Enzyme Activity, Cold Hardiness, and Supercooling Point in Developmental Stages of Acrosternum Arabicum (Hemiptera: Pentatomidae)
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Journal of Insect Science (2016) 16(1): 64; 1–6 doi: 10.1093/jisesa/iew045 Research article Enzyme Activity, Cold Hardiness, and Supercooling Point in Developmental Stages of Acrosternum arabicum (Hemiptera: Pentatomidae) Mozhgan Mohammadzadeh1,2 and Hamzeh Izadi1 1Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran (P93352001@post. vru.ac.ir; [email protected]) and 2Corresponding author, e-mail: [email protected] Subject Editor: Oliver Martin Received 13 February 2016; Accepted 15 May 2016 Abstract Several species of pentatomid bugs feed on pistachio fruits in Iran. Acrosternum arabicum Wagner (Hemiptera: Pentatomidae) is one of the most important pests of pistachio in Rafsanjan, Iran. This study was carried out to investigate the carbohydrase activities, supercooling points, and cold hardiness profiles of different develop- mental stages of A. arabicum under laboratory conditions. The midgut amylolytic of A. arabicum showed an optimal pH at 7.0. The highest amylolytic activity was found in the female adults (35.41 6 0.90 nmol/min/gut). The mean amylolytic activity measured in first instar nymph was 6.75 6 0.54 nmol/min/gut. Midgut a- and b- glucosidase showed an optimal activity at pH 5 and 7, respectively. These activities increased from first (83 6 5 and 54 6 5 nmol/min, respectively) to fifth (881 6 17 and 237 6 14 nmol/min, respectively) instar nymphs. The enzyme activities increased in the adults. Midgut a- and b-galactosidase showed an optimal activity at pH 5. a- and b-galactosidase activities were low in the first instar nymphs (73 6 5 and 21 6 3 nmol/min, respectively). The level of a- and b-galactosidase activities in the female adults (533 6 18 and 246 6 6 nmol/min, respectively) was higher than the nymphs. The lowest super cooling points (À19 and À18.2 C, respectively) and the highest cold hardiness (22 and 18% following 24 h exposure at À 20 C, respectively) were recorded for the eggs and adult females. Key words: Acrosternum arabicum; carbohydrase; stage specific digestion; super cooling point Pistachio as a major agricultural product in Iran attacks by several and physiology of digestive enzymes during on-growing of insect pests. Different species of hemipteran bugs (e.g., Acrosternum hee- pest (Kazzazi et al. 2005). geri, A. millieri, A. arabicum, Apodiphus amygdali, Brachynema Carbohydrases are widespread in nature, being found in animals, germari, Brachynema segetum) have been reported as pistachio pests microorganisms, and plants (Franco et al. 2000). Activity of carbo- throughout pistachio producing regions of Iran (Mehrnejad 2001). hydrases such as a-amylase, glucosidases, and galactosidases has A. arabicum Wagner (Hemiptera: Pentatomidae) is one of the key been demonstrated and described in the gut of a number of hemip- pests of pistachio since attacks nuts from early spring to the time of teran insects (Zeng and Cohen 2000, Kazzazi et al. 2005, Oliveira harvest, causing severe losses in production as well as affecting the et al. 2006, Swart et al. 2006, Vatanparast et al. 2011, Mehrabadi product quality (Mehrnejad et al. 2013). Control of this pest is thor- et al. 2012, Ghamari et al. 2014). oughly dependent of chemical application. However, many prob- Cold hardiness has been defined as the ability of an insect to re- lems are associated with using the pesticides (Subramanyam and sist injury during long or short term exposure to low temperature. Hagstrum 1995, Hagstrum and Subramanyam 1996, Regnault- Several factors such as developmental stage, genetic potential, sea- Roger et al. 2012, Macfadyen et al. 2014, Guedes et al. 2016), and son, duration of exposure, and nutritional status may influence the it is necessary to use alternative methods for control of pentatomid cold hardiness of insects. Cold hardiness is a graded response in pests. which capacity of insect to tolerate harsh environmental conditions The digestive tract of insects is an excellent target for control is proportional to the intensity of the environmental stimulus. Some agents that in general are not toxic to other organisms (Nauen et al. physiological adaptations (e.g., cryoprotectants accumulation) may 2001, Lwalaba et al. 2010). Therefore, for developing alternative be associated with cold hardiness. Insects at temperate zones usually methods of insect control, it is essential to understand the function utilize different strategies to avoid temperature below 0C. These VC The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2016, Vol. 16, No. 1 strategies describe how insect resist temperatures where