Revista de la Sociedad Entomológica ISSN: 0373-5680 [email protected] Sociedad Entomológica Argentina Argentina

SALDÚA, Luciana; TACALITI, María S.; TOCHO, Erica; DIXON, Anthony F. G.; CASTRO, Ana M. Genetic analysis of greenbug populations of Schizaphis graminum (Hemiptera: Aphididae) from Argentina and Chile based on enzyme variability Revista de la Sociedad Entomológica Argentina, vol. 70, núm. 1-2, 2011, pp. 83-92 Sociedad Entomológica Argentina Buenos Aires, Argentina

Available in: http://www.redalyc.org/articulo.oa?id=322028488008

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative ISSN 0373-5680 (impresa), ISSN 1851-7471 (en línea) Rev. Soc. Entomol. Argent. 70 (1-2): 83-92, 2011 83

Genetic analysis of greenbug populations of Schizaphis graminum (Hemiptera: Aphididae) from Argentina and Chile based on enzyme variability

SALDÚA, Luciana**1, María S. TACALITI*1, Erica TOCHO**, Anthony F. G. DIXON*** and Ana M. CASTRO*

* Cátedra de Genética, Facultad de Ciencias Agronómicas, CC31, (1900) , Argentina; e-mail: [email protected] ** Instituto de Fisiología Vegetal (INFIVE)-CONICET, CC 327, (1900) La Plata, Argentina *** School of Biological Sciences, University of East Anglia, Norwich, U.K., NR4, 7TY, U.K. 1- L. Saldúa and M. S. Tacaliti contributed equally to this paper

Análisis genético de las poblaciones del pulgón verde Schizaphis graminum (Hemiptera: Aphididae), colectadas en Argentina y Chile, basado en su variabilidad enzimática

„ RESUMEN. Veintitrés poblaciones de Schizaphis graminum (Rondani) y sesenta clones colectados en regiones muy contrastantes de la Argentina y Chile fueron investigadas electroforéticamente. Se encontró un alto grado de polimorfismo enzimático. La estructura enzimática fue descripta para el sistema estearasa y se encontraron nueve loci diferentes. Se determinó que existe estratificación latitudinal, las poblaciones fueron asociadas en tres grupos de acuerdo a la latitud donde fueron colectadas. El 90% de los loci resultaron polimórficos en el primer grupo y el 100% de los loci lo fueron en el resto. La heterocigosidad observada fue menor que la esperada. Ningún alelo fue fijado, de acuerdo con el valor del Fsr. El primer grupo tuvo un ligero exceso de heterocigosis, mientras que los demás grupos mostraron valores ligeramente positivos de Fsr, debido al exceso de homocigosis. Con respecto a Frt, el tercer grupo mostró un valor alto que estuvo en concordancia con el alto valor de flujo génico. Tres poblaciones no pudieron ser incluidas en ningún grupo. Las α-carboxil- estearasas siempre estuvieron presentes incluso en clones y poblaciones colectadas en zonas no agrícolas, lo que implica que la resistencia a insecticidas está ampliamente extendida a lo largo del territorio argentino y chileno. No se encontró relación entre el patrón enzimático y el biotipo, así como tampoco con la especie hospedera de donde fueron colectados los áfidos.

PALABRAS CLAVE. Schizaphis graminum. Enzima. Flujo génico. Estructura genética. Asociación geográfica

„ ABSTRACT. Twenty-nine Schizaphis graminum (Rondani) populations and sixty clones collected from very contrasting regions of Argentina and Chile were investigated electrophoretically. A high degree of enzymatic polymorphism was found. The enzymatic structure was described for the esterase system, finding nine different loci. Latitudinal stratification was determined and populations were associated into three groups, according to Recibido: 14-IX-2010; aceptado: 2-V-2011 84 Rev. Soc. Entomol. Argent. 70 (1-2): 83-92, 2011 the latitude they were collected from. The 90% of loci resulted polymorphic in the first group and 100% of loci were polymorphic in the rest. Observed heterozygosity was lower than expected. No group had fixed alleles according to Fsr values; group one had a slight heterozygous excess, while the other groups showed slight positive Fsr values, because there was a homozygous excess. According to Frt, the third group showed the highest value that was in concordance with the highest gene flow value. Three populations could not be included in any group. The α-carboxyl-esterase was always present, also in clones and populations which were located in non agricultural zones, implicating that the insecticide resistance is well spread throughout the Argentinean and Chilean territory. No relationship was found between the enzymatic patterns and the biotype or with the host species where the aphids were collected from.

KEY WORDS. Schizaphis graminum. Enzyme. Genetic flow. Genetic- structure. Geographical association.

