Pak. J. Biotechnol. Vol. 11 (2) 163-171 (2014) ISSN print: 1812-1837 www.pjbt.org ISSN Online: 2312-7791 EFFECT OF INDOL-3 ACETIC ACID ON THE BIOCHEMICAL PARAMETERS OF Achoria grisella HEMOLYMPH AND Apanteles galleriae LARVA Fevzi Uçkan, Havva Kübra Soydabaş* Rabia Özbek Kocaeli University, Faculty of Arts and Science, Department of Biology, 41380, Kocaeli, Turkey * E-mail : [email protected]; [email protected] Article received November 15, 2014, Revised December 12, 2014, Accepted December 18, 2014 ABSTRACT Biochemical structures such as lipid, protein, sugar and glycogen are known to play a pivotal role on the relationship between host and its parasitoid. Any changes in these parameters may have potential to alter the balance of the host-parasitoid relation. Taking this into account, the effects of plant growth regulator, indol-3 acetic acid (IAA) on the biochemical parameters of the host and parasitoid were investigated. Achoria grisella Fabricus (Lepidoptera: Pyralidae) is a serious pest and causes harmful impacts on honeycomb. Endoparasitoid Apanteles galleriae Wilkinson (Hymenoptera: Braconidae) feeds on the hemolymph of the A. grisella larva and finally causes mortality of the host. Different concentrations (2, 5, 10, 50, 100, 200, 500, and 1,000ppm) of IAA were added to the synthetic diet of host larvae. Protein, lipid, sugar, and glycogen contents in hemolymph of host and in total parasitoid larvae were determined by Bradford, vanillin-phosphoric acid, and hot anthrone tests using UV-visible spectrophotometer, respectively. Protein level in host hemolymph increased upon supplement of each doses of IAA except for 10ppm. IAA application enhanced the level of sugar at 100 and 200ppm whereas a decrease was observed in lipid at 5, 10, 200, and 1,000ppm doses in host. All doses were effective on the parasitoid larvae. Nevertheless, the most effective dose was 50ppm, which increased glycogen but decreased lipid amount. Similarly, trearment of 1000ppm IAA increased protein level and 100ppm reduced level of sugar. Our study indicated that application of IAA resulted in different effects on the amount of biochemical structures associated with the hemolymph of pest species and its natural enemy. Therefore, results showed that not only the target but also the non-target organisms exposed to widely-used plant growth regulators may be affected and this, in turn may also change the host-parasitoid interaction. Key words: Achoria grisella, Apanteles galleriae, Indol-3-acetic acid, biological control. INTRODUCTION Plants are major sources for the erns of growth and reproduction in her- production of various natural compounds bivorous insects (Isman et al., 1983; which can be used for many purposes. Kaur and Rup, 2002). Nowadays focus Among them, the plant growth increases on natural products due to the regulators (PGRs) which directly or disadvantages of synthetic pesticides, indirectly affects the development of because they cause environmental pollu- plants via various biochemical path- tions and pesticide-induced resistance ways. PGRs have influence on the patt- as well as they reflects adverse effects 164 Uçkan, F. et al., Pak. J. Biotechnol. on nontarget organisms. Eco-friendly insects and they take part in fuel, egg compounds may also support the yield production and diapause (Hahn et al., and therefore can be used in integrated 2008; Suarez et al., 2005; Bernstein pest management (IPM) programs to and Jervis, 2008; Hahn and Denlinger, control the pests (Laher, et al 2000; 2007). To maintain the survival of Akol, et al., 2002; Kaur and Rup, 2002; insects, energy is required in different Uçkan et al., 2011a). It is reported that processes such as moulting, adult, PGRs have the capability to inhibit anti- gonadal and reproductive growth. oxidative defense system and block or Muscular activity and fasting also activate immune potential enzymes in increase the metabolism. The biological rat´s spleen and lung tissue (Celikand effects of IAA on the biochemical Tuluce, 2007). It is evident that fecu- parameters in different developmental ndity, longevity and egg viability have stages, number of off spring produced also been changed in insects with the and sex ratio of the koinobiont, supplement of PGRs (Visscher, 1980; solitary, larval endo-parasitoid A. 1983). Auxins are the main class of galleriae reached on it shost A.grisella PGRs, responsible for regulating many which is a serious pest because it developmental processes, such as apical feeds on combs, wax, and honey as dominancy, phototropism, and cell reported by Uçkan et al., (2011a). division in plants. Among them indol Additionally, a correlative study of host- -3-acetic acid (IAA) is the most parasitoid biochemical parameters like important natural auxin produced in total protein, lipid, sugar and glycogen plants, microorganisms, insects, and need attention to investigate. animals (Sugawara, et al., 2009; Pugine, MATERIALS AND METHODS etal., 2007). It