Salicylic Acid Induced Resistance in Drought-Stressed Pistachio Seedlings Influences Physiological Performance of Agonoscena pistaciae (Hemiptera: Aphalaridae).

Induced host plant resistance is a potential approach to insect and disease management. Salicylic acid (SA) acts as a signal molecule to induce resistance in plants against sap-sucking insects. The effects of salicylic acid-induced resistance against common pistachio psylla, Agonoscena pistaciae Burckhardt and Lauterer, were investigated in well-watered and drought-stressed pistachio, Pistacia vera L. cv. Akbari, seedlings. Agonoscena pistaciae exhibited a significant preference for plants treated with SA as compared with untreated controls or those subjected to drought stress. Plants subjected to both drought stress and SA treatment were equivalently colonized as compared with control plants but were more attractive than those subjected to drought stress alone. Psyllid mortality increased on plants subjected to simultaneous drought stress and SA treatment as compared with controls. Salicylic acid treatment mediated production of defensive enzymes in plants, including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX) and polyphenol oxidase (PPO), as well as that of other metabolites such as phenol, malondialdehyde (MDA), H2O2, free amino acids, and pigments via phenylpropanoid pathways under conditions of drought. Despite increases in activity of detoxification (glutathione S transferase, carboxylesterase) and antioxidative (SOD, CAT, APX, phenoloxidase, GPX) enzymes in psyllids, reduced survival of A. pistaciae on drought stressed and SA-treated plants was likely caused by excessive H2O2 and high phenolic content in treated plants. Based on our results, we postulate that salicylic acid-induced defense against A. pistaciae under drought conditions could be manipulated to enhance antibiosis against this key pest in pistachio orchards.

Evaluation of physiological and biochemical responses of pistachio plants (Pistacia vera L.) exposed to pesticides.

Pesticides may manipulate plant physiology as non-target organisms. In this study, we examined biochemical responses of pistachio plants (Pistacia vera L.) to imidacloprid and phosalone as common pesticides used to control pistachio psyllids. Enzymatic characterization in treated plants with pesticides showed greater specific activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, phenylalanine ammonia-lyase, glutathione reductase, and glutathione S-transferase compared with untreated plants during 14 days after treatment. Further experiments displayed elevated levels of total phenols and total proteins coupled with significant increases in proline and total soluble carbohydrate contents in treated plants in comparison to untreated plants. Moreover, pesticide treatment leads to a significant decrease in polyphenol oxidase activity. Nevertheless, no significant changes in contents of hydrogen peroxide, malondialdehyde, total chlorophyll, and electrolyte leakage index were obtained in treated plants. Pesticides' impacts on host plant physiology resulted in similar responses between two pesticides with differences in peak days. Overall, the findings of this study provide an insight into the side effects of phosalone and imidacloprid, chemicals with no specific target site in plants, on the physiology and biochemistry of pistachio plants at recommended rates.

Effect of Age-Dependent Parasitism in Eggs of Tuta absoluta (Lepidoptera: Gelechiidae) on Intraguild Predation Between Nabis pseudoferus (Hemiptera: Nabidae) and Trichogramma brassicae (Hymenoptera: Trichogrammatidae).

The tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a destructive pest of tomato that can cause up to 100% yield loss. The predatory bug Nabis pseudoferus (Remane) (Hemiptera: Nabidae) and the parasitoid Trichogramma brassicae (Bezdenko) (Hymenoptera: Trichogrammatidae) are natural enemies of this pest. Since the interaction between predators and parasitoids in different trophic levels including intraguild predation (IGP) can decrease or increase the efficiency of natural enemies, the effects of age-dependent parasitism of host eggs on IGP between these two species were investigated under laboratory conditions. In no-choice and choice preference tests, the predatory bug was exposed to 40 parasitized and nonparasitized eggs of different ages (24, 48, and 72 h old). Investigation of switching behavior was conducted using various combinations of tomato leafminer eggs (30:90, 45:75, 60:60, 75:45, and 90:30 nonparasitized:parasitized eggs) using eggs of different ages (24, 48, and 72 h old). In no-choice tests, the highest feeding rate of the predatory bug was 39.21 ± 0.36 eggs on 24-h-old nonparasitized eggs and the lowest feeding rate was 1.4 ± 0.80 eggs on 72-h-old parasitized eggs. In choice tests, comparison of the Manly's ß indices indicated that the predatory bug preferred to feed on nonparasitized eggs with 48- and 72-h-old eggs, but there was no significant preference for the 24-h-old eggs. Results of switching test showed that the linear regression between Manly's ß index and different ratios of nonparasitized eggs to parasitized and nonparasitized eggs was not significant in 72-h-old eggs. However, this regression was significant with 24- and 48-h-old eggs and the predator's preference was dependent upon the ratio of nonparasitized and parasitized tomato leafminer eggs. Results of the current study showed that the increasing age of parasitized egg decreased intensity of IGP between N. pseudoferus and T. brassicae.