its body flu- Glucosidases and Galactosidases Assay ids might be expected to freeze (Lee 2010, Storey and Storey 2012, Glucosidases and galactosidases activities were determined as de- Sinclair et al. 2015). scribed by Nakonieczny et al. (2006) and Ghamari et al. (2014). Supercooling point (SCP) is the temperature at which freezing The substrates were p-nitrophenyl-a-D-glucopyranoside (5 mM, for begins, and ice formation will usually then proceed from the site of a-glucosidase), p-nitrophenyl-b-D-glucopyranoside (5 mM, for b- nucleation to other parts of the insect’s body. The SCP is experimen- glucosidase), p-nitrophenyl-a-D-galactopyranoside (5 mM, for a- tally determined by detecting the released latent heat of fusion as galactosidase), and p-nitrophenyl-b-D-galactopyranoside (5 mM, for body water freezes (Bemani et al. 2012). Many investigations of in- b-galactosidase). Reaction was started by the addition of 5 ll sub- sect cold tolerance begin with preliminary measurements of the SCP strate and stopped 30 min later by adding 100 ll of NaOH (2 M) because this provides an anchor point about which the cold toler- and released p-nitrophenols were determined at the ABS405nm after ance strategy can be determined (Sinclair et al. 2015). 5 min. In the blank, the enzyme extract was added to the reaction Our investigations focus on the presence and activity level of di- mixture after NaOH treatment. All experiments were replicated gestive carbohydrases (a-amylase, glucosidases, and galactosidase) three times. in different instar nymphs and adults of A. arabicum and develop- mental changes in SCPs and cold hardiness of A. arabicum, to gain a Determination of SCPs better understanding of the developmental physiology of the insect. The SCPs of individual egg, nymph, and adult (n ¼ 6) were mea- sured using a thermocouple (NiCr–Ni probe) connected to an auto- matic temperature recorder, Testo 177-T4 (Testo, Germany), so Material and Methods that the cooling could be recorded at 0.5 min. intervals and the data Insect Culture were read using Comsoft 3 Software. The specimens were attached Adults of A. arabicum were collected from a pistachio garden in to the thermocouple by adhesive tape and placed inside a program- Kerman, Iran. A colony of different developmental stages was fed mable refrigerated test chamber (Gotech, GT-7005-A, Taiwan). The on Salsola kali L. and maintained at 26 6 1C, relative humidity of temperature was lowered at a rate of 0.5 C/min, starting at 20 C 65 6 5% and a photoperiod of 16:8 h (L:D cycle), for two and ending at À30 C. The temperature at which an abrupt increase generations. occurred with the liberation of the latent heat of freezing was taken as the SCP (Khani and Moharamipour 2010). Preparation of Samples Survival at Low Temperature Adults and different instar the nymphs were sampled 1 d after molt- Eggs, nymphs, and adults of A. arabicum were transferred (n ¼ 7– ing when an active feeding behavior of insect was observed. 10) to a programmable refrigerated test chamber. The temperature Nymphs of different instars and adults were dissected following the was lowered at a rate of 0.5C/min, from 20C to the desired treat- procedure described by Cohen (1993). In brief, insect midguts were ment temperature (0, À5, À10, À15, and À20C). The eggs, removed in 0.15 mM NaCl solution from nymphal and adult’s nymphs, or adults were held at these temperatures for 24 h and then stages previously immobilized on ice. For each sample, 10 midguts slowly (0.5C/min) heated to 25C and held at that temperature for were homogenized in 1.0 ml of cooled distilled water (at 4 C) and 24 h. Live and dead eggs, nymphs, and adults were counted. The then centrifuged at 15,000 Â g for 15 min at 4 C(Borzoui et al. blacked eggs and the nymphs or adults showing no movement were 2015). The supernatant was recovered and frozen (À20 C) for enzy- considered as dead (Khani and Moharamipour 2010). matic assays. In these conditions, enzyme activity remained constant for at least 1 month. Statistical Analysis Statistical analysis were performed using one-way analysis of vari- Determination of Luminal pH of Midgut ance (ANOVA) followed by a post-hoc Tukey’s test (P ¼ 0.05). Appropriate optimal pH for a-amylase, glucosidases, and galactosi- Data were initially tested for normality (Kolmogorov–Smirnov test) dases was determined in starved adult females by monitoring RDA and homoscedasticity (Levene’s test) before subjecting them to activity at various pH ranging from 3.0 to 10.0. Buffer used were ANOVA. A non-parametric test (Mann–Whitney U test and 100 mM glycine HCl (pH 3.0–4.0), sodium acetate (pH 5.0–6.0), Kruskal–Wallis test) was used to test for differences in cold hardi- and sodium phosphate (pH 7.0–10.0).