INTRODUCTION plants (Puterka & Peters, 1988; Wood et al., 1961; Teetes et al., 1975; Porter et al., Schizaphis graminum (Rondani) 1982; Harvey & Hackerrot, 1969). These (Hemiptera: Aphididae) is the most widely biotypes have been designated A-K based spread aphid pest in Argentina, and has on host plants containing various resistance received much attention because of its history genes, and their origin and evolution are still of overcoming host plant resistance and for controversial subjects (Porter et al., 1997; its wide range of host species. Greenbug was Shufran et al., 2000). Greenbug biotypes C first detected in 1914 in La Pampa province and E were detected in 1988 in Argentina (Ves in Argentina, currently the aphid is spread Losada, 1987). Some biotypes were found throughout the cereal region. Important to be distinct genetic entities that probably outbreaks were recorded in the center of diverged through years of reproductive Santa Fe, Córdoba, La Pampa and south- isolation (Shufran et al., 2000). Noriega et al west of (Noriega et (2000) identified B and C biotypes in Buenos al., 2000). Aires and Córdoba provinces, they also Greenbugs damage their hosts producing reported consistent evidence that biotype E chlorosis, reduction of root volume in wheat is present in Buenos Aires. Finally, biotypes F (Burton, 1986), barley (Arriaga, 1969; Castro and A were found infesting wheat (Almaraz et al., 1988; Gerloff & Orttman, 1971) oat et al., 2001). and sorghum (Castro, 1987; Castro et al., Extensive use of insecticides has led 1990). It also reduces the aerial biomass to widespread development of resistance of wheat (Burton, 1986), barley and rye to one or more classes of insecticide in (Arriaga, 1956). When early attacks occur in many insect species including some aphid oats and barley a decrease in the movement species. The relationship between aphid of protein reserve from seed to aerial part of resistance to insecticides and esterases has the plant has been recorded (Castro et al., been thoroughly confirmed for several aphid 1987 b and Castro and Rumi, 1987). species (Siegfried & Zera, 1994; Devonshire, Biotypes have been defined as 1989; Field et al., 1988). A major cause of populations within an arthropod species that insecticide resistance in Myzus persicae differ in their ability to utilize a particular (Sulzer) (Hemiptera: Aphididae) is the plant genotype (Smith, 2005). Since 1961, amplification of one or two closely related eleven different greenbug biotypes had been genes encoding the carboxilesterases E4 identified in the USA by means of injury and FE4, which hydrolyse and sequester levels determined in different resistant host insecticides (Field & Devonshire, 1998). SALDÚA, L. et al. Greenbug genetic variability 85 A wide range of copy numbers can exist 10% acrylamide with both tris-chlorhydric for both genes, which give a proportionate (pH 8.65) and tris-borate (pH 7.9) buffers in increase in esterase level and consequent a vertical electrophoresis system, running at resistance (Field et al., 1999; Devonshire et 16 volts for 15 minutes and subsequently at al., 1998). 8 volts for 2 hours. In order to obtain indirect information The gels were developed for alpha and about the existence and intensity of genetic beta esterase activity, several subtracts exchange between different regions and were used to differentiate isozymes from about the presence of insecticide resistance, allozymes (20 ug alfa-naftil acetato, 20 ug the purpose of the current research is to beta-naftil acetato, 20 ug alfa-naftil-mirstate, determine the variability in esterase activity 20 ug beta-naftil-miristate, 20 ug alfa-naftil- in greenbug populations collected from very butirate, 20 ug beta-naftil-butirate, 20 ug alfa- contrasting regions of Argentina and Chile in naftil-laurate and 20 ug beta-naftil-laurate autumn and spring since 1987. dissolved in acetone) added to 50 mg of fast blue RR salt and diluted in 100 ml 0.1M sodium phosphate buffer (pH 7.2). The gels MATERIAL AND METHODS were scanned for analytical studies. Esterase patterns were determined Aphid material and identified the allelic forms. These Aphids were collected from contrasting models were determined by means of high regions of Argentina and Chile (Clúa et significant correlation coefficients between al., 2004), Table I. They were reared on electrophorms (PROCOR, SAS, 1998) and susceptible barley (‘Malteria Eda’) and the genotypes. Data was analyzed with maintained at 16:8 LD photophase, 20ºC and POPGENE 3.2 (Yeh et al., 1997) and Shanon’s 50% HR in growth cabinets. Clones were index was obtained, observed and calculated initiated by allowing a single isolated female heterozygosities, Fsr, Frt, Fst and gene flow to reproduce by parthenogenesis, assuming were obtained. The average Hardy-Weinberg that the progeny obtained is genetically expected and observed heterozygosities were identical. Populations and the derived calculated among clones within populations, clones were kept separately by covering populations within regions and populations pots with transparent plastic glass with voile within total area. on the top and ladder. These aphids were Preliminary analysis showed that there collected on different hosts in the field and was a regional structure (Castro, 1994), maintained during several months isolated in consequently the inbreeding effect of the insectary. population substructure was assessed with Approximately 200 individuals from every the fixation index (F). The F index is a useful clone were separated and collected into an index of genetic differentiation because it eppendorf tube where they were smashed allows an objective comparison of the overall with a capillary tube sealed with heat on effects of population substructures among its tip. The samples were diluted 1:1 with different clones. This index explains the trisglicina buffer (pH 8.3) and centrifuged at reduction in the expected heterozygosity with 13.500 rpm for 3 minutes. The supernatant random mating at any level of a population was collected and diluted 1:1/4 with glycerol hierarchy, related to another, more inclusive and frozen at -20ºC. level of the hierarchy. Fsr is the fixation index of the populations relative to the regional Procedures aggregates. Frt is the proportional reduction In order to determine the genetic base of in heterozygosity of the regional aggregates different isozyme and to separate these from relative to the total combined population. allozymes, several specific substrates were Finally, Fst compares the least inclusive to used simultaneously. Activity of esterase the most inclusive levels of the population was performed in poliacrylamide gel using hierarchy measuring all effects of population 86 Rev. Soc. Entomol. Argent. 70 (1-2): 83-92, 2011 substructure combined. F values vary from Table I. Region of collection, geographical 0 to 1, the last value indicates fixation for coordinates (GPS Data) and host plant of each alternative alleles in different populations greenbug (Schizaphis graminum) population. Coordinates (Hartl et al., 1997). Population Host Plants S. L. W.L.

