Host plant association and genetic differentiation of corn and rice strains of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) in Colombia

ECOLOGY, BEHAVIOR AND BIONOMICS

Host plant association and genetic differentiation of corn and rice strains of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) in Colombia

Clara I Saldamando^I; Ana M Vélez-Arango^I,II

^IFacultad de Ciencias, Depto de Biociencias, Univ Nacional de Colombia, UNALMED, Calle 59A No 63-20, Medellín, Colombia; [e-mail]

^IILab de Biotecnología Vegetal, Corporación para Investigaciones Biológicas CIB, Cra 72 No 78B-141, Medellín, Colombia

ABSTRACT

Spodoptera frugiperda (Smith) is a polifagous insect of major economic impact in the western hemisphere and exhibits two strains (i.e., corn and rice) that are morphologically identical but differ in ecology, genetics and physiology. In this work we identified these strains and their respective hybrids by using a PCR-RFLP of the COI gene and PCR of the tandem region FR. Moreover, we performed a population structure analysis by using 253 larvae from Tolima, a region where S. frugiperda is a pest on corn, rice, sorghum and cotton. Corn strain was found on 42% in corn, 34% in cotton, 19% in sorghum and 0.04 % in rice and rice strain on 35% in corn, 0.06% in cotton, 0.06% in sorghum and 53% in rice, demonstrating that corn strain specificity is superior to rice strain. Hybrids between these strains were more abundant in corn. The distributions on their host plants reflect a population genetic differentiation in S. frugiperda with values of PhiPT (COI) = 0.31, P < 0.0001, PhiPT (FR) = 0.17, P < 0.0001 for all crops and PhiPT (COI) = 0.42, P < 0.01, PhiPT (FR) = 0.13, P < 0.01 for the sixteen sampled farms. The dendrograms showed two clusters representing both strains. The results obtained in this study suggest that the management of this insect must differ on each host plant, given the specialization that both strains present, particularly in corn and rice.

Key words: Strain, population genetics, pest

Among insects, speciation mainly involves the evolution of distinct populations due to local adaptation to certain host plants (Feder 1998). Examples for speciation through host plant adaptation are the apple maggot Ragolethis pomonella (Walsh) on hawthorn and domestic apple (Bush 1969, Bush & Smith 1998, Feder 1998), the larch budmoth Zeiraphera diniana (Guénée) on European larch and cembran pine (Emelianov et al 2001), or the european corn borer Ostrinia nubilalis (Hübner) on corn and mugwort (Martel et al 2003).

The fall armyworm (FAW) Spodoptera frugiperda (Smith) is a key pest of several crops such as corn, cotton, sorghum and rice (Nagoshi & Meagher 2003b, 2004, Busato et al 2004, Prowell et al 2004, Vélez-Arango et al 2008). The fall armyworm equally exhibits two strains associated to its main host plants: corn and rice (Prowell 1986, Prowell et al 2004). However, the corn strain has also been collected from cotton and sorghum and the rice strain has been found in pasture grasses (Nagoshi & Meagher 2003a). Speciation in S. frugiperda merits special attention for several reasons: 1) S. frugiperda strains exhibit varying resistance to chemical and biological control agents (Adamczyk et al 1997); 2) FAW strains are particularly interesting to explore (sympatric) speciation process through host plant adaptation (Dres & Mallet 2002); 3) both strains hybridize under laboratory and field conditions (Nagoshi & Meagher 2003b, Prowell et al 2004); 4) FAW presents evidence for prezygotic isolation (Prowell & Martin 1987, Prowell et al 1992, Lu et al 1994); and 5) reproductive isolation may be associated to X linked traits or maternally inherited traits (Prowell 1998).

The presence of S. frugiperda strains has been reported in various countries in the western hemisphere (Busato et al 2004, Prowell et al 2004), including Colombia (Vélez-Arango et al 2008). Strains are morphologically identical, but show vast genetic differences in several markers, such as allozymes, esterases, AFLP's, and ND4 gene (Prowell 1986, McMichael & Prowell 1999, Prowell et al 2004). A PCR-RFLP method based on the restriction enzyme MspI identifies S. frugiperda strains by amplifying products of 569 bp of the COI gene and producing two cleavage sites of 497 bp and 72 bp on the corn strain only (Levy et al 2002, Nagoshi & Meagher 2003a,b). Another method used to differentiate the strains is the FR (For Rice) tandem repeated fragment, that produces amplifications (mass smears) above 500 bp in the rice strain and from 0 to 3 faint bands on corn strain (Lu et al 1994, Nagoshi & Meagher 2003a).

