Molecular identification for Helicoverpa spp. in Bt and non-Bt crops in central Argentina

‒ The objective of this work was to identify, by molecular analysis, the species of the genus Helicoverpa in corn, soybean, and lucerne Bt and non‑Bt crops, in central Argentina. A total of 1,343 carterpillars were analyzed by PCR‑RFLP. Helicoverpa zea was the only species detected on non‑Bt and on most Bt corn hybrids. The species H. gelotopoeon predominated on non‑Bt soybean and on lucerne, and H. zea larvae occurred in low proportion. Bt soybean did not show Helicoverpa spp. infestations. The exotic H. armigera was not found, though adult moths of this species were light‑trapped in a complementary sampling.

Argentina ranks among the world's top exporters of soybean and corn, and it is also one of the largest lucerne producers. These crops are all affected by species of Helicoverpa (Lepidoptera: Noctuidae: Heliothinae). Two native bollworms, the polyphagous H. zea and H. gelotopoeon, are particularly frequent in damaged crops in the region. They have been traditionally controlled using chemical insecticides and, when available, transgenic cultivars expressing Bacillus thuringiensis (Bt) toxins. In recent years, the invasive "Old World bollworm", H. armigera, has entered the South American continent. It was first reported in Brazil in 2013, but its introduction in that country is believed to have occurred in 2008 or earlier (Sosa-Gómez et al., 2016). In Argentina, the presence of H. armigera has been recorded since 2013 (Murúa et al., 2016). The correct identification of Helicoverpa spp. is problematic because of their similar morphology and overlapping host range. Adult moths can be distinguished based on their genitalia; however, larvae are practically identical. Therefore, a PCR-RFLP method has been developed to identify the main Helicoverpa spp. in the country (Arneodo et al., 2015).
Routine monitoring and management of Helicoverpa caterpillars in Argentina usually do not imply species discrimination, which is costly and time-consuming. Thus, the identity of Helicoverpa spp. (including the exotic H. armigera) feeding on the different crops remains poorly documented.
The objective of this work was to identify, by molecular analysis, the species of the genus Helicoverpa in corn, soybean, and lucerne Bt and no Bt crops, in central Argentina.
The research was carried out in the agricultural core region of central temperate Argentina, where most (˃ 70%) of the country's soybean, corn, and lucerne are produced. Sampling areas included agricultural and mixed production systems (agriculture/livestock or agriculture/dairy farms) in five counties: Rafaela, Manfredi, Marcos Juárez, Laboulaye, and Pergamino ( Figure 1). Plots of different Bt and non-Bt corn hybrids, Bt and non-Bt soybean, and non-Bt lucerne were sampled in each county, during February 2016 (Table 1), when Helicoverpa larval infestations became noticeable. Corn and lucerne plots were visited at the flowering stage; soybean plots were visited at the vegetative and flowering stages. In each plot, two transects were randomly sampled. If available, 50 Helicoverpa spp. larvae were collected and conserved at -20°C until processing. A total of 1,450 larvae were sampled in this survey. DNA was individually extracted from the last abdominal segment of worms using CTAB. Partial sequences of cytochrome oxydase I (COI) gene were amplified by PCR with primers H3Fw (5'-CGAGCAGAATTAGGTAAYCC-3') and H3Rv (5'-GCTGATGTRAAATAAGCTCGAG-3').  Amplicons were digested with Hinf I endonuclease, the resulting fragments were separated by agarose gel electrophoresis (1%), and species were identified according to their differential RFLP patterns (Arneodo et al., 2015). Adults of H. zea, H. gelotopoeon and H. armigera, identified by microscopic examination of the genitalia, were included as controls in every batch of tests. A log-linear maximum likelihood analysis of variance was performed with a model of the main effects for the variables location, crop and species. To confirm transgenic events, pieces of leaves of the different Bt corn hybrids (15 per plot) were analyzed at the Genetically Modified Organisms Detection Laboratory, Biotechnology Institute, CICVyA-National Institute of Agricultural and Livestock Technology, Argentina, ISO 17025. In pest-infested plots, GMO detection analyses were performed on the same leaves from which the larvae had been collected. The methods implemented were based on Sybr Green qPCR. In addition to the main survey (that is, Helicoverpa spp. larvae directly collected from the crops), a smaller sampling of adult moths was undertaken in the same counties. Fifty Helicoverpa spp. specimens per location (total=250) were captured using light-traps and identified by PCR-RFLP (Table 1). All soybean, corn and lucerne crops surveyed in central Argentina, in the 2016 summer, showed Helicoverpa spp. infestations, except for Bt soybean ('Intacta RR2'), 'Viptera 3' Bt corn, and a non-Bt soybean plot in Pergamino (Table 1). COI fragments were amplified from most Helicoverpa specimens collected. Amplification failures were likely due to poor DNA quality. In total, 1343 Helicoverpa spp. larvae (out of the 1,450 collected ones) were successfully analyzed by the PCR-RFLP method for species discrimination (Figure 1).
