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Protein profiles of Gonipterus scutellatus (Coleoptera: Curculionidae) larvae fed on leaves from three Eucalyptus species

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Gonipterus scutellatus (Coleoptera: Curculionidae) is a pest of eucalyptus in Chile. Susceptibility of Eucalyptus camaldulensis, E. globulus ssp. globulus, and E. robusta to larval feeding were studied by determining foliar area loss. The effect of
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  357Huerta et al.—Proteins of Gonipterus scutellatus larvae fed on  Eucalyptus  leavesAMANDA HUERTADepartamento de Silvicultura Facultad de Ciencias ForestalesUniversidad de Chile Casilla 9206, Santiago, Chile email: ahuerta@uchile.cl, ahuertaf@gmail.comITALO CHIFFELLEDepartamento de Agroindustria Facultad de Ciencias AgronómicasUniversidad de ChileCasilla 1004, Santiago, ChileMARYI SERRANOTATIANA VÁSQUEZDepartamento de Silvicultura Facultad de Ciencias ForestalesUniversidad de ChileCasilla 9206, Santiago, ChileJAIME E. ARAYADepartamento de Sanidad Vegetal Facultad de Ciencias AgronómicasUniversidad de ChileCasilla 1004, Santiago, Chile Abstract   Gonipterus scutellatus  (Coleoptera: Curculionidae) is a pest of eucalyptus in Chile. Susceptibility of  Eucalyptus   camaldulensis ,  E. globulus  ssp.  globulus , and  E. robusta  to larval feeding were studied by determining foliar area loss. The effect of feeding on larval protein proles was analysed by electrophoresis.  E. camaldulensis  was more susceptible (11.21% foliar area consumed) than  E. globulus  (6.47%) and  E. robusta  (3.62%). Nineteen proteins were common in larvae fed the three kinds of food, whereas 11 exhibited variations (marker proteins). As all larvae came from  E. globulus  providing the same nutrition, relatively few Protein proles of Gonipterus scutellatus (Coleoptera: Curculionidae) larvae fed on leaves from three Eucalyptus  species (20) proteins were detected in them. More proteins (30) were found in larvae feeding on  E. robusta . The greatest number of marker proteins occurred in  E. robusta  (11), followed by  E. camaldulensis  (9), and  E. globulus  (1). Thus, larvae fed on three eucalyptus species had three different protein proles. Keywords  eucalyptus weevil;  Eucalyptus camal-dulensis ;  Eucalyptus globulus ;  Eucalyptus robusta ; foliar damage; marker protein INTRODUCTION The eucalyptus weevil, Gonipterus scutellatus  Gyllenhal is an Australian insect specialised for eucalyptus (Withers 2001), where it is considered one of its major pests because of the important damage caused to plantations in many countries in Africa and Europe (e.g . , Arzone & Meotto 1978; Rabasse & Perrin 1979; Richardson & Meakins 1986; Mansilla 1992; Cordero et al. 1999), California, United States (Cowles & Downer 1995; Hanks et al. 2000), and New Zealand (Cadahía 1980). In South America, the species was rst reported for Argentina in 1926 by Marelli (Rosado 1993). It then spread to other countries, and was found later in Uruguay (1943), Brazil (1955), and Chile (1998) (Zanuncio et al. 1998; Beéche et al. 1999). Both adults and larvae feed on growing eucal-yptus foliage. Leaves damaged by larvae have characteristic tracks (Elliott & De Little 1984). The dramatic growth of weevil populations within a few years is favoured by abundance of preferred hosts, adequate mild weather, and absence of natural enemies in certain areas (Tooke 1953). The high reproductive potential of the insect, together with its capacity for intense defoliation, can cause growth loss and/or deformities (Santolamazza & Cordero 1998). Continued defoliation may lead to tree death (Elliott & De Little 1984). In central Chile,   localised infestations of G. scutellatus  were found in 1998 in the 5th and Metropolitan regions (Beéche et al. 1999), which  H06065; Online publication date 21 August 2007  Received 11 July 2006; accepted 9 March 2007     New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35 : 357–3630014–0671/07/3503–0357 © The Royal Society of New Zealand 2007  358 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35generated much concern over the future of the country’s 525 057 ha of  Eucalyptus  spp. (INFOR 2006). Afterwards, this pest has reached the 4th, 7th, and 8th regions (SAG 2005). In the countries where it has spread, G. scutellatus  has showed marked preferences for several species of  Eucalyptus  (Cordero & Santolamazza 