Hybridization between both FAW strains has been evidenced in different US states (Prowell 1986, Nagoshi & Meagher 2003a), French Guyana, Ecuador, Guadalupe, and certain Caribbean islands (Prowell et al 2004). These results indicate that interbreeding readily occurs in nature (Prowell et al 2004).

To prevent hybridization, certain prezygotic isolation barriers have been evidenced between the two strains, such as temporal isolation (Prowell et al 1992), partial assortative mating strategies (Prowell & Martin 1987, Lu et al 1994), ecological isolation (Prowell 1986 1998, Prowell et al 2004), and differences in pheromone composition, suggesting chemical isolation (Groot et al 2008). However, opposite results related to assortative mating have been obtained under laboratory conditions (see Withford et al 1988, Quinsenberry 1991, Nagoshi & Meagher 2003a), possibly due to geographic origin of the strains or age of S. frugiperda populations used to test fitness components of the species (Prowell 1988). Genetic analysis of FAW populations has been performed in only few countries of South America (Busato et al 2004, Martinelli et al 2007, 2007, Clark et al 2007). However, none of these studies has covered Colombia, and only a limited number of studies in the US have investigated host plant associations (Nagoshi & Meagher 2004, Prowell et al 2004).

In Colombia, S. frugiperda is a key pest in corn and a secondary pest on cotton, sorghum and rice, particularly at the department of Tolima, a region where most of the studies are focused on its control (Alvarez y Sanchez 1983) by using transgenic cotton crops, with no effects on the mortality of this species (Zenner de Polania unpublished). To prevent life cycle synchrony between FAW and other key pests, all four crops included in this study are rotated. Although rotation does not reduce FAW densities in the field, knowledge of the strain identification of this insect is critical for the integrated pest management of FAW. Moreover, an analysis of population differentiation between these two strains is also important to determine whether they represent genetically differentiated units with restricted gene flow. For these reasons, the purposes of this work were: a) to identify S. frugiperda strains and b) to analyse the population differentiation in this department of Colombia by using a PCR-RFLP of the mitochondrial cytochrome oxidase subunit I (COI) gene and a PCR for the tandem repeated unit FR (FAW rice strain) already standardised by Nagoshi & Meagher (2003a) and Lu et al (1994).

Material and Methods

Insect collection. In northern Tolima, FAW larvae were collected from corn, cotton, and rice fields that were positioned at close distance (< 5 km apart). In south and central Tolima, larval collections were done in closely positioned cotton, sorghum and corn fields (Table 1). Collections were made during late 2006 and early 2007. Upon collection, larvae were stored in 2.5 ml plastic tubes with 70% ethanol, labelled according to the collecting site, and sent to the laboratory Biotecnología Vegetal UNALMED-CIB (Corporación para Investigaciones Biológicas), where they were subsequently kept at -70ºC until processing.

Insect genotyping. Genotyping was performed on 253 individuals by using a PCR-RFLP of the COI gene at the mitochondrial DNA and a PCR for the nuclear region FR (Nagoshi & Meagher 2003a). DNA extraction was performed following modified protocols of Sambrook -& Russell (2001). Details on PCR-RFLP's on COI gene and PCR of the tandem FR region are described elsewhere (Vélez-Arango et al 2008).

Data analysis. A binary data matrix was created for each marker, representing presence (1) or absence (0) of a certain band. Both MspI and FR markers were considered neutral and dominant, since the first marker is haploid and is part of the mitochondrial cytochrome oxidase subunit I (COI) gene and is maternally inherited (Levy et al 2002), while the second marker produces either a smear pattern higher than 500 bp or 0-3 faint bands with molecular weight below 500 bp. This marker is thought to be linked to the Y and X chromosomes in S. frugiperda (Nagoshi & Meahger 2003a) and hence considered diploid. Since both markers are dominant, Hardy Weinberg equilibrium was not assumed (Excoffier et al 1992, Hedrick 2004), and an AMOVA (Analysis of Molecular Variance) was used to determine genetic differentiation of S. frugiperda strains. Population structure analyses were conducted separately between crops and between individual farms for each molecular marker using the software GenAlEx 6 (Peakall & Smouse 2006), following methods of Excoffier et al (1992), and Michalakis & Excoffier (1996).