1. Salta 24°40´ 65°03´ Sorghum halepense 2. Tucumán 26°50´ 65°12´ Sorghum halepense RESULTS 3. Santiago del Estero 27°48´ 61º 35´ Sorghum halepense 4. Frías 28°39´ 65°09´ Sorghum halepense Hordeum sp 5. La Cumbre 30°59´ 64°29´ Bands in the gels were distributed in Bromus sp. 6. Rafaela 31°10´ 61°28´ Sorghum sp. seven regions (A-G) each one representing Triticum sp. 7. Paraná 31°52’ 60° 29’ Sorghum sp. different loci (Fig. 1) and a total of ten Bromus sp. 8. Córdoba Sorghum sp. different electrophenotypes were revealed. 31°40’ 62° 20’ Norte Avena sp. Hordeum vulgare All studied populations and clones showed 9. Córdoba Sur 32°40’ 60° 53’ Avena sp. esterase activity. 10. Villa María 32°25’ 63° 15’ Avena sp. Sorghum sp. 11. Baradero 33°49´ 59°30´ The first region (A), comprised by A1, A2, Avena sp. A3, revealed a brownish coloration that did Avena sp. 12. Los Hornos 34°55’ 57° 57’ Hordeum vulgare not show enzymatic variability. Three bands Poa sp Avena sp. appeared in all the studied genotypes (Fig. 1). 13. B. Bavio 35°05’ 57° 44’ Sorghum sp. Hordeum sp. This enzyme represented a dimorphic protein, Secale cereale being every individual heterozygous. 14. Malargüe 35°30’ 69° 35’ Bromas sp. Hordeum sp. Hordeum sp. The second region (B) is represented 15. Ayacucho 37°08’ 58° 29’ Poa sp. by the presence or absence of bands. Two Triticum sp. 16. Balcarce 37°50’ 58° 17’ Hordeum vulgare alleles were detected, being one of them Triticosecale null. The B1 band represented the null allele. Hordeum vulgare Avena sp. Secale cereale The B2 bands stained brownish. The model 17.Bordenave 37°51’ 63° 01’ Triticosecale that matches this pattern is a monomorphic Triticum sp. Bromus sp. protein. Avena sativa 18. G. Chaves Hordeum vulgare 38°02’ 60° 05’ The third region (C) comprised C1 to C4 Triticum aestivum bands, stained reddish and presented seven Triticum durum Secale cereale 19. Osorno (CH)1 38°13’ 72° 20’ different patterns composed by none, one or Triticum sp. Triticum sp. two electrophorms each. A monomorphic 20. Miramar 38°15’ 57° 50’ Hordeum vulgare Bromus sp. protein with four alleles (C1, C2, C3 and C4) T.aestivum T. durum could explain the activity of this enzyme. 21. 38°23’ 60° 17’ Triticosecale The homozygous individuals presented Hordeum vulgare Avena sp. one or none band, while heterozygous 22. La Dulce 38°25’ 58° 42’ Hordeum vulgare Triticum sp. individuals revealed two bands. All possible 23. Lonquimay (CH) 1 38°26’ 71° 21’ Avena sp. Triticum sp. combinations between alleles were presented 24.Temuco (CH)1 38°20´ 72°20´ Avena sp. Triticum sp. in the current survey (Fig. 1, genotype 5 has 25. Cabildo 38°30’ 61° 54’ Tritordeum C2- C3, genotype 6 has C3- C4, genotype 7 TRITICUM SP. 26. Teka 43°28’ 70° 50’ showed C2- C4). BROMUS SP. 27. Bahía Blanca 42°29’ 61° 55’ Triticum sp. The fourth region, D, (D1 to D5) revealed 28. Castro (CH)1 42°29´ 73°45´ Dactylis sp. the activity of alpha esterase bands by a 29. Los Alerces 42°55’ 71° 20’ Triticum sp. brownish coloration. Every genotype with in our clones, representing heterozygous none, one or two bands showed five different individuals (Fig. 1, genotypes 6-10). allelic forms within all the homozygous Similarly, the fifth region (E) ranging from individuals. Ten electrophoretic patterns E1 to E4, revealed none, one or two bands. indicating the activity of monomorphic Seven different patterns were detected. proteins with five alleles (Fig. 1, genotypes: The activity of a monomorphic protein 1, 2, 3, 4 and 5) were found. Although, two with four alleles, one of them considered band patterns were also registered, only null, explained this pattern. Those clones five combinations of alleles were present with none activity represented the null SALDÚA, L. et al. Greenbug genetic variability 87

