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

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

 

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) La Plata, Argentina; e-mail: msilviatacaliti@yahoo.com.ar
^** 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

Recibido: 14-IX-2010
Aceptado: 2-V-2011

 

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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 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.

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

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INTRODUCTION

Schizaphis graminum (Rondani) (Hemiptera: Aphididae) is the most widely spread aphid pest in Argentina, and has received much attention because of its history of overcoming host plant resistance and for its wide range of host species. Greenbug was first detected in 1914 in La Pampa province in Argentina, currently the aphid is spread throughout the cereal region. Important outbreaks were recorded in the center of Santa Fe, Córdoba, La Pampa and south­west of Buenos Aires province (Noriega et al., 2000).
Greenbugs damage their hosts producing chlorosis, reduction of root volume in wheat (Burton, 1986), barley (Arriaga, 1969; Castro et al., 1988; Gerloff & Orttman, 1971) oat and sorghum (Castro, 1987; Castro et al., 1990). It also reduces the aerial biomass of wheat (Burton, 1986), barley and rye (Arriaga, 1956). When early attacks occur in oats and barley a decrease in the movement of protein reserve from seed to aerial part of the plant has been recorded (Castro et al., 1987 b and Castro and Rumi, 1987).
Biotypes have been defined as populations within an arthropod species that differ in their ability to utilize a particular plant genotype (Smith, 2005). Since 1961, eleven different greenbug biotypes had been identified in the USA by means of injury levels determined in different resistant host plants (Puterka & Peters, 1988; Wood et al., 1961; Teetes et al., 1975; Porter et al., 1982; Harvey & Hackerrot, 1969). These biotypes have been designated A-K based on host plants containing various resistance genes, and their origin and evolution are still controversial subjects (Porter et al., 1997; Shufran et al., 2000). Greenbug biotypes C and E were detected in 1988 in Argentina (Ves Losada, 1987). Some biotypes were found to be distinct genetic entities that probably diverged through years of reproductive isolation (Shufran et al., 2000). Noriega et al (2000) identified B and C biotypes in Buenos Aires and Córdoba provinces, they also reported consistent evidence that biotype E is present in Buenos Aires. Finally, biotypes F and A were found infesting wheat (Almaraz et al., 2001).
Extensive use of insecticides has led to widespread development of resistance to one or more classes of insecticide in many insect species including some aphid species. The relationship between aphid resistance to insecticides and esterases has been thoroughly confirmed for several aphid species (Siegfried & Zera, 1994; Devonshire, 1989; Field et al., 1988). A major cause of insecticide resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) is the amplification of one or two closely related genes encoding the carboxilesterases E4 and FE4, which hydrolyse and sequester insecticides  (Field  &  Devonshire,  1998).
A wide range of copy numbers can exist for both genes, which give a proportionate increase in esterase level and consequent resistance (Field et al., 1999; Devonshire et al., 1998).
In order to obtain indirect information about the existence and intensity of genetic exchange between different regions and about the presence of insecticide resistance, the purpose of the current research is to determine the variability in esterase activity in greenbug populations collected from very contrasting regions of Argentina and Chile in autumn and spring since 1987.

MATERIAL AND METHODS

Aphid material
Aphids were collected from contrasting regions of Argentina and Chile (Clúa et al., 2004), Table I. They were reared on susceptible barley (‘Malteria Eda’) and maintained at 16:8 LD photophase, 20ºC and 50% HR in growth cabinets. Clones were initiated by allowing a single isolated female to reproduce by parthenogenesis, assuming that the progeny obtained is genetically identical. Populations and the derived clones were kept separately by covering pots with transparent plastic glass with voile on the top and ladder. These aphids were collected on different hosts in the field and maintained during several months isolated in the insectary.

Table I. Region of collection, geographical coordinates (GPS Data) and host plant of each greenbug (Schizaphis graminum) population.

Approximately 200 individuals from every clone were separated and collected into an eppendorf tube where they were smashed with a capillary tube sealed with heat on its tip. The samples were diluted 1:1 with trisglicina buffer (pH 8.3) and centrifuged at 13.500 rpm for 3 minutes. The supernatant was collected and diluted 1:1/4 with glycerol and frozen at -20ºC.