On the other hand, Popgene 1.31 (Yeh et al 1997) was used to calculate Nei's genetic distances and produce two dendrograms with UPGMA algorithm for each marker (Sneath & Sokal 1973). Dendrograms were constructed on the four crops and on the sixteen farms sampled with Mega 4.0 (Tamura et al 2007). Nei genetic distance was chosen on this study because it does not assume Hardy Weinberg equilibrium (Hedrick 2004). In addition, since S. frugiperda strain data are categorical, three contingency tables (Sokal & Rohlf 1995) were performed in Genstat 5.0 (2003) to asses host plant association of S. frugiperda strains to their respective host plants. Also, a logistic regression was used to test three effects on S. frugiperda strains distributions in Tolima: a) an effect of the region were collections were made, b) an effect of the host plant where larvae were collected and c) an effect of surrounding crops of the sampling location. Logistic regression was carried out in Minitab 15 (2007).

Results and Discussion

From all 253 collected FAW larvae, a total of 143 individuals were genotyped as corn strain, and 49 individuals were genotyped as rice strain (Table 1). Electrophoretic patterns for each marker of corn and rice strains were similar to those defined by Nagoshi & Meagher (2003a).

Two types of hybrids were found: a) 37 individuals that presented both digestions of 497 bp and 72 bp with the enzyme MspI and smear amplifications higher than 500 bp with FR primers (subsequently termed hybrids + /+) and b) 24 individuals that did not present neither a digestion of 497 bp and 72 bp with the enzyme MspI or smear amplification products >500 pb with FR primers (named hybrids -/-). The hybrids could be the product of bidirectional crosses between the strains, producing F1 generations, or backcrosses of F1 individuals to parentals, suggesting that interstrain mating occurs easily in Tolima. However, opposite observations have been made in FAW populations of Florida where Nagoshi & Meagher (2003b, 2004) found that hybrids were the product of unidirectional interstrain matings between rice females and corn males. Similarly, Lu et al (1994) found restricted interstrain mating in nature on FAW populations in Georgia, USA arguing that bi-directional crosses are rare or absent in nature. Contrary to above findings and in correspondence with our work, Prowell et al (2004) found evidence of bi-directional crosses in nature in a multitude of FAW populations, with 54% possible offspring of rice strain females mated with corn strain and 46% reciprocal cross. In our study, 41% of hybrids were collected from corn, 26% on cotton, 9% on sorghum and 23% on rice, supporting Prowell et al (2004) who reported that the majority (i.e., 62%) of presumptive hybrids in the corn habitat.

AMOVA results show genetically differentiated FAW populations between the four crops (corn, cotton, sorghum and rice) with significant PhiPT values for both markers: PhiPT = 0.309 (df = 3, 249; P < 0.0001) for the COI region and PhiPT = 0,168 (df = 3, 249; P < 0.0001) for the FR marker. Indeed, pair wise comparisons between crops showed restricted gene flow between corn and rice, cotton and rice and sorghum and rice (Table 2). Nevertheless, this gene flow is existent since hybrids between both strains were found in Tolima, perhaps due to the coexistence of corn and rice strains in corn, one of the crops where hybrids were more abundantly collected. Moreover, AMOVA tests support the result mentioned above when FAW populations amongst sixteen farms were considered: PhiPT = 0.414 (df = 15, 237; P < 0.01) for the COI region and PhiPT = 0,129 (df = 15, 237; P < 0.01) for the FR marker.