Fig. 1. Enzymatic patterns for nine loci of Schizaphis graminum with the description of the found genotypes in sampled populations and clones. homozygous individuals. Other clones have reported above. In region E1 six clones only one band (Fig. 1, genotypes 2- 4), while (Bordenave 8, Gonzales Chaves 2, Gonzales heterozygous individuals presented two Chaves 1, Villa María 2, Teka 4 and Bavio 2) bands combining alleles E2, E3 and E4 (Fig. showed a three band pattern that represented 1, genotypes 5, 6, 7). the activity of a dimorphic protein. These

The sixth region, F (F1 to F3), showed individuals were heterozygous (E122- E123- also none, one or two bands, indicating the E133). In region F1, four clones (Gonzales activity of monomorphic proteins. The zone Chaves 2, 4, 1 and Bordenave 9) revealed a stained light reddish. Probably, homozygous three band pattern (F122- F123- F133), being individuals were represented by none or one all individuals heterozygous. band and heterozygous by two bands, by The allelic frequencies of all the studied combining allele F2 and F3. regions were estimated (Table II). Hardy- The last region (G), comprised G1 to Weinberg equilibrium was only found

G7, revealed by dark-brownish bands for region F1. All studied loci were found characteristic of alpha carboxyl esterase polymorphic, nonetheless not all the activity. This region was present in all the possible allelic combinations were found in studied genotypes with only one band. our study. Seven different allelic forms were found First analysis of genetic distance showed representing the activity of a monomorphic that populations could be joined into three protein. groups by means of similitude index values Ten clones did not share the pattern (Table III). First group was conformed by commonly found in regions E and F. These populations: Salta, Tucumán, Santiago two new patterns were considered as two del Estero, Frías, La Cumbre, Rafaela, and new proteins different from the previously Córdoba Norte (that were collected from 88 Rev. Soc. Entomol. Argent. 70 (1-2): 83-92, 2011 Table II. Allelic frequencies of esterase loci determined for populations and clones of Schizaphis graminum

Table III. Percentage of gene polymorphism, observe and expected heterozygocity, Fsr, Frt, Fst and gene flow for the three groups of Schizaphis graminum, following Nei (1987).

%Gene Shannon Gene Group Obser. Hetrozygosity Expec.Heterozygosity Fsr Frt Fst Polymorphism Index Flow 1 90 0.82 0.28 0.47 -0.15 0.49 0.41 0.26 2 100 0.90 0.28 0.50 0.18 0.23 0.38 0.80 3 100 0.95 0.28 0.54 0.07 0.46 0.41 0.35

