using a Teflon tissue homogenizer.After homogenation, supernatants were kept in a deep freezer at -20 o Ctill use for biochemical assays.The insects were homogenized in distilled water (50 mg./1 ml.)The homogenates were centrifuged at 8000 rpm for 15 min at 5 o C in refrigerated centrifuge.The deposits were discarded and the supernatant was kept in a deep freezer till used for the determination of the following: 2. Main Contents: -Total soluble protein as described by Bradford (1976).
-Free Amino acid assayed by ninhydrin reagent according to Lee and Takabashi (1966).

Enzymes Assay:
The Following Enzymes Activities Were Determined: -Proteases activity was measured as described by Tatchell et al., (1972).-Carbohydrates hydrolyzing enzymes; Amylase, Trehalase and Invertase were determined by the method of Ishaaya and Swiriski (1976), using starch, trehalose and sucrose as substrates.-Lipase activity was measured as described by Tsujitaet al. (1989).
-Acetyl choline-esterase activity was determined using acetylcholine bromide (AChBr) as substrate according to the method described by Simpson et al. (1964).
-Non-specific α and β esterase activities were measured as described by Asperen, (1962) using α naphthyl acetate and β naphthyl acetate, respectively, as substrates.-Glutathione S-transferase activity (GST) was determined spectrophotometrically at 340 nm according to the method of Habig et al. (1974).-Acid and alkaline phosphatase activities were measured from the larval haemolymph as described by Laufer and Schin (1971).

Statistical Analysis:
Data from all experiments were subjected to analysis of variance(ANOVA) Using the computer program SAS (Statistical Analysis Systems).

RESULTS AND DISCUSSION
Several studies done on various insect species have shown that temperature rise leads to increased metabolic rate, thus decreasing the development period and causing an increase in stored foods (Lale et al., 2003;Taveras et al., 2004;Khrüt et al., 2006;Coracini et al., 2007).In addition, a number of physiological stress responses occur in insects as a result of variations in temperature.One reaction to thermal stress is the generation of reactive oxygen species (ROS), which can be harmful by causing oxidative damage.(Ali et al., 2017).