2000). In Chile, the most abundant eucalyptus species is  E. globulus ssp . globulus Labill. Other species planted include  E. viminalis  Labill. and  E camaldulensis Dehnh. on semi-arid areas, and  E. nitens  (Deane & Maiden) Maiden in colder locations (INFOR 2006). In the Chilean region of San Felipe (5th region), G. scutellatus  has 3–4 generations a year (Estay et al. 2002). According to Klein & Waterhouse (2000), its importance as a pest on eucalyptus is surpassed only by the eucalyptus longicorn beetle, Phoracantha semipunctata  F. The variation in susceptibility in  Eucalyptus  spp., including  E. melliodora ,  E. globulus ,  E. sideroxylon ,  E. camaldulensis ,  E. nitens ,   and  E. viminalis , has been reported (Farrow et al. 1994; Floyd et al.  1994). Furthermore, plant resistance in eucalyptus species to their pests can be based on leaf chemical composition (Floyd & Foley 2001). Some insects develop diverse strategies to elude the vegetal plant defense: they increase their proteolytic activity, induce resistance enzymes proteolytic to protease inhibitors or express proteases that degrade specically to inhibitors of plant proteases, for which they do not have inhibitors (Vivanco et al. 2005). This work attempted to determine the suscepti-bility of three  Eucalyptus  spp. to infestation of G. scutellatus  larvae by measuring foliar area loss of plants infested. The effect of feeding them on larval protein proles was characterised by electrophoresis, comparing size and intensity of bands on gels after protein separation. MATERIALS AND METHODSAssessment of foliar area loss Gonipterus scutellatus  larvae were collected at the end of the summer from a heavily infested stand of young  E. globulus  plants in the San Felipe province (5th region in central Chile). Only second instar larvae were used as rst instar larvae were too delicate. These larvae were taken in cloth bags in coolers to the Forest Entomology Laboratory, College of Forestry Sciences, University of Chile, in Santiago. Larvae were kept in 5-litre plastic boxes and fed regularly in the laboratory with fresh leaves of  E. globulus (Santolamazza & Cordero 1998), which were replaced every   other day. One-year-old plants (c. 1 m high) of  E. camal-dulensis ,  E. globulus , and  E. robusta  Smith, on a mixture of soil, sand, and peat treated with Captan 2 g/litre and urea were placed on 19 cm diameter plastic pots, in 1.8 ×  1.5 ×  1.8 m cages with a cloth bag placed around them to prevent the insects from escaping, under normal Mediterranean climate type eld conditions, and periodic irrigation. Data sets were distributed normally. Two second instar  G. scutellatus  larvae were placed on all plants (treatments) and left to feed for a month. Foliar area was measured using a square (0.5 cm) point template at the beginning and end of the study to obtain the area change on each plant. The foliar-area ratio was used, which is the quotient of the nal and initial measurements by plant. Data are expressed as means   ± SD. A model I, xed factors, two-way factorial experimental design was used (factor 1 = presence or absence of larvae; factor 2 = eucalyptus species). A design using ve replicates of plants of each species with insects (treatments) plus a control without insects was used. To maintain consumption by larvae, dead ones were substituted by others of similar development, an event which occurred in only two instances.  A Student’s t   test was used to determine if the presence of the insect inuenced the loss of leaf foliar area on the eucalyptus species evaluated. Electrophoresis of protein proles After a month of feeding, three samples (replications) of two larvae by eucalyptus species from the essay described above were analysed by electrophoresis of protein proles. These larvae were kept 24 h in Petri dishes without food to clean their gut content and homogenised at 4 ° C in buffer (TRIS-HCl 50 m  M   pH 8.5, EDTA 5 m  M  , SDS 0.07%, with protease inhibitor phenyl methyl sulfonyluoride 0.1 m  M  ). Homogenisation was achieved by forcing the larvae through a narrow gage between a Teflon pestle and a glass container (Fleischer et al. 1979) and 10 min centrifugation at 1250 g . This material was immediately frozen and stored at –20 ° C. Bradford’s (1976) method was used to determine the protein concentration of extracts of each sample. The extracts were used to determine the electrophoresis proles in one-dimensional gel of denatured polyacrylamide (Laemmli 1970) and Bollag’s et al. (1996) protocol. The molecular weight ranges of proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis  359Huerta et al.