26º 50´ S to 31º 40´ S; Fig. 2A). Second studied groups the observed heterozygosity group joined the following populations: was lower than the expected heterozygosity, Paraná, Córdoba Sur, Villa María, Baradero, this could be explained by the effect of the Bavio, Malargüe and Temuco (from 31º homozygous individuals that were the most 52´S to 35º 30´S; Fig. 2B). The third group frequent genotype and consequently, the was represented by Ayacucho, Balcarce, observed heterozygosity values dropped due Bordenave, Gonzales Chaves, Osorno, to the lack of heterozygous individuals. Miramar, Tres Arroyos, La Dulce, Lonquimay, None group had fixed alleles according Cabildo, Bahía Blanca, Los Alerces, Teka, to Fsr values (Table III); group one had a and Castro (from 37º 08´ S to 43º 28´S; Fig. slight heterozygous excess, while groups two 2C). In the first group, La Cumbre showed and three showed slight positive Fsr values, significant differences with the rest of the because there was a homozygous excess. populations. Baradero belongs to the first Comparing Frt values (Table III) the first group according to its geographical location, and third groups showed the highest values, however it joined the third group. Similarly, while the second group differed significantly Lonquimay joined the rest of the third group from the total, showing the lowest values of at high genetic distance. Temuco fit into the Frt. Fst values (Table III) showed significant second group but is geographically located differences related to the total expected in the strip corresponding to the third group heterozygosity (higher than 0.25) of the total (38º 20´S). populations in all the studied populations. The second and third groups revealed These results agreed with the calculated gene 100% of the loci polymorphic while the first flow values. The second group had the highest group has 90% of polymorphic loci (Table III). value contrasting with the first group which Consistently with the values of polymorphic had the lowest value, and simultaneously loci the lowest Shannon’s index was found showed the highest Fst with very great in the third group, followed by the first differences with the total populations based group and the second raised 0.90. In all the on the expected heterozygosity under Hardy- SALDÚA, L. et al. Greenbug genetic variability 89