Procedures
In order to determine the genetic base of different isozyme and to separate these from allozymes, several specific substrates were used simultaneously. Activity of esterase was performed in poliacrylamide gel using 10% acrylamide with both tris-chlorhydric (pH 8.65) and tris-borate (pH 7.9) buffers in a vertical electrophoresis system, running at 16 volts for 15 minutes and subsequently at 8 volts for 2 hours.
The gels were developed for alpha and beta esterase activity, several subtracts were used to differentiate isozymes from allozymes (20 ug alfa-naftil acetato, 20 ug beta-naftil acetato, 20 ug alfa-naftil-mirstate, 20 ug beta-naftil-miristate, 20 ug alfa-naftil-butirate, 20 ug beta-naftil-butirate, 20 ug alfa-naftil-laurate and 20 ug beta-naftil-laurate 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 were scanned for analytical studies.
Esterase patterns were determined and identified the allelic forms. These models were determined by means of high significant correlation coefficients between electrophorms (PROCOR, SAS, 1998) and the genotypes. Data was analyzed with POPGENE 3.2 (Yeh et al., 1997) and Shanon’s index was obtained, observed and calculated heterozygosities, Fsr, Frt, Fst and gene flow were obtained. The average Hardy-Weinberg expected and observed heterozygosities were calculated among clones within populations, populations within regions and populations within total area.
Preliminary analysis showed that there was a regional structure (Castro, 1994), consequently the inbreeding effect of population substructure was assessed with the fixation index (F). The F index is a useful index of genetic differentiation because it allows an objective comparison of the overall effects of population substructures among different clones. This index explains the reduction in the expected heterozygosity with random mating at any level of a population hierarchy, related to another, more inclusive level of the hierarchy. Fsr is the fixation index of the populations relative to the regional aggregates. Frt is the proportional reduction in heterozygosity of the regional aggregates relative to the total combined population. Finally, Fst compares the least inclusive to the most inclusive levels of the population hierarchy measuring all effects of population substructure combined. F values vary from 0 to 1, the last value indicates fixation for alternative alleles in different populations (Hartl et al., 1997).

RESULTS

Bands in the gels were distributed in seven regions (A-G) each one representing different loci (Fig. 1) and a total of ten different electrophenotypes were revealed. All studied populations and clones showed esterase activity.

Fig. 1. Enzymatic patterns for nine loci of Schizaphis graminum with the description of the found genotypes in sampled populations and clones.

The first region (A), comprised by A1, A2, A3, revealed a brownish coloration that did not show enzymatic variability. Three bands appeared in all the studied genotypes (Fig. 1). This enzyme represented a dimorphic protein, being every individual heterozygous.
The second region (B) is represented by the presence or absence of bands. Two alleles were detected, being one of them null. The B1 band represented the null allele. The B2 bands stained brownish. The model that matches this pattern is a monomorphic protein.
The third region (C) comprised C1 to C4 bands, stained reddish and presented seven different patterns composed by none, one or two electrophorms each. A monomorphic protein with four alleles (C1, C2, C3 and C4) could explain the activity of this enzyme. The homozygous individuals presented one or none band, while heterozygous individuals revealed two bands. All possible combinations between alleles were presented in the current survey (Fig. 1, genotype 5 has C2- C3, genotype 6 has C3- C4, genotype 7 showed C2- C4).
The fourth region, D, (D1 to D5) revealed the activity of alpha esterase bands by a brownish coloration. Every genotype with none, one or two bands showed five different allelic forms within all the homozygous individuals. Ten electrophoretic patterns indicating the activity of monomorphic proteins with five alleles (Fig. 1, genotypes: 1, 2, 3, 4 and 5) were found. Although, two band patterns were also registered, only five combinations of alleles were present in  our  clones,  representing heterozygous individuals (Fig. 1, genotypes 6-10).
Similarly, the fifth region (E) ranging from E1 to E4, revealed none, one or two bands. Seven different patterns were detected. The activity of a monomorphic protein with four alleles, one of them considered null, explained this pattern. Those clones with none activity represented the null homozygous individuals. Other clones have only one band (Fig. 1, genotypes 2- 4), while heterozygous individuals presented two bands combining alleles E2, E3 and E4 (Fig. 1, genotypes 5, 6, 7).
The sixth region, F (F1 to F3), showed also none, one or two bands, indicating the activity of monomorphic proteins. The zone stained light reddish. Probably, homozygous individuals were represented by none or one band and heterozygous by two bands, by combining allele F2 and F3.
The last region (G), comprised G1 to G7, revealed by dark-brownish bands characteristic of alpha carboxyl esterase activity. This region was present in all the studied genotypes with only one band. Seven different allelic forms were found representing the activity of a monomorphic protein.
Ten clones did not share the pattern commonly found in regions E and F. These two new patterns were considered as two new proteins different from the previously reported above. In region E1 six clones (Bordenave 8, Gonzales Chaves 2, Gonzales Chaves 1, Villa María 2, Teka 4 and Bavio 2) showed a three band pattern that represented the activity of a dimorphic protein. These individuals were heterozygous (E₁22- E₁23-E₁33). In region F₁, four clones (Gonzales Chaves 2, 4, 1 and Bordenave 9) revealed a three band pattern (F₁22- F₁23- F₁33), being all individuals heterozygous.
The allelic frequencies of all the studied regions were estimated (Table II). Hardy-Weinberg equilibrium was only found for region F₁. All studied loci were found polymorphic, nonetheless not all the possible allelic combinations were found in our study.