—Proteins of Gonipterus scutellatus larvae fed on  Eucalyptus  leaves(SDS-PAGE) in reducing conditions, according to the Laemmli method using minigels (6 cm ×  8 cm ×  0.75 mm). The stacking gels consisted of T = 5% and C = 2.7% and the separating gels of T = 12.5% and C = 2.7%. A vertical electrophoresis apparatus (mini-protein, Bio-Rad) was used to run the gel in electrophoresis buffer (Tris 25 m  M  , glycine 192 m  M  ,   pH 8.8) at a constant voltage setting at 120 V until the bromophenol blue tracker dye reached the bottom of the gel (usually 125 min at room temperature). Standard proteins from 20 to 220 kDa were used as molecular weight markers (LMW BioChile). Fining agents and standard proteins were treated in the same way with Laemmli buffer (v/v) containing β -mercaptoethanol, and 20 µg proteins were loaded in each well. After electrophoresis, separated proteins were stained with 1.5% Coomassier brilliant blue in 50% (v/v) methanol, and destained in acetic acid/methanol/water (1:1:8). The area of marker proteins was found using gel densitometry (BioCaptMW software, Microsoft, Redmond, United States). Chi-square test was used to determine if signicant differences in size and intensity of bands on gels of marker proteins existed between extracts. Only representative gels are shown in Fig. 1. Fig. 1  PAGE-SDS representing protein extracts obtained from Gonipterus scutellatus  fed three species of eucalyptus. A , 1 and 2 replications; B , 1 and 3 replica-tions. (G =  Eucalyptus globulus , R =  E. robusta , C =  E. camaldu-lensis , L = larvae. Numbers 1–3 are replications. PM1 and PM2 are standard molecular mass (kDa).)  360 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35 RESULTSFoliar area loss caused by G. scutellatus  larvae All the control plants had an increase in foliar area. However, in plants with larvae, all species had a decrease in foliar area.  E. robusta  grew the most, followed by  E. globulus , and then  E. camaldulensis , on which they inicted the most damage, reducing its foliar area by 11.21%.  E. globulus  and  E. robusta  had foliar area losses of 6.47% and 3.62%, respectively.  E. robusta  plants under treatment attained greater leaf area growth than  E globulus  and  E. camaldulensis  (Table 1).  The Student’s t   test applied to differences in the average ratio of foliar area of plants with and without insects indicates that G. scutellatus  larvae reduced this parameter in the three species, but signicantly only in  E. camaldulensis  (Table 1). Protein electrophoresis associated with G. scutellatus  larvae Representative gels of G. scutellatus  larvae are shown in Fig. 1. Larvae with the greatest number of proteins were those fed on  E. robusta  (30 proteins), followed by  E. camaldulensis  (28 proteins), and then  E. globulus  (20 proteins) (Fig. 1, 2). There were 19 proteins common to larvae fed all three kinds of food, whereas 11 exhibited variations (protein markers). The differences in protein extracts of G. scutellatus  larvae fed  E. robusta  or  E. camaldulensis  were related to protein 9, which was absent in larvae fed on  E. camaldulensis , and protein 29, which only appeared in larvae fed on  E. robusta . The other nine proteins were common in these two species (proteins 5, 11, 17, 18, 20, 21, 25, 27, and 28 in Fig. 2). In addition to the proteins observed, individuals feeding on  E. robusta  or  E. camaldulensis  exhibited equivalent band areas (Fig. 1). Therefore,  E. globulus   caused signicant differences in number of larval marker proteins ( P  < 0.05). The Chi-square test ( χ 2 = 48.46; P  < 0.05) indicated no difference between the marker proteins (size and intensity) of G. scutellatus  larvae that fed on  E. robusta  versus  E. camaldulensis .  E. globulus  was the only plant that produced different larval marker proteins. DISCUSSIONFoliar area loss caused by G. scutellatus  larvae The reduction observed in foliar area (Table 1) caused by G. scutellatus  larvae was produced, under the conditions of the study, in the month of herbivory. A longer feeding period would likely result in further defoliation, producing a concomitant reduction in plant growth and survival.  