Pop3

Pop6

Pop8

Pop2 Pop1

Pop11 Pop4

Pop5 Figure 2A: Group 1

Pop14 Pop9

Pop13 Pop24

Pop10

Pop7 Pop12

Figure 2B: Group 2

Pop22

Pop26

Pop17 Pop18

Pop29 Pop21

Pop27 Pop28

Pop15

Pop19

Pop16

Pop20

Pop25

Figure 2C: Group 3

Fig. 2. Genotypes grouping of greenbug clones and populations collected in Argentina and Chile, by means of similitude index values A: Sampled collected between 26º 50´ S to 31º° 40´ S. B: Aphids collected between 31º° 52´S to 35º 30´S. C: Aphids collected between 37º 08´ S to 43º 28´S. 90 Rev. Soc. Entomol. Argent. 70 (1-2): 83-92, 2011 Weinberg equilibrium. because the number of sexual individuals is not enough to successfully carry out them. Previous studies have shown that greenbug DISCUSSION populations collected from very contrasting ecological regions of Argentina showed The present study revealed the existence of significant differences in reproduction variability for esterase enzyme in Schizaphis behavior, lifespan, host preference (Ramos graminum in populations of Argentina and et al., 1998; Clúa et al., 2004), even the Chile, contrasting with previous results genetic variation of aphid aggressiveness has where no distinguishable differences were been studied among biotypes by means of found (Steiner et al., 1985; Beregovoy the coleoptile tests (Giménez et al., 1991a, & Shark, 1986; Abid et al., 1989) and in b, 1992; Castro, 1994; Castro et al., 1991). agreement with Giménez et al. (1991a) It is also important to mention that all and Castro et al. (1996). Nine different loci the populations were collected from a were found, only one showed no variability. very great variety of host species including These loci were found to be reliably resolved cultivated and non-cultivated host species. and polymorphic in preliminary surveys of No relationship was found between isozyme variation (Castro et al., 1987a). the host species where the insects were Kephart (1990) provided helpful collected from and the current genetic information for interpreting isozyme patterns. analysis of sampled populations. Clones Infrequent multimeric association or low and populations have been maintained in enzyme activity may generate faint bands. In insectary conditions reared on susceptible plants with null alleles that lack enzymatic barley since their collection date, this fact activity, homozygotes and heterozygotes could have conditioned the enzymatic become visually indistinguishable in patterns and probably as it was found by monomorphic proteins, heterozygotes show Anstead et al. (2002) a relationship between unexpected two or four banded phenotypes recent captured population of Schizaphis in dimorphic and tetramorphic proteins. graminum and its hosts might be found. Furthermore, under certain experimental In the present study, populations and conditions, extra ghost bands that show clones were associated in three groups primary bands are present. The proposal of the latitudinally structured. Similarly, Li (2011) existence of a null allele in the heterozygous found in Oxya hyla intricata Stál (Orthoptera: individual is based on the intensity of the Catantopidae) three monophyletic clades bands in the gels (Kephart, 1990). In the latitudinally structured corresponding to current results it is not possible to confirm the three geographical regions separated by enzymatic patterns from regions B, C, D, E, F high mountains. In the current survey, it and F1 because it is difficult to differentiate would be possible that day-light conditions whether the individuals that showed only (photoperiod) and temperature regimes one band are homozygous for this gene or could have been conditioning the genetic heterozygous carrying the null allele because similitude of the studied populations. There no band intensities differences could be were four exceptions, La Cumbre, Baradero, found. In region A the model is not adjusted Lonquimay and Temuco populations. La to the presence of three monomorphic Cumbre could be considered as a different proteins, due to the lack of discontinuity group and a correlation between the absence between the bands, although this could be a of eggs (Clúa et al., 2004) and the isolation dimorphic protein. from the rest of the studied populations could Kephart (1990) proposed that the genetic be considered. Lonquimay was collected basis of observed patterns should be verified over 2800 msl with a high rainfall and very by isozyme analysis of progeny arrays from cold winters with a wide range in day-night controlled crosses. In fact, crosses in our temperature, although no discontinuity aphid populations are extremely difficult of host species was found between the SALDÚA, L. et al. Greenbug genetic variability 91 valley and the agriculture parcels situated collected in Argentina and Chile. It was also high in the Andes. This population was possible to identify latitudinal stratification characterised as sexually reproductive by of greenbug populations. It is worth to Clúa et al. (2004). Nonetheless, there were note that α-carboxyl-esterase was present, parthenogenetic individuals together with also in clones and populations collected in others that induced sexual individuals. non agricultural zones, implicating that the Temuco population genetically joined with insecticide resistance in our local populations the second group (from 31º 52´S to 35º is well spread throughout the Argentinean 30´S), but geographically it is located into and Chilean territories. Finally, there was the third group (from 37º 08´ S to 43º 28´S). no association between the biotype and the Temuco is located into the typical cereal electrophoretic esterase patterns. winter production region of Chile (central valley) with very dry summers and a rainfall below 700 mm. Populations from Baradero LITERATURE CITED and Parana were highly similar to each other due to their geographical location, 1. ABID, H. S, S. D. KINDLER, S. G. JENSEN, M. A. THOMAS- COMPTON & S. M. SPOMER. 