Table II. Allelic frequencies of esterase loci determined for populations and clones of Schizaphis graminum

First analysis of genetic distance showed that populations could be joined into three groups by means of similitude index values (Table III).

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).

First group was conformed by populations: Salta, Tucumán, Santiago del Estero, Frías, La Cumbre, Rafaela, and Córdoba Norte (that were collected from 26º 50´ S to 31º 40´ S; Fig. 2A). Second group joined the following populations: Paraná, Córdoba Sur, Villa María, Baradero, Bavio, Malargüe and Temuco (from 31º 52´S to 35º 30´S; Fig. 2B). The third group was represented by Ayacucho, Balcarce, Bordenave, Gonzales Chaves, Osorno, Miramar, Tres Arroyos, La Dulce, Lonquimay, Cabildo, Bahía Blanca, Los Alerces, Teka, and Castro (from 37º 08´ S to 43º 28´S; Fig. 2C). In the first group, La Cumbre showed significant differences with the rest of the populations. Baradero belongs to the first group according to its geographical location, however it joined the third group. Similarly, Lonquimay joined the rest of the third group at high genetic distance. Temuco fit into the second group but is geographically located in the strip corresponding to the third group (38º 20´S).

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.

The second and third groups revealed 100% of the loci polymorphic while the first group has 90% of polymorphic loci (Table III). Consistently with the values of polymorphic loci the lowest Shannon’s index was found in the third group, followed by the first group and the second raised 0.90. In all the studied groups the observed heterozygosity was lower than the expected heterozygosity, this could be explained by the effect of the homozygous individuals that were the most frequent genotype and consequently, the observed heterozygosity values dropped due to the lack of heterozygous individuals.
None group had fixed alleles according to Fsr values (Table III); group one had a slight heterozygous excess, while groups two and three showed slight positive Fsr values, because there was a homozygous excess.
Comparing Frt values (Table III) the first and third groups showed the highest values, while the second group differed significantly from the total, showing the lowest values of Frt. Fst values (Table III) showed significant differences related to the total expected heterozygosity (higher than 0.25) of the total populations in all the studied populations. These results agreed with the calculated gene flow values. The second group had the highest value contrasting with the first group which had the lowest value, and simultaneously showed the highest Fst with very great differences with the total populations based on the expected heterozygosity under Hardy-Weinberg equilibrium.