The population differentiation was mainly produced between the farms Hacienda Pajonales (rice farm, n = 33), Algodonera Andina (cotton farm, n = 28) and CI Nataima (corn farm, n = 14) with the other thirteen farms (Table 3, 4). This can be explained due to FAW population composition, with the rice strain and hybrids predominant on the first farm, and corn strain and hybrids principally found on the second and third farm. Furthermore, pairwise comparisons amongst farms for both markers were mostly not significant, particularly between corn, sorghum and cotton farms, since corn strain was predominant on them. All these results are in line with findings of Busato et al (2004), Clark et al (2007) and Martinelli et al (2007), because in all these separated studies there were differentiated populations of S. frugiperda in Argentina, Brasil, and US. However, only in the present work this analysis was focused on strain identification previously to a population structure analysis. It is important to mention that the molecular markers used here are more useful for strain identification than for population analysis, since they do not provide much information about variation within the strain. However they were adequate to provide the required information for the proposed analysis of the strain and hybrid composition within farms and crops.

Contingency tables indicated a differential distribution of both FAW strains on the four host plants (Table 5), with corn strain mainly present in corn, sorghum and cotton, and rice strain predominantly in rice and in lower proportions on the other crops (Fig 1). Similar results were reported by Prowell et al (2004). We also obtained clustering for dendrograms obtained from each marker based on Nei genetic distances for the four studied crops, with a first cluster composed of S. frugiperda larvae from corn, sorghum and cotton and a second cluster from rice crops (Fig 2a). Similar findings were made by Prowell (1988). In addition, we recorded differences in host distribution, with (+/+) hybrids being more abundant on corn and cotton, while (-/-) hybrids on rice and cotton. Clustering of hybrids on dendrograms was not possible since Nei genetic distances were separately calculated for both markers.

Nei genetic distances and dendrograms obtained for each marker and all sixteen farms analyzed (Fig 2b) failed to separate corn, sorghum and cotton farms from rice farms, the explanation for this result being that more molecular markers are needed for the construction of dendrograms between these farms or simply that the association between corn and rice strains to corn and rice as hosts is not so strong, so both strains coexist in both crops, but their frequency is different, given that corn strain is more abundant in corn and rice strain in rice.

Finally, logistic regression (ML = -273,775; G = 22,047; df = 3, P < 0.0001) indicates that S. frugiperda strain distribution was not affected by sampling region or by surrounding crops, but by the host plant (i.e., crop) on which larvae were collected.

In general, our study shows that host plant association is the main cause of the genetic differentiation of FAW populations in Tolima, Colombia. This finding is comparable to the association of O. nubilalis with corn and mugwort in France (Martel et al 2003, Malausa et al 2007). Both lepidopterans may be in a similar stage of speciation as host plant associated populations' origin relative to other insects, such as R. pomonella (Feder 1998). Identification of S. frugiperda strains is crucial for regional FAW integrated pest management, as the department of Tolima is considered a major producer of all main FAW hosts plants. In addition, since corn strain is apparently more resistant than rice strain to several insecticides and to the endotoxin Cry1Ac on transgenic cotton crops (Adamczyk et al 1997), corn, cotton and sorghum crops may have to be managed in a different way that rice crops are.

Finally, current crop rotation may not be the most effective management strategy for FAW in the region, as corn strains will likely shift from corn or sorghum to cotton, while the rice strain will shift from rice to pasture grasses from the first to the second semester of the year. We suggest corn, sorghum and cotton crops to be sawed in allopatric sites if they are produced at the same semester of the year in order to prevent migration of corn strain amongst them, and production of rice crops at separated distances from pasture grasses, to reduce movement of rice strain between them. In rice, S. frugiperda is not a major pest in Tolima as the other three crops, since it is produced in wetlands where FAW larvae are incapable to survive.

Acknowledgements

We would like to thank Krys Kwyckhuys for the revision of an early version of the manuscript and to Elizabeth Aguilera and Jhon Alexander Agudelo for assistance in collecting S. frugiperda larvae; Universidad Nacional de Colombia (Medellín) and the Corporación de Investigaciones Biológicas (UNALMED-CIB), Unidad de Biotecnología Vegetal for production of this work. We are grateful to CORTOLIMA for the permitting collection and genetic access in 2007. Research was funded by Universidad Nacional de Colombia grants number 20101006109 and 20101007063.

Received 10/VI/09.

Accepted 30/IV/10.

Edited by Fernando L Cônsoli - ESALQ/USP

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Publication Dates

• Publication in this collection
  17 Jan 2011
• Date of issue
  Dec 2010

History

• Accepted
  30 Apr 2010
• Received
  10 June 2009