is investigated that IAA Insects: experimantal colonies of the has various effects on the develop- host A.grisella and the parasitoid A. ment and reproduction of the solitary galleriae were established from succe- koinobiont larva endoparasitoid Apanteles ssive colonies in Kocaeli University galleriae Wilkinson (Hymenoptera: animal physiology research laboratory. Bracomidae) reared on the lesser wax The host colony was maintained by moth, Achoria grisella Fabr. (Lepido- feeding the insects with a synthetic diet ptera: Pyralidae) (Uçkan et al., 2008; modified by Sak et al., (2006) and bit Uçkan et al., 2011b). Investigations has of honeycomb was added for egg not yet completed without estimating deposition and feeding of the newly the changes in the amount of four hatched larvae. Both host and important biochemical components parasitoid species were reared in the (i.e. lipids, free sugars, glycogen and same environmental chamber at 25±2 proteins) in the same individual (Foray °C, 60±5 % relative humidity, and photo- et al., 2012). Proteins be represent an period of 12 : 12 (L : D) hours. Early important energy producing source in Vol. 11 (2) 2014 Effect of ındol-3 acetıc acıd .... .. 165 instars of A. grisella were provided for sitoids were reared in the same environ- A. galleriae adults to lay their eggs in mental conditions at 25±2 °C, 60±5 RH host larvae. For feeding of A. galleriae and 12:12 (L:D) hours photoperiod. adults a piece of cotton soaked in a Sample Collection: To investigate the honey solution (50 %) was placed inside effects of different doses of IAA on the the jars (Uçkan and Ergin (2003). The hemolymph of host larvae, 0.02-0.04 g method, which was described by Uçkan individuals were randomly selected and Gülel (2000) used to establish and from different colonies at different times. maintain successive colonies of both Host larvae were washed with 70% host and parasitoid species. ethanol and punctured on the first hind Exposing to Indol-3-asetic acid: One leg with a steril insect pin (Altuntaş, et to two days old A. grisella adults were al., 2012). From each host larvae 3µl left in 210 ml jars containing 01 g of hemolymph was collected with a glass honey comb for mating and oviposition. micro capillary tube (Sigma Aldrich). On the 5 th day of incubation host adults Sterilized microcentrifuge tubes having were removed from the jars. These series 0.005 g N-phenylthiourea (Sigma Aldrich) were used to take hymolymph from host were used to store hemolymph at -20 °C larvae. For collecting parasitoid larvae, (Vogelweith, et al., 2014). Parasitized the same incubating steps were applied host larvae were randomly selected from as previously described for host larvae. different colonies at different times to Additionally in 7 th day, 1-2 days old obtain parasitoid larvae. Under stereo parasitoids were placed into the jars. microscope parasitized host larvae disse- Honey:distilled water (1:1) feed was cted between head capsule and first supplemented to parasitoid adults. The thoracic segment, so parasitoid larvae parasitoids were removed from the jars were moved outside from the host body. on the 9 th day. Early instars of the host Sterilized microcentrifuge tubes contai- were exposed to 5 g of host diet (Sak et ning 500 µl 10 % trichloroacetic acid al., 2006) supplemented with 2, 5, 10, was used to store the larvae, which were 50, 100, 200, 500 and 1000ppm IAA used for protein assay. Parasitoid larvae (Merck) at the dark period of time and stored at -20 °C with out any treatment same feeding procedure repeated once were used to assay the glycogen, total at week until collection of both hemo- sugar and lipid. lymph from non-parasitized host and Biochemical assays: parasitoids from parasitized host larvae. Protein: Plummer (1971) method was IAA was indirectly taken by the para- applied for the extraction of protein. sitoid larvae from host hemolymph. Parasitoids larvae were homogenized The controls of both non-parasitized (QIAGEN Tissue Lyser LT) in 500 μl of and parasitized host larvae were fed on 10 % trichloroacetic acid solution at 50 a IAA-free diet. Both host and para- Hz for 30 min and centrifuged (Beck- 166 Uçkan, F. et al., Pak. J. Biotechnol. man Coulter Microfuge 22R) at 3500 absorbance was taken at 525 nm through rpm at room temperature for 15 min. The UV-vis spectrophotometer. For the deter- supernatant was discarded and 500 μl of mination of glucose amount 200 μl of 96 % ethanol was added to the tubes. the supernatant was transferred to a 2 These were then vortexed for 5 min and ml microcenrifugetube and heated at centrifuged at 3500 rpm for 15 min. After 90 °C until 50 μl remained. 950 μl of centrifugation the supernatant was dis- anthrone reagent (vanHandel, 1985b) carded and the pellet wasre disolved in was added to supernatant, heated at 90 distilled water. The quantity of protein °C for 15 min., cooled on ice and absor- was determined by using Bradford bance was noted at 625 nm.