Biochemical Analysis:
Temperature has profound effects on chemical and biochemical reactions (Hochachka and Somero, 2002).Moreover, temperature increases, the higher kinetic energy of biochemical reactions speed up the rate of metabolic processes, scaling up to affect the physiology and behaviour of individual organisms (Angilletta, 2009).Protein has always been interesting biochemical tool for insect biochemists because of their potential role in growth, development, morphogenesis and many intermediaries of the metabolic pathway of insects Kar et al., (1994).In the current study as seen in (Table .1&Fig.1), it is clear that the levels of total proteins recorded, a gradual increase in haemolymph during the development of fourth larval instars by raising temperature at15, 20,25 and30 o C.They were (47.67,57.67,62.33 and 59.33 mg/g.b.wt.respectively, but there were no significant differences between the levels of total protein in the last three degrees of temperature.Meanwhile, at 35 o C the amount of total protein (22.67mg/g.b.wt)decreased significantly in comparison with total protein levels at previous degrees of temperature.In addition, free amino acid increased gradually by increasing of temperatures at15, 20, 25and 30 o C, they were 376.33, 627.0, 732.3 and776.3Ug alanine/ml.respectively.There were no significant changes between amounts of free amino acids at 20 and 30 o C, while, they decreased significantly at 35 o C it was 73.33 Ug alanine/ml.These results may be revealed to Caterpillar growth rates increase with raising the temperature and the larvae consumed more protein rich diets.Our results agree with the finding of (Kingsolver,2006 andLee andRoh2010).In the previous study, Nagata and Kobayashi (1990) had reported an increase in protein synthesis during feeding stage in bombyx mori in haemolymph and increase the soluble protein level during pupal development could be attributed to compensatory replacement of protein that utilized for formation of pupation.Moreover, Martin et al., (1969) reported that the increase of protein levels in haemolymph during oncogenic development of fifth larval instars of silkworm bombyx mori.The increase of protein levels of haemolymph due to the synthesis of new proteins by tissue and release into haemolymph.Hachiya et al., (2007) reported that proteins denature more rapidly at higher temperatures, which, in turn, requires greater rates of protein synthesis and repair to maintain basic cellular function.On contrast Malik and Malik (2009) were found when the larvae and pupae exposed to selected higher temperatures (31 °C and 36 o C), a significant decrease in the protein levels of haemolymph was noticed and the order of decrease was to be more at 36°C than at 31°C.Relatively higher increase in the free amino acid levels in the haemolymph presumably provides protective cover to tissues against high temperature by an increase in osmolality and reduction in evaporative water loss.On the other hand Kiranet al., (1998)reported that the fat body synthesizes a number of protein and release them into the haemolymph during active larval period.This clearance was confirmed by Willmer et al., (2004) they reported that high temperature affected all biological process including the structure of proteins and biological members and rates of biochemical and physiological reactions.Moreover, (Gullan and Cranston, 2005) concluded the high temperature tended to kill insect's cells by denaturing proteins, altering membrane, enzyme structures and properties, by the loss of water (dehydration) and they offer a rich potential for pest management strategies.While the previous study carried by Neven (2000) on the codling moth were reared at different temperature 10,15, 20, 25 and30 o C, the moth subjected to acute heat treatments had a maximal carbon dioxide evolution rate, respiration increase in response to increasing temperatures to critical upper limit.After this point, respiration decreased.So, death occurs soon after the respiration rate drop even if the insect returned to normal thermal condition indicating that death cells ,death is occurring and denatured protein.
In contrast, the recent study carried out by Lemoine and Shantz (2016) on Spodoptera exigua cleared the decreasing of protein at 30 o C due to, nitrogen digestibility declined by almost 25% at 30 ∘ C compared with 25 ∘ C. Reduced nitrogen digestibility, coupled with increased cellular division and somatic growth rates, may be responsible for the observed strengthening of protein limitation at high temperatures.
Lepidopteran larvae may be particularly vulnerable to environmental warming because their rapid growth rates demand high protein foods (Lee et al., 2004) Ehsan et al., (2011) were revealed the fourth larval instars of Pistachio white leaf borer reared at 25 o C had a lower level of glycogen and higher level of protein in comparison with those larvae reared at 35 o C. Glycogen is storage form of energy, therefore temperature rise leads to increase metabolic rate, decrease the development period and cause an increase in stored foods.Protein content in larvae reared at 25 o C was significantly higher than those reared at 35 o C, but glycogen content in the larvae reared at 35 o C was more than larvae reared at 25 o C.
In our study the amount of total lipids as seen in (Table .1&Fig. 1) were recorded an increasing significantly in haemolymph at lower temperature 15 o C and 20 o C.They were 14.98 and 10.33mg/.g.b.wt.However, the recorded decrease in total lipid levels by gradual increase of temperature at25,30 and 35 o C they were 6.7, 6.80 and 7.03mg/g.b.wt.,respectively.No significant changes in the total lipid levels recorded between these degrees of temperatures.These results may be due to increasing in food consumption at lower degrees of temperatures to provide energy.These results agree with findings of.Hochachka and Somero, (1973), they revealed the increase in the contents of total lipids, free fatty acids and phospholipids to increase in the food consumption.The increase in free fatty acids may provide the lipoprotein enzymes, with an environment to modulate the latter's activity at low temperature.On the other hand, many authors (Ellis et al., 2002;Costamagna and Landis, 2004) reported that the orders Lepidoptera and orthoptera use lipids and stored carbohydrates as the main energy source.Lee and Roh (2010) concluded that lipids storage efficiency was lower in larvae of Spodoptera exigua at 18 o C than at 26 ºC, and was similar to those at 34 ºC.Dooremalen et al., (2011) found that in Philosamia ricini, lipid composition may be an important trait under lying fitness response to temperature, because it affects membrane fluidity as well as a viability of stored energy reserves.The observed increase in the phospholipid content, larvae showed greatly increased activity of those mitochondrial enzymes which were membrane associated, and thus the former retained their reticular structures intact during cold exposure.Therefore, Philosamia ricini larvae were exposed to higher temperature of 35°C, monovalent cations like Na + and K + increased whereas divalent cations like Ca 2+ and Mg 2+ decreased, the percent changes observed being more at 36°C than at highest.At 31°C.The haemolymph monovalent cations were hyper feeding larvae and divalent cations were hyporegulated at the quantity of silk synthesized by their silk glands.
In current study, the amount of total carbohydrates as seen in (Table .1.& Fig. 1) increased significantly at 15 o C and20 o C they were 20.43 and 26.19mg/g.b.wt.There was significant difference in the total carbohydrates between these temperatures while decreased at 25 o C, 30 o C and 35 o C they were 9.63, 9.40 and 4.77mg/g.b.wt.There were no significant changes between 25 o Cand 30 o C while the amount of total carbohydrates decrease significantly at35 o C.These results disagree with Sonmez and Gulel (2008), they concluded that a low temperature decreases the total carbohydrates and protein amounts of the pest Acanthoscelides obtectus.They recommended that Storage should be kept at 10-15 o C so that, A.obtectus and other possible pests give minimal damage crops.