DISCUSSION

The present study revealed the existence of variability for esterase enzyme in Schizaphis graminum in populations of Argentina and Chile, contrasting with previous results where no distinguishable differences were found (Steiner et al., 1985; Beregovoy & Shark, 1986; Abid et al., 1989) and in agreement with Giménez et al. (1991a) and Castro et al. (1996). Nine different loci were found, only one showed no variability. These loci were found to be reliably resolved and polymorphic in preliminary surveys of isozyme variation (Castro et al., 1987a).
Kephart (1990) provided helpful information for interpreting isozyme patterns. Infrequent multimeric association or low enzyme activity may generate faint bands. In plants with null alleles that lack enzymatic activity, homozygotes and heterozygotes become visually indistinguishable in monomorphic proteins, heterozygotes show unexpected two or four banded phenotypes in dimorphic and tetramorphic proteins. Furthermore, under certain experimental conditions, extra ghost bands that show primary bands are present. The proposal of the existence of a null allele in the heterozygous individual is based on the intensity of the bands in the gels (Kephart, 1990). In the current results it is not possible to confirm the enzymatic patterns from regions B, C, D, E, F and F1 because it is difficult to differentiate whether the individuals that showed only one band are homozygous for this gene or heterozygous carrying the null allele because no band intensities differences could be found. In region A the model is not adjusted to the presence of three monomorphic proteins, due to the lack of discontinuity between the bands, although this could be a dimorphic protein.
Kephart (1990) proposed that the genetic basis of observed patterns should be verified by isozyme analysis of progeny arrays from controlled crosses. In fact, crosses in our aphid populations are  extremely  difficult because the number of sexual individuals is not enough to successfully carry out them.
Previous studies have shown that greenbug populations collected from very contrasting ecological regions of Argentina showed significant differences in reproduction behavior, lifespan, host preference (Ramos et al., 1998; Clúa et al., 2004), even the genetic variation of aphid aggressiveness has been studied among biotypes by means of the coleoptile tests (Giménez et al., 1991a, b, 1992; Castro, 1994; Castro et al., 1991).
It is also important to mention that all the populations were collected from a very great variety of host species including cultivated and non-cultivated host species. No relationship was found between the host species where the insects were collected from and the current genetic analysis of sampled populations. Clones and populations have been maintained in insectary conditions reared on susceptible barley since their collection date, this fact could have conditioned the enzymatic patterns and probably as it was found by Anstead et al. (2002) a relationship between recent captured population of Schizaphis graminum and its hosts might be found.
In the present study, populations and clones were associated in three groups latitudinally structured. Similarly, Li (2011) found in Oxya hyla intricata Stál (Orthoptera: Catantopidae) three monophyletic clades latitudinally structured corresponding to three geographical regions separated by high mountains. In the current survey, it would be possible that day-light conditions (photoperiod) and temperature regimes could have been conditioning the genetic similitude of the studied populations. There were four exceptions, La Cumbre, Baradero, Lonquimay and Temuco populations. La Cumbre could be considered as a different group and a correlation between the absence of eggs (Clúa et al., 2004) and the isolation from the rest of the studied populations could be considered. Lonquimay was collected over 2800 msl with a high rainfall and very cold winters with a wide range in day-night temperature, although no discontinuity of  host  species  was  found  between  the valley and the agriculture parcels situated high in the Andes. This population was characterised as sexually reproductive by Clúa et al. (2004). Nonetheless, there were parthenogenetic individuals together with others that induced sexual individuals. Temuco population genetically joined with the second group (from 31º 52´S to 35º 30´S), but geographically it is located into the third group (from 37º 08´ S to 43º 28´S). Temuco is located into the typical cereal winter production region of Chile (central valley) with very dry summers and a rainfall below 700 mm. Populations from Baradero and Parana were highly similar to each other due to their geographical location, although they joined different groups. It was not possible to collect any greenbug sample in those locations situated in-between those places, probably there would have been a common origin for these aphids or a high genetic flux.
Some biotypes can be characterized by diverse methods. Shufran et al. (2000), studying the biotypes of S. graminum by a molecular phylogenetic analysis based on the cytochrome oxidase I mitochondrial gene, combined with earlier studies of mDNA (Power et al., 1989), rDNA (Black, 1993) and RAPD (Black et al., 1992), concluded that the biotypes of S. graminum are probably host-adapted races. The C biotype was the predominant agricultural type infesting sorghum and wheat found in our populations (Noriega et al., 2000; Clúa et al., 2004). By analyzing the relationship between the isozymes profiles and greenbug biotypes distinctive patterns were found, agreeing with Abid (1989), Ramos et al. (1998) and Noriega (2000). On the contrary, Beregovoy and Starks (1986) established that few isozyme differences exist between biotypes B, C, or E. In our study, biotype C that gathered Balcarce population, Bavio 6, La Dulce 9, Bahia Blanca 1 and 2, Bordenave 6 and 9 (Noriega et al., 2000; Clúa et al., 2004), showed different electrophenotypes represented in Figure 1 (i.e. genotypes 2, 1, 1, 3, 2, 5, 5 for the C esterase, respectively).
Current results show there is genetic variability   on   the   greenbug   populations collected in Argentina and Chile. It was also possible to identify latitudinal stratification of greenbug populations. It is worth to note that α-carboxyl-esterase was present, also in clones and populations collected in non agricultural zones, implicating that the insecticide resistance in our local populations is well spread throughout the Argentinean and Chilean territories. Finally, there was no association between the biotype and the electrophoretic esterase patterns.

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