1989. Isozymes although they joined different groups. It was characterization of Sorghum aphid species and not possible to collect any greenbug sample greenbug biotypes (Homoptera: Aphididae). Annals of the Entomological Society of America 82: 303-306. in those locations situated in-between those 2. ALMARAZ, L., H. O. CHIDICHIMO & A. M. CASTRO. 2001. places, probably there would have been a Biotype composition of Argentinean populations of Schizaphis graminum. Actas XXIII Congreso Chileno common origin for these aphids or a high de Entomología, Temuco, Chile, p 2. genetic flux. 3. ANSTEAD, J. A., J. D. BUND & K. A. SHUFRAN. 2002. Some biotypes can be characterized Mitochondrial DNA sequence divergence among Schizaphis graminum (Hemiptera: Aphididae) clones by diverse methods. Shufran et al. (2000), from cultivated and non-cultivated hosts: haplotype studying the biotypes of S. graminum by a and host associations. Bulletin of Entomological Research 92: 17-24. molecular phylogenetic analysis based on 4. ARRIAGA, H. O. 1956. El centeno Insave FA. Híbrido the cytochrome oxidase I mitochondrial sintético resistente a la toxemia del pulgón verde de los cereales. Revista de la Facultad de Agronomía La gene, combined with earlier studies of Plata XXXII, pp. 190-209. mDNA (Power et al., 1989), rDNA (Black, 5. ARRIAGA, H. O. 1969. Resistance to greenbug toxemia. 1993) and RAPD (Black et al., 1992), Barley news 13: 58-60. 6. BEREGOVOY, V. H. & K. J. L. STARKS. 1986. Enzyme concluded that the biotypes of S. graminum patterns in biotypes of greenbug Schizaphis graminum are probably host-adapted races. The C (Rondani). (Homoptera:Aphididae). Journal of the Kansas Entomological Society 59: 517-523. biotype was the predominant agricultural 7. BLACK, W., N. DUTEAU, G. PUTERKA, J. NECHOLS type infesting sorghum and wheat found in & J. PELLORINI. 1992. Use of random amplified polimerasa chain reaction (RAPD-PCR) to detect DNA our populations (Noriega et al., 2000; Clúa polymorphism in aphids (Homoptera: Aphidiae). et al., 2004). By analyzing the relationship Bulletin of Entomological Research 82: 151-159. between the isozymes profiles and greenbug 8. BLACK, W. C. 1993. Variation in the ribosomal RNA cistron among host-adapted races of an aphid (Schizaphis biotypes distinctive patterns were found, graminum). Insect Molecular Biology 2: 59-69. agreeing with Abid (1989), Ramos et al. 9. BURTON, R. L. 1986. Effect of greenbug (Homoptera: Aphididae) damage on roots and shoots biomass in (1998) and Noriega (2000). On the contrary, wheat seedlings. Journal of Economic Entomology 79: Beregovoy and Starks (1986) established 633-636. 10. CASTRO, A. M. Inéd. Uso de Hordeum chilensis en la that few isozyme differences exist between mejora de la resistencia a áfidos en cereales. Thesis, biotypes B, C, or E. In our study, biotype C Universidad de Valencia, 1994, 150 pp. that gathered Balcarce population, Bavio 6, 11. CASTRO, A. M & C. P. RUMI. 1987. Greenbug damage on the aerial vegetative growth of two barley cultivars. La Dulce 9, Bahia Blanca 1 and 2, Bordenave Environmental and Experimental Botany 23 (3): 263-271 6 and 9 (Noriega et al., 2000; Clúa et al., 12. CASTRO, A. M., C. P. RUMI & H. O ARRIAGA. 1987a. Host plant isozymes profiles of greenbug susceptible and 2004), showed different electrophenotypes tolerant barley, oats and sorghum cultivars. Annals represented in Figure 1 (i.e. genotypes 2, 1, Plant Resistance to Insects Newsletter 13: 39-40. 13. CASTRO, A. M, C. P. RUMI & H. O. ARRIAGA. 1987b. 1, 3, 2, 5, 5 for the C esterase, respectively). Oat production losses under greenbug (Schizaphis Current results show there is genetic graminum, Rondani) infestation and forward recovery. variability on the greenbug populations Turrialba 39: 97-105. 92 Rev. Soc. Entomol. Argent. 70 (1-2): 83-92, 2011 14. CASTRO, A. M., C. P. RUMI & H. O. ARRIAGA. 1988. 31. LI, T., M. ZHANG, Y. QU, Z. REN, J. ZHANG, Y. GUO, K. Influence of greenbug on root growth of resistance L. HEONG, B. VILLAREAL, Y. ZHONG & E. MA. 2011. and susceptible barley genotypes. Environmental and Population genetic structure and phylogeographical Experimental Botany 28: 61-72. pattern of rice grasshopper, Oxya hyla intricata, across 15. CASTRO, A. M., C. P. RUMI & H. O. ARRIAGA. 1990. Southeast Asia. Genetica 139: 511-524. Alteraciones en el crecimiento radical de cultivares 32. NEI, M. 1987. Molecular Evolutionary Genetics/ Masatoshi de sorgo infestados con pulgón verde. Turrialba 41: Nei. Columbia University Press, New York, pp. 176- 166-171. 187. 16. CASTRO, A. M, C. P. RUMI & H. O. ARRIAGA. 1991. 33. NORIEGA, A., H. CHIDICHIMO & A. M. CASTRO. 2000. Greenbug effect on grain yield and seed protein Determination of biotypes in three populations content. Annals Plant Resistance to Insect Newsletters of greenbug collected in three localities of cereal 16: 67-68. producing area in Argentina. Revista de La Facultad 17. CASTRO A. M., S. RAMOS, P. FRANGI, D. GIMÉNEZ, A. de Agronomía, La Plata, 104(2): 85-92. VASICEK & M. MUÑOZ. 