Gonipterus scutellatus  larvae inicted the most damage upon  E. camaldulensis , and caused a reduction of foliar area on all plants. This indicates that  E. camaldulen s is  is more vulnerable to infestation by this insect than the other two species. The greater growth rate of  E. globulus  and  E. robusta  plants (Table 1) may be a strategy aimed at reducing insect attack, as in Floyd & Foley (2001), who indicated that tolerance and rapid growth are pest-resistance mechanisms for eucalyptus, whereas more resistant plants may be able to tolerate damage and/or grow rapidly enough to reduce the damage inicted by the insects.  The decrease in foliar area of  E. robusta  may be owing to this plant being more susceptible to damage by G. scutellatus.  Farrow et al. (1994) have shown that plants in the same gender and still of the same species present considerable variations in Table 1  Change in foliar-area caused by second stage Gonipterus scutellatus  larvae on plants of three species of eucalyptus, after month feeding. Treatment means with same letter do not differ statistically according to Student’s t   test (t 8  = 1.8595; P  < 0.05). Foliar-area ratio *,†  Larvae of Foliar-area  Eucalyptus  species Control G. scutellatus reduction (%)  E. camaldulensis  1.062±0.065 0.943±0.035 a 11.21  E. globulus  1.066±0.035 0.997±0.068 b 6.47  E. robusta  1.050±0.019 1.012± 0.049 b 3.62 * Data are expressed as average   ± SD. † Foliar-area ratio is the average quotient of the nal and initial measurements.  361Huerta et al.—Proteins of Gonipterus scutellatus larvae fed on  Eucalyptus  leavessusceptibility to damage by insects, as related to their genetic characteristics. The low rates of foliar area reduction in the plant species evaluated may be related to the supposition that larvae eat and grow only on the tree where they emerge. Specically, the larvae might not completely adapt to the food received on  E. globulus because the chemical composition of young and old leaves is different (Floyd et al. 2001). Electrophoresis protein profles associated with G. scutellatus  larvae The protein markers probably varied owing to the food received by G. scutellatus  larvae. The reduced number of proteins associated with  E. globulus  is related to the srcin of the larvae because they did not need to modify their protein pool. They did not experience a change in diet because they were collected from a stand of this species as rst instar larvae (Fig. 2). It is instructive to note that, of proteins that varied, the only one present in larvae fed on  E. globulus  was number 9. As mentioned, this may be related to the maintenance of food acquisition of the larvae feeding on this species. As such,  E. robusta  caused the greatest variation in protein in G. scutellatus  larvae. The 11 proteins that exhibited variations in the extracts (Fig. 2) constitute marker proteins that indicate changes in larval metabolism, which may be caused by the food they received. Thus, the absence of proteins 9 and 29 in larvae feeding on  E. camaldulensis  may indicate that their number of marker proteins is different than in larvae feeding on  E. robusta . The differences between larvae fed on  E. globulus  compared with  E. camaldulensis  and  E. robusta , combined with the exclusiveness of protein 29 in  E. robusta larvae, allows us to identify when a larva has been fed on one of these species. A possibility proposed to explain the greater number of protein bands in individual larvae feeding on  E. camaldulensis  and  E. robusta  is that during electrophoresis, a protein could have fractionated because of an error in the process and appeared in the gel as if it were two small proteins. This hypothesis was discarded when comparing replicate gels because marker proteins displayed the same behavior in all the extracts taken from larvae feeding on the same species. Presence or absence of marker proteins in G. scutellatus  larvae may be inuenced by chemical composition of the leaves they ate during their development, indicating a physiological reaction to changes in diet. Although it was not studied here, composition of the essential oils of eucalyptus can cause differences in susceptibility to insect damage, which may be reected in its chemical composition Fig. 2  Protein markers in Gonipterus scutellatus  larvae ac-cording to their food source. (PM, standard molecular mass; hori- zontal lines are protein proles, numbered according to their ap-pearance in the gel.)
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