Molecular and morphological identification of pistachio armored scale insects (Hemiptera: Diaspididae), with description of a new species.

Members of the family Diaspididae (Hemiptera: Coccomorpha) can be devastating pests that suck parenchyma cell contents from crops and cause severe damage to pistachio trees (Pistacia vera L.). The current research collected and characterized diaspidid species from pistachio orchards in Kerman province, Iran, according to their morphological and molecular features. Lepidosaphes pistaciae Archangelskaya, Suturaspis davatchi (Balachowsky & Kaussari) and Melanaspis inopinata (Leonardi) are redescribed and a new species, Melanaspis pistaciae Hosseininaveh & Kaydan sp. n., is described. Phylogenetic trees based on molecular analysis of COI and 28S rDNA fragments placed all the species in separated clades and confirmed M. pistaciae as a new taxon which is concluded by morphological differences. Molecular analysis suggests non-monophyly of the populations of each species. Melanaspis pistaciae sp. n. has spread to most cultivated pistachio areas in Iran and has probably been misidentified as M. inopinata in the past. Further investigation of the biology of this species may lead to development of more effective approaches for controlling this pest.

Circadian Rhythmicity of Diazinon Susceptibility, Detoxifying Enzymes, and Energy Reserves in Aphis gossypii (Hemiptera: Aphididae).

The daily susceptibility rhythm of the cotton aphid, Aphis gossypii Glover, to diazinon and the corresponding changes in the activity of three xenobiotic detoxifying enzymes-cytochrome P450 monooxygenases (P450), glutathione S-transferases (GSTs), and esterases-were investigated. Bioassays were conducted to estimate the median lethal doses (LD50) of diazinon at six different zeitgeber times (ZT0, 4, 8, 12, 16, and 20) under constant light (LL) and lighting conditions of 16 h of light and 8 h of darkness (LD). The results showed that the highest susceptibility occurred at the onset of night (ZT16) and 4 h before this time point (ZT12) under the LD condition. The endogenous rhythmicity of susceptibility was ensured, as the highest susceptibility occurred at the same time under the LL condition. The circadian changes in susceptibility to diazinon were almost coincident with changes in esterase and GSTs activity, but not in P450 activity. We also found rhythmic changes in energy components of whole-body aphids, with similar patterns of circadian changes of proteins, lipids, and soluble carbohydrates, but not glycogen, under LL and LD conditions. These photoperiod conditions (LD and LL) showed different fluctuation in trends of energy resources and of course, different quantities. Our study represents the first report of circadian control of insecticide susceptibility in aphids and provides insights into more efficient control of these pests by unveiling the times of day during which aphids are more susceptible to insecticides with attention to endogenous physiological phenomena.

Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N-methyl carbamates.

Carbohydrases in the digestive system of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae).