1996. Variability determined 34. PORTER, K. G., G. L. PETERSON & O. VISE. 1982. A new by isozymes between and within Schizaphis graminum greenbug biotype. Crop Science 22: 847-850. (Rondani) populations. Actas XVIII Congreso Chileno 35. PORTER, D. R., J. D. BURD, SHUFRAN, K. A, J. A. WEBSTER de Entomología, Temuco, Chile, p. 132. & G. L. TEETES. 1997. Greenbug (Homoptera: 18. CLÚA, A., A. M. CASTRO, S. RAMOS, D. O. GIMÉNEZ, A. Aphididae) biotypes: select by resistance cultivars VASICEK, H. CHIDICHIMO & A. F. G DIXON. 2004. or preadapted opportunists? Journal of Economic The biological characteristics and distribution of Entomology 90: 1055-1065. Schizaphis graminum and Diuraphis noxia (Hemiptera: 36. POWER, T. O, S. G. JENSEN, S. D. KINDLER, C. J. STRYKER Aphididae) in Argentina and Chile. European Journal & L. J. SANDAL. 1989. Mitochondrial DNA divergence of Entomology 101(1): 193-198. among greenbug (Homoptera: Aphididae) biotypes. 19. DEVONSHIRE, A. L. 1989. Resistance of Aphids to Annals of the Entomological Society of America 82: Insecticides. In: A. K. Minks and P. Harrewijn 298-302. (eds.) Aphids: Their Biology, Natural enemies and 37. PUTERKA, G. J & D. C. PETERS. 1988. Rapid techniques Control. Volume 2C. Elsevier Science Pulishers B.V., for determining greenbug (Homoptera: Aphididae) Amsterdam. biotypes B, C, E and F. Journal of Economic Entomology 20. DEVONSHIRE, A. L., L. M. FIELD, S. P. FOSTER, G. D. 81: 396-399. MOORES, M. S. WILLIAMSON & R. L. BLACKMAN. 38. RAMOS, S., H. O. CHIDICHIMO & A. M. CASTRO. 1998. The evolution of insecticide resistance in the 1998: Evaluation of antibiosis to isolates of greenbug peach-potato aphid, Myzus persicae. Philosophical (Schizaphis graminum, Rond.) in differential cultivars. Transactions of the Royal Society B 353: 1677-1684. Actas XIX Congreso de Entomología, Rio de Janeiro, 21. FIELD, L. M., A. L. DEVONSHIRE & B. G. FORDE. 1988. Brasil, p. 300. Molecular evidence that insecticide resistance in 39. SHUFRAN, K. A, J. D. BURD, J. A. ANSTEAD & G. LUSHAI. peach-potato aphids (Myzus persicae (Sul.) results 2000. Mitochondrial DNA sequence divergence from amplification of an esterase gene. Biochemical among greenbug (Homoptera: Aphididae) biotypes: Journal 251: 309-321. evidence for host-adapted races. Insect Molecular 22. FIELD, L. M. & A. L. DEVONSHIRE. 1998. Evidence that the Biology 9 (2): 179-184. E4 and FE4 esterase genes responsible for insecticide 40. SIEGFRIED, B. D. & A. J. ZERA. 1994. Partial purification resistance in the aphid Myzus persicae (Sulzer) are part and characterization of a greenbug (Homoptera: of a gene family. Biochemical Journal 330: 169-173. Aphididae) Esterase associated with resistance to 23. FIELD, L. M., R. L. BLACKMAN., C. TYLER-SMITH & A. L. parathion. Pesticide Biochemistry and Physiology, 49: DEVONSHIRE. 1999. Relationship between amount 132-137. of esterase and gene copy number in insecticide- 41. SMITH, C. M. 2005. Plant resistance to arthropods: resistant Myzus persicae (Sulzer). Biochemistry Journal molecular and conventional approaches. Springer 339: 737-742. Science & Business, Manhattan. 24. GERLOFF, E. & E. ORTMAN. 1971. Physiological changes 42. STEINER, W. W. M., D. J. VOEGTLIN & M. E. IRWIN. in barley induced by feeding stress. Crop Science 11: 1985. Genetic differentiation and its bearing on 174-176. migration in North America population of corn leaf 25. GIMÉNEZ, D. O., G. N. BROCCHI, A. M. CASTRO, L. aphid, Rhopalosiphum maidis (Fitch) (Homoptera: B. ALMARAZ & H. O ARRIAGA. 1991a. Greenbug Aphididae). Annals of the Entomological Society of variability evaluated by isozymes polymorphisms. America 78: 518-525. Annals Plant Resistance to Insects Newsletter 17: 68-691. 43. TEETES, G. A., C. A. SCHAEFER, J. R. GIPSON, R. C. 26. GIMÉNEZ, D. O., G. N. BROCCHI, A. M. CASTRO & L. B. MCINTYRE & E. E. LATHAN. 1975. Greenbug ALMARAZ. 1991b. Biological test: screening greenbug resistance to organophsphorus insecticide on the Texas aggressiveness and barley cultivar resistance by the high plains. Journal of Economic Entomology 68: 214- coleoptile growth. Actas XIX Reunión Nacional de 216. Fisiología Vegetal, Córdoba, Argentina, pp 33-34. 44. VES LOSADA, J. C. 1987. Determinación de un nuevo 27. GIMÉNEZ D. O, G. O BROCCHIO, A. M. CASTRO, L. B. biotipo del pulgón verde de los cereales en Argentina. ALMARAZ & H. O. ARRIAGA. 1992. The development Estación Experimental Agropecuaria Anguil. Anguil, of new biological test. Annals Plant Resistance to La Pampa. Publicación Técnica 40: 1-11. Insect Newsletters 18: 53. 45. WOOD, E. A. 1961. Biologycal studies of a new greenbug 28. HARTL, D.L. & A. J. CLARK. 1997. Principles of Population biotype. Journal of Economic Entomology 54: 1171- Genetics. 3rd editon. Sunderland, MA, Sinauer 1173. Associates. 46. YEH, F. C., R. C. YANG, T. B. J. BOYLE, Z. H. YE & J. X. 29. HARVEY, T. K & H. L. HACKEROTT. 1969. Recognition of MAO. 1997. POPGENE, the user-friendly shareware a greenbug biotype injurious to sorghum. Journal of for population genetic analysis. Molecular Biology and Economic Entomology 12: 776-779. Biotechnology Centre, University of Alberta, Canada. 30. KEPHART, S. R. 1990. Starch gel electrophoresis of plant isozymes: a comparative analysis of techniques. American Journal of Botany 77 (5): 693-712.