COMPARISON OF α-AMYLASE ISOFORMS FROM THE MIDGUT OF CERAMBYX CERDO L . ( COLEOPTERA : CERAMBYCIDAE ) LARVAE DEVELOPED IN THE WILD AND ON AN ARTIFICIAL DIET

α-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI < 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa.

C. cerdo larvae feed on the inner bark, sapwood and heartwood along the stem.Their development lasts 3 -4 years in the wild while in the laboratory the period of development is approximately one third shorter (Nenadović et al., 1999).
C. cerdo is polyphagous and can be found on deciduous mature, weakened trees and occasionally on young and healthy trees, especially those growing in open and sunny locations.It is distributed in Europe, the Caucasus, Asia Minor and northern Africa (Kimoto and Duthie-Holt, 2004).In Central Europe, only trees of the genus Quercus (oaks) are hosts to C. cerdo, while in more southern parts they can develop in some other trees (www.arkive.org).Outside Europe C. cerdo can be found on Carpinus, Castanea, Ceratonia, Fagus, Fraxinus, Juglans, Pyrus, Robinia, Salix and Ulmus (Kimoto and Duthie-Holt, 2004).From this point of view, it was interesting to find out how many isoforms of amylase are present in the midgut of C. cerdo larvae and clarify whether there is any connection between this enzyme and polyphagy in C. cerdo.It was also interesting to establish if there are differences in the amylase isoforms between the C. cerdo larvae developed in nature and those reared in the laboratory since it is possible that all insects have the full complement of ordinary digestive enzymes, the relative amounts of which change in response to diet composition (Terra and Fereira, 1994).
In the present paper the amylase isoforms of C. cerdo larvae from nature and those reared in the laboratory were compared.Two groups of C. cerdo larvae were examined to find out whether seasonal changes in the environment have an impact on this enzyme.

Insects
Tree groups of C. cerdo larvae were examined in this study.The first larvae group was collected from the wild in March -ML (March larvae).The second larvae groups was also collected from the wild in September -SL (September larvae); the third group was collected from the wild in March and then reared under laboratory conditions and fed an artificial diet -ADL (artificial diet larvae).All the larvae were collected from oak trees (quercus species) in the Fruška Gora Mountains in Serbia.

Rearing conditions of larvae
Individual larvae were confined in round-bottomed plastic boxes (diameter 6 cm) with ventilation holes on top.During development, the larvae were kept in the dark at 23°C and RH 50%.Dietary media was replaced with a fresh one weekly, and the weight of the larvae was measured.Larval molting was examined daily.

Preparation of crude midgut extracts
After decapitation the midguts were dissected on ice, weighed and homogenized using a pre-chilled mortar and pestle in ice-cold 0.9% NaCl, 0.1 M acetate buffer pH 6.0 with the addition of quartz powder.The homogenates were centrifuged for 10 min at 5000 x g (Božić et al., 2003).
α-Amylase activity assay α-Amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure (Bernfeld, 1955) using soluble starch as a substrate.Samples (50 μl) were incubated in 450 μL 50 mM acetate buffer pH 5.0 containing 1.0% (w/v) starch, 2.0 mM NaCl and 0.1 mM CaCl 2 at 35 o C for 10 min.Maltose was used as a standard.Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).One unit of αamylase activity was defined as the amount of enzyme required to produce 1 μmol maltose in 1 min at 35 o C.
After the electrophoretical separations, α-amylase activity was detected according to a previously published method (Dojnov et al., 2008).The gels were transferred first to a buffered substrate solution (1.0% (w/v) starch, 50 mM acetate, 2.0 mM NaCl and 0.1 mM CaCl 2 pH 5.0) for 30 min at 30 o C and then incubated in a buffer solution (50 mM acetate, 2.0 mM NaCl and 0.1 mM CaCl 2 pH 5.0) at 30 o C for 30 min.After rinsing in water, amylolytic activity was stopped by adding a staining solution (1.3% (w/v) I 2, 3% (w/v) KI).The α-Amylase activity appeared as clear bands on a dark background.

Determination of protein concentrations
Protein concentrations were determined by the Bradford method (Bradford, 1976) using bovine serum albumin as the protein standard.Each data point represents the mean of three independent assays ± SEM (the standard errors were less than 5% of the means).