The spined soldier bug, Podisus maculiventris, is a generalist predator of insects and has been used in biological control. However, information on the digestion of food in this insect is lacking. Therefore, we have studied the digestive system in P. maculiventris, and further characterized carbohydrases in the digestive tract. The midgut of all developmental stages was composed of anterior, median, and posterior regions. The volumes of the anterior midgut decreased and the median midgut increased in older instars and adults, suggesting a more important role of the median midgut in food digestion. However, carbohydrase activities were predominant in the anterior midgut. In comparing the specific activity of carbohydrases, α-amylase activity was more in the salivary glands (with two distinct activity bands in zymograms), and glucosidase and galactosidase activities were more in the midgut. Salivary α-amylases were detected in the prey hemolymph, demonstrating the role of these enzymes in extra-oral digestion. However, the catalytic efficiency of midgut α-amylase activity was approximately twofold more than that of the salivary gland enzymes, and was more efficient in digesting soluble starch than glycogen. Midgut α-amylases were developmentally regulated, as one isoform was found in first instar compared to three isoforms in fifth instar nymphs. Starvation significantly affected carbohydrase activities in the midgut, and acarbose inhibited α-amylases from both the salivary glands and midgut in vitro and in vivo. The structural diversity and developmental regulation of carbohydrases in the digestive system of P. maculiventris demonstrate the importance of these enzymes in extra-oral and intra-tract digestion, and may explain the capability of the hemipteran to utilize diverse food sources.

Insect inducible antimicrobial peptides and their applications.

Antimicrobial peptides (AMPs) are found as important components of the innate immune system (host defense) of all invertebrates. These peptides can be constitutively expressed or induced in response to microbial infections. Indeed, they vary in their amino acid sequences, potency and antimicrobial activity spectra. The smaller AMPs act greatly by disrupting the structure or function of microbial cell membranes. Here, the insect innate immune system with emphasis on inducible antimicrobial peptide properties against microbial invaders has been discussed.

Food stress prompts dispersal behavior in apterous pea aphids: do activated aphids incur energy loss?

The pea aphid, Acyrthosiphon pisum (Hem: Aphididae), has been repeatedly used as a model species in a wide range of biological studies including genetics, ecology, physiology, and behavior. When red pea aphids feed on low quality plants in crowded conditions, some individuals lose their color shade and become pale yellowish, while other individuals on the same host plants remain changeless. The pale aphids have been shown to walk significantly faster and migrate more frequently to neighboring plants compared to the original red ones. We hypothesized that the color change and higher activity of pale aphids are directly associated with their suboptimal nutritional status. We showed that the pale aphids have significantly lower wet and dry weights than red ones. Analyses of energy reserves in individual aphids revealed that the pale aphids suffer a significant loss in their lipid and soluble carbohydrate contents. Our results provide a strong link between host quality, body color, dispersal rate, and energy reserves of pea aphids. Apparently, utilization of energy reserves resulted from an imbalance in food sources received by the aphids stimulates them to walk more actively to find new hosts and restore their lost energy. This reversible shift enables aphids to quickly respond to deprived host plants much earlier than the appearance of winged morph and restore their original status when they find appropriate host.

Effect of midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. Kurstaki.

Bacillus thuringiensis (Bt) is the most effective microbial control agent for controlling numerous species from different insect orders. All subspecies and strains of B. thuringiensis can produce a spore and a crystalline parasporal body. This crystal which contains proteinaceous protoxins is dissolved in the alkaline midgut, the resulting molecule is then cleaved and activated by proteolytic enzymes and acts as a toxin. An interesting aspect of this activation process is that variations in midgut pH and protease activity have been shown to account for the spectrum of some Bt proteins activity. Thus, an important factor that could be a determinant of toxin activity is the presence of proteases in the midgut microenvironment of susceptible insects. Reciprocally, any alteration in the midgut protease composition of the host can result in resistance to Bt. Here in this paper, we reviewed this processes in general and presented our assays to reveal whether resistance mechanism to Bt in Diamondback Moth (DbM) larvae could be due to the function of the midgut proteases? We estimated LC50 for both probable susceptible and resistant populations in laboratory and greenhouse tests. Then, the midgut protease activities of the B. thuringiensis induced-resistant and susceptible populations of the DbM were assayed on Hemoglubin and on N-alpha-benzoyl-DL-arginine-p-nitroanilide (BapNA) for total and tryptic activities, respectively. Six hours after feeding on Bt treated and untreated canola leaves, the midguts of instar larvae of both populations were isolated. Following related protocols, peptides released through the activity of proteinases on Hemoglubin and BApNA were recorded using microplate reader. Control (Blank) was also considered with adding TCA to reaction mix before adding enzymatic extract. Data analysis indicated that there are significant differences for tryptic activity on BApNA and also for total proteolytic activity on Hemoglubin between susceptible and resistant populations fed on Bt treated leaves. But these differences were not significant for larvae fed on healthy canola leaves between these two populations. These results which supported the role of DbM's proteolytic system in development of resistance to Bt, will be discussed in details.