A strong association between the pest and the host plant was observed, regardless of the sampling site. The variables insect species and crop had a significant effect on population distribution (Pr> ChiSq <0.00001). The species H. zea was the only one found on corn [non-Bt and transgenic 'MG' (Cry1Ab), 'VT Triple Pro' (Cry1A.105 + Cry2Ab + Cry3Bb1), and 'Power Core' (Cry1A.105 + Cry2Ab + Cry1Fa2) hybrids. In 'Viptera 3' (Vip3Aa20) corn hybrids, lepidopteran larvae were not detected. The transgenic condition of the Bt corn hybrids was confirmed in all cases. The analyzed leaf samples contained the expected Bt gene(s) related to the insecticidal characteristics of each corn hybrid. Resistance of H. zea to Cry 1Ab and Cry1A.105 + Cry2Ab toxins have been previously reported throughout the American continent (Dively et al., 2016). Recently, a reduced susceptibility to Vip3Aa toxin was reported in the USA (Yang et al., 2020). Concerning Fabaceae, H. gelotopoeon was largely the most abundant species of the genus in both  (Pearson et al., 1989;Swenson et al., 2013). Neither Helicoverpa spp. nor other lepidopteran pests were found on transgenic Bt soybean. None of the 1343 sampled larvae from the different crops examined by PCR-RFLP corresponded to the introduced species H. armigera. Nevertheless, H. armigera represented a small share of the additional adult moth sampling in some locations (up to 4%), where H. zea constituted the vast majority of the specimens, followed by H. gelotopoeon (Table 1, Figure 1). These data suggest that H. armigera occurred in low abundance in central Argentina, during the evaluation period. In this line, a thorough study on the abundance of H. armigera and H. zea moths, caught in light traps in Distrito Federal (central Brazil), showed that H. armigera populations declined in the growing seasons following the big outbreak of 2012/2013. The authors found a significant negative correlation between the average maximum temperature and relative humidity (boosted by El Niño events) and the number of moths collected, with the highest Oceanic Niño Index (ONI) values registered in 2015/2016 (Specht et al., 2021). In addtion to the small population size, other factors could also explain the absence of H. armigera larvae in the samples. For instance, other plants might be serving as hosts for this insect. A survey conducted during 2015/2016 in corn, in the state of Rio Grande do Sul (southern Brazil) showed similar results. Even though H. armigera has been reported since 2011, in that region, no Helicoverpa larvae other than H. zea were recovered from infested corn ears. This was possibly due to the advantage of H. zea over H. armigera in intraguild interactions in corn, and to reduced H. armigera populations because of the absence of cotton crops, which is a preferred host (Bentivenha et al., 2016). In northern Argentina, infestations with H. armigera larvae have already been recorded on chickpea, soybean, sunflower, and spiny plumeless thistle (Arneodo et al., 2015;Murúa et al., 2016). Given the damage potential of H. armigera, and because meteorological and agroecological conditions vary from year to year, the presence of this pest across the country should be periodically monitored. The economically important genus Helicoverpa contains species with worldwide (for instance, H. armigera), continental (H. zea), and regional (H. gelotopoeon) distribution. The presence of Helicoverpa bollworms in central Argentina is well known to farmers, but this is the first large-scale molecular survey to clearly identify, at the species level, larvae feeding on the main Bt and non-Bt crops in the region. The information provided here will be useful for analyzing possible changing scenarios for the Helicoverpa complex in the core agricultural lands of Argentina.