Enzyme Assay:
The optimum temperature for enzyme reactions is generally defined as the temperature at which the maximum reaction rate is achieved Takaya (2005).The fact that each enzyme behaves differently under various temperature regimes highlights the fact that each enzyme has an optimum temperature activity.Carbohydrates, proteases, and lipases are the three main enzymes involved in the digestion of food by insects (Callaghan et al., 2002).
In our study, it is clear in (Table 2 & Fig. 2) the activities of the carbohydrate hydrolyzing enzymes amylase, and invertase levels were increased by the gradual increase of temperatures at 15, 20, 25 o C.They were 81.0, 117.0, 268.66 Ug glucose/min/g.b.wt., for amylase and 4079.3, 5943.3, 8502.7 Ug glucose/min/g.b.wt.,for invertase.They reached their maximum activities at 30 C, there were 341.0 and 9012.0Ugglucose/min/g.b.wt., in both amylase and invertase enzymes respectively.Their levels were decreased at 35 o C which were 67.66 and 1580.0Ugglucose/min/g.b.wt.,respectively.There were significance differences between levels of amylase at all degrees of Temperature.Meanwhile there were significant differences in levels of invertase enzyme in all degrees of temperature except 25 and 30 o C. In contrast, Sultan et al., (2009) showed the effects of temperature on amylase were presented in salivary gland and mid gut at optimum temperature 35-40 o C.Meanwhile, in our study the activity levels of Trehalase increased at 20 o C, they were1800.0mg/g.b.wt.While activity levels decreased at 15, 25,30 and 35 o C, there were 573.33, 1370.0,1305.0 and 1016.67 mg/g.b.wt., respectively.Proteases, which are also known as endopeptidases, enroll an important function in protein digestion.These enzymes begin the protein digestion process by breaking internal bonds in proteins.The amino acid residues vary along the peptide chain, therefore, different kind of proteinases are necessary to hydrolyze them.Based on active site group and their corresponding mechanism, digestive proteinases can be classified as serine, cysteine, and aspartic proteases (Terra and Ferreira, 2012).
In the current study in (Table 2& Fig. 2) the activity levels of Protease Enzymes were increased by increasing temperature.They, were 58.33, 85.33, 117.0 and 207.66 ug alanine/min/g.b.wt at 15, 20, 25and 30 o C respectively.There was significant reduction in enzyme level at 35 o C it was 23.33ug alanine/min/g.b.wt Pant & Gupta (1979) noted that a high proteolytic activity in second larval instar of Philosamia.Ricini declined steadily till late fifth instar development.The decrease was attributed to the composition of food ingested.The low proteolytic activity was due to the host plants which are rich sources of amino acids and which are consumed in large quantities by this insect.The two major proteases classes in the digestive systems of phytophagous insects are the serine and cysteine proteases (Haq et al., 2004).Moreover, Srinivasan et al., (2008) had reported on the midgut enzymes of various pests belonging to Lepidoptera.Serine proteases are known to dominate the larval gut environment and contribute to about 95 % of the total digestive activity in Lepidoptera.
Lipases (triacylglycerol-acyl-hydrolase EC 3.1.1.3),which catalyzes the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms (Naumff, 2001).In current study as seen in (Table 2 & Fig. 2),the activity levels of lipase in mid gut of larvae increased significantly at lower temperatures 15 and 20 o C, they were1306.33and 1131.0Ug oleic acid/g.b.wt respectively.There were no significant changes between them.On the other hand, their activity levels decreased significantly when temperature raised at 25, 30and 35 o C.They were820.33,810.0 and 789.0 Ug oleic acid/g.b.wt.respectively.There was no significant change between them.These results agree with finding of some previous authors Ishaaya et al.,(1971) concluded that enzymatic activity increased when the temperature rose from 10 to 32°C.At 10°C (i.e.below the threshold of larval development), both proteolytic and amylolytic activities in the midgut wall were less than 10 percent of that obtained at 32°C.
Detoxification enzymes in insects are generally demonstrated as the enzymatic defense against foreign compounds and play a significant role in maintaining their normal physiological functions (Mukanganyama et al., 2010).Several defensive mechanisms and biochemical reactions are involved in the detoxification processes against any temperature Stress.These mechanisms predominantly involve either metabolic detoxification of the Temperature stress before it reaches to their damage, or the sensitivity changes of the larvae.The most common metabolic resistance mechanisms involve esterases, glutathione S-transferases (GSTs).Generally speaking, increase of activity of detoxification enzymes is the most universal resistant mechanism in insects.An elevation in the activity of such enzymes but surprisingly, this assumption couldn't be achieved as GST relatively decreased exposed to different constant temperature.GSTs also play an important role in stress physiology, and have been implicated in intracellular transport and various biosynthetic pathways (Wilce and Parker, 1994).
Table (2 In our study, it is clear in (Table .3.& Fig. 3) the level of glutathione Stransferases (GSTs)enzyme increase significantly at 30 o C. It reached its maximum activity which was 131.66 m mole sub conjugated/min/g.b.wt, while its activity level decreased at 15and 35 o C, there were 27.0 and 16.0 mole sub conjugated/min /g.b.wt.respectively.There was no significant difference between them.Whereas, there were significant differences between20 and25 o C.