Digestive proteolytic and amylolytic activities of Helicoverpa armigera in response to feeding on different soybean cultivars.

BACKGROUND: Digestive proteolytic and amylolytic activities of the larvae of Helicoverpa armigera (Hübner) fed either on artificial diet or on different soybean cultivars (356, M4, M7, M9, Clark, Sahar, JK, BP, Williams, L17, Zane, Gorgan3 and DPX) and response of the larvae to feeding on some soybean-based protease inhibitors were studied. RESULTS: The highest general and specific proteolytic activities were in artificial-diet-fed larvae. Although the highest general proteolytic activity was in the larvae fed on L17, M4 and Sahar cultivars, the lowest tryptic activity was on L17 and Sahar, which may be due to the presence of some serine protease inhibitors in these two cultivars, resulting in hyperproduction of chymotrypsin- and elastase-like enzymes in response to the inhibition of these enzymes. The highest amylolytic activity was on M4, and the lowest was on Williams and DPX. General proteolytic activity of SKTI-fed larvae was the highest compared with SBBI- and STI-fed larvae. CONCLUSION: The findings demonstrated that the cultivars L17 and Sahar were partially resistant to this pest, probably because of some secondary chemicals or proteinaceous protease inhibitors of these cultivars.

Partial biochemical characterization of alpha- and beta-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae).

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of alpha- and beta-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of alpha- and beta-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 micromol(-1) min(-1) mg protein(-1), respectively. Activity of alpha- and beta-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of alpha- and beta-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 micromol(-1) min(-1) mg protein(-1), respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 micromol(-1) min(-1) mg protein(-1), respectively. Results showed that the optimal pH for alpha- and beta-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for alpha- and beta-glucosidase activity in the midgut was obtained as 45 degrees C. The addition of CaCl(2) (40 mM) decreased midgut beta-glucosidase activity whereas alpha-glucosidase activity was significantly increased at this concentration. The alpha-glucosidase activity, in contrast to beta-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive beta-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.

Digestive proteolytic activity in the Sunn pest, Eurygaster integriceps.

The Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the most important pests of wheat and causes considerable damage to this valuable crop annually. Digestive proteinase activity of adult insects was investigated using general and specific substrates and inhibitors. Proteolytic activity was low when the common conventional substrates, azoalbumin, azocasein and hemoglobin were used to assay salivary glands and midguts. Using the fluorescent casein substrate (BODIPY FL casein), total proteolytic activity was measured at different pH. Maximum proteolytic activity was detected at pH 7 (100%) and 8(65%) which suggested the presence of serine proteinases in the salivary glands. There was no detectable proteolytic activity in midgut extracts. The inhibitors; PMSF (inhibitor of serine proteinases) and TPCK (a specific chymotrypsin inhibitor) showed greater than 50% inhibitory effect on total proteolytic activity, however, TLCK (specific trypsin inhibitor) and E-64(specific cysteine proteinase inhibitor) did not inhibit total proteolytic activity. Using fluorescent specific substrates for serine and cysteine proteinases (Z-Arg-AMC, Z-Arg-Arg-AMC, Z-Arg-Phe-AMC and Suc-Ala-Ala-Pro-Phe-AMZ) revealed the presence of tryptic and chymotryptic activity in the salivary gland extract. Zymogram analysis under non-reducing SDS-PAGE conditions and using the substrate APNE showed at least 8 tryptic and chymotryptic activity bands in salivary gland extracts. A single high molecular weight band with tryptic activity (165 kDa) was detected using the substrate BApNA in a zymogram analysis using native-PAGE. Kinetic studies showed a k(m) value of 0.6 mM for this enzyme against the substrate BApNA .The inhibitor TLCK decreased activity of the trypsin-like enzyme up to 73% and almost completely eliminated the only band related to this proteinase in the zymogram. Soybean Kunitz type trypsin inhibitor showed no effect on proteolytic activity of the trypsin-like serine proteinase. In general, the results revealed the presence of chymotrypsin- and trypsin-like serine proteinases in the salivary gland of E. integriceps, and it seems that the major total proteolytic activity is due to chymotrypsin proteinases.