RESULTS
The α-Amylase activity and protein concentration in C. cerdo midgut extracts were compared and the results are shown in Table 1.The highest amylase activity and specific activity was observed in the ADL midgut extract, while the ML extract had a higher amylase activity than the SL extract.
Zymography detection of the amylase isoforms of C. cerdo midgut extracts after IEF is show in Fig. 1.Three amylase isoforms were found in the SL midgut extract as well as in the ML midgut extract, with one intensive and two less visible bands with higher pI values in the latter.Two strong bands of amylase isoforms were detected in the ADL extract, both had similar pI values (near to that as the major isoform of ML).The pI values of all the C. cerdo amylase isoforms were less than 3.5.
Seven amylase isoforms of C. cerdo (ACC 1-7) larvae were found in the ML midgut extract, six isoforms in the SL midgut extract, while in the ADL extract there were four major isoforms (ACC 1-4) according to the native PAGE zymogram (Figs 2/B, 3/B and 4/B).The Amylase isoform on the ACC 6 position in the ML extract was missing in the SL crude extract.
Fractionation of proteins from the ML midgut extract is shown in Fig. 2. Amylase activity was detected in fractions 9 -13 with a maximum in 11 fraction (Ve = 13.5 mL).ACC 1-4 were found in all eluted fractions, while ACC 5 and 7 were presented in fractions 11 and 12 (Fig. 2/B).ACC 5 was the most visible in fraction 11, while ACC 6 was not present in the eluted fractions, only in the crude extract.
Fractionation of the proteins from the SL midgut extract is shown in Fig. 3. Amylase activity was detected in fractions 9 -13 with a maximum in the 12 fraction (Ve = 14.0 mL).ACC 1-4 isoforms were detected in fractions 10, while all isoforms (6) were presented in fractions 11 and 12 (Figure 3/B).
Fractionation of the proteins from the ADL midgut extract is shown in Fig. 4.There were two amylase activity peaks in the elution profile.The first peak corresponded to fraction 5 (Ve = 10.5 mL), where only one amylase isoform was found (Fig. 4/B).The second amylase activity peak corresponded to fraction 11 and contained all isoforms presented in the ADL crude midgut extract.
The molecular mass of the ACC was estimated to be 34 kDa, calculated from the plot of log molecular mass versus K av using standard proteins as markers after elution from a Superose 12 HR FPLC column.healthy trees and this is a characteristic of pest insects.The tunnels that are produced in a tree by the action of C. cerdo larvae are places of fungal infection by Biscogniauxia mediterranum which can causes charcoal disease (Martin et al., 2005).Although not a pest in its original form, C. cerdo might have negative economical and medical impact.
It has been postulated that isoenzymes may be important for insects as they could provide an increased capability to adapt to different sources of food and to overcome the activities of inhibitors of plant origin (Wagner et al., 2002).In all the larvae groups of C. cerdo examined, multiple forms of amylase were detected.Seven amylase isoforms were detected in the ML midgut extracts of C. cerdo.Similarly, five amylase isoforms were detected in the midgut of M. funereus larvae from the wild (Dojnov et al., 2008).Since C.cerdo can be found on wider geographical region and the larvae feed on the inner bark, sapwood and heartwood along the stem, the existence of more amylase isoforms in the midgut extracts of the C. cerdo larvae from the wild might be due to this.Two major isoforms of α-amylase have been reported in Prostephanus truncatus (Mendiola-Olaya et al., 2000), three isoforms in Rhyzopertha dominica (Cinco -Moroyoqui et al., 2008) and four isoforms of α-amylase have been identified in Callosobruchus maculatus (Campos et al., 1989).C. cerdo leucyl -aminopeptidase also exist in multiple forms (Božić et al., 2004).
The seasonal variability in amylase isoform presence in C. cerdo larvae presented in this paper is in agreement with other Cerambycidae, and was observed in M. funereus larvae as well (Ivanović and Nenadović, 1999;Ivanović et al., 1987;and in our previous work: Nenadović et al., 1982).Amylase activity is in correlation with the overall higher activity of larvae in spring.
Induction of this enzyme by different diets has been described in Zabrotes subfasciatus larvae (Silva et al., 2001).The artificial diet used in this work induced C. cerdo larval midgut amylases.Although there were fewer isoforms in the ADL group, the amylases had a higher overall activity as well as specific activity.The artificial diet used in this work induced amylase (results not published) and peptidases in M. funereus larvae as well (Lončar et al., 2009).Thus, C. cerdo larvae reared in the laboratory can be used for the purification and characterization of α-amylase and other digestive enzymes.
The effects of food on the expression of proteins can be seen not only by the levels and presence of amylase isoforms but also the overall protein profiles after SDS-PAGE.The obtained differences between the protein profiles of the ML, SL and ADL midguts are in correlation with results obtained for M. funereus protein profiles reared on different diets (Ilijin et al., 2004).
In terms of molecular weights, C. cerdo αamylase bears a resemblance to M. funereus amylase (31 kDa) (Dojnov et al., 2008) and to the α-amylase isoforms from C. maculatus midgut (Campos et al., 1989).We found that C. cerdo α-amylase was an acidic protein which compares well with the pI values of α-amylases from other Coleoptera (Terra and Ferreira, 1994).

УПОРЕДНА АНАЛИЗА ИЗОФОРМИ
DISSCUSION C. cerdo is classified as "Vulnerable" on the list of protected species by the World Conservation Monitoring Centre from 1996 (In: IUCN 2009.IUCN Red List of Threatened Species).The polyphagy of C. cerdo comprises the plasticity of the species which enables it to inhabit new hosts.Nevertheless C. cerdo can occasionally inhabit

Table 1 .
Amylase activity, protein concentration and specific activity in midgut extracts of C. cerdo larvae.ML -larvae collected from nature in March, SL -larvae collected from nature in September, ADL -larvae from artificial diet.Values are represent as Mean ± SEM (n=3).