Table (3):
The activity levels of metabolic enzymes of 4 th larval instars S. littoralis at different constant temperature.Our results disagree with Debora et al., (2016) who concluded that AChE activity decreased at higher temperatures and corroborating the results of Domingues et al., (2007) who observed that the activity of the AChE of Chironomus.riparius Meigen, is higher at 6 °C and 16 °C than at 26 °C.These results also disagree with finding of Débora et al.,(2016) who studied effects of different temperature on Chironomus sancticaroli where he found AChE activity decreased with increasing temperatures: at 20 and 25 °C, it was 69% and 59% lower than at 30 °C, respectively.No significant changes in enzyme activity were detected between 20 and 25 °C.No changes in the activity of EST-α were observed between 20 and 25 °C.However, at 30 °C the enzyme activity increased by 44% and 45% when compared to 20 and 25 °C, respectively.The enzyme activity of EST-β was high at the intermediate temperature of 25 °C.At this temperature, EST-β activity was 24% higher than at 20 °C and 18%higher than at 30 °C.In contrast, enzyme activity at 20 and 30 °C did not differ.Singh et al., (2013) concluded that Pricini when exposed to low temperature cause alterations in the activity and kinetics of the tissue.Thus AChE activity appears to be a potential biomarker towards the evaluation of the impact of cold stress on silk worms.
Fig (2): The activity levels of digestive enzymes of 4 th larval instars S. littoralis at different constant temperatures.

Fig
Fig (3): The activity levels of metabolic enzymes of 4 th S. littoralis larval instars at different constant temperatures.

Table ( 1
): Effect of different constant temperatures on biochemical of S. littoralis