The Heliothinae (Lepidoptera: Noctuidae) complex encompasses 381 described species, distributed in 28 genera ([@iew052-B39]). The most economically important species included in *Helicoverpa* Hardwick and *Chloridea* Duncan and (Westwood) are polyphagous. The females lay eggs, and the larval stage can survive and feed on a very wide range of host plant species. Many of these hosts are crops, including many field crops: cotton (*Gossypium hirsutum* L.; Malvales: Malvaceae), sorghum (*Sorghum* spp. Moench; Poales: Poaceae), sunflower (*Helianthus annuus* L.; Asterales: Asteraceae), chickpea (*Cicer arietinum* L.; Fabales: Fabaceae), alfalfa (*Medicago sativa* L.; Fabales: Fabaceae), lupins (*Lupinus* spp. L.; Fabales: Fabaceae), soybean (*Glycine max* L.; Fabales: Fabaceae), tobacco (*Nicotiana tabacum* L.; Solanales: Solanaceae), corn (*Zea mays* L.; Poales: Poaceae), wheat (*Triticum* spp. L.; Poales: Poaceae), horticultural crops such as tomatoes (*Solanum lycopersicum* L.; Solanales: Solanaceae), lettuce (*Lactuca sativa* L.; Asterales: Asteraceae), capsicum (*Capsicum annuum* L.; Solanales: Solanaceae), various bean crops, and flowers such as chrysanthemum (*Chrysanthemum* spp. L.; Asterales: Asteraceae), gladiolus (*Gladiolus* spp. L.; Asparagales: Iridaceae), and rose (*Rosa* spp. L.; Rosales: Rosaceae) ([@iew052-B10]).

The native species of *Helicoverpa* in South America are divided into two groups: (a) gelotopoeon, including *Helicoverpa* *gelotopoeon* (Dyar), *Helicoverpa* *titicacae* Hardwick, *Helicoverpa* *bracteae* Hardwick, and *Helicoverpa* *atacamae* Hardwick; and (b) zea, including only *H. zea* (Boddie). Species of *Chloridea* are represented by *Chloridea* *virescens* (Fabricius), *Chloridea* *tergemina* (Felder and Rogenhofer), and *Chloridea* *subflexa* (Guenée) ([@iew052-B28], [@iew052-B36], [@iew052-B39]).

In the gelotopoeon group, species can only be separated by morphological characters of the adults, since the biology and morphology of the immature stages remain unknown ([@iew052-B35]). Recently, *H. armigera* (Hübner) has been detected in Brazil ([@iew052-B11], [@iew052-B47], [@iew052-B49]), Paraguay ([@iew052-B43]), Argentina ([@iew052-B29]), Bolivia, Uruguay ([@iew052-B20]), Puerto Rico ([@iew052-B33]), and the United States ([@iew052-B34]). Thus, the presence of *H. armigera* in northwestern Argentina (NOA) has led to the inclusion of this species in the Heliothinae complex, together with *H. gelotopoeon*, *H. zea*, and *C. virescens*.

*Helicoverpa gelotopoeon* is a polyphagous pest and has been reported in cotton, alfalfa, sunflower, soybean, chickpea, and corn, to name a few crops it attacks. Larvae cause damage in the vegetative and reproductive plant growth stages. This species occurs in Argentina, Chile, southern Brazil, Paraguay, and Uruguay ([@iew052-B36], [@iew052-B35]). In Tucumán and other provinces of Argentina, *H. gelotopoeon* causes severe damage to soybean crops and can be difficult to control with insecticides ([@iew052-B35], [@iew052-B42]. Consequently, it causes a significant economic impact, because Argentina is the third major soybean producer in the world, covering an area of 20.1 million hectares ([@iew052-B9]). This insect species also affects cotton and chickpea crops, causing more severe problems when they are grown in succession cropping systems ([@iew052-B36], [@iew052-B15], [@iew052-B35], [@iew052-B42].

The differentiation of the Heliothinae complex in the egg, larva, and pupa stages is very difficult, but adults can be distinguished by the pattern of wing veins and male genitalia using traditional taxonomic methods ([@iew052-B38], [@iew052-B35]). A further complication is distinguishing between *H. armigera* and the native *H. zea* (North and South America), since the latter is estimated to have diverged from the former only approximately 1.5--2 million years ago, and the same pheromone compounds, although in different concentrations, are found in both species ([@iew052-B38], [@iew052-B51], [@iew052-B7]). Thus, males of both species are attracted to sex pheromone lures released by females of both species in the field. Yet another complication is the fact that *H. armigera* and *H. zea* have been shown to hybridize in the laboratory and could well be doing the same in the field ([@iew052-B18], [@iew052-B21], [@iew052-B22]). It is unclear whether *H. armigera* can hybridize with other endemic Heliothinae species such as *H. gelotopoeon.* Therefore, genitalic dissection or molecular techniques are necessary to accurately identify adult males collected in pheromone traps ([@iew052-B38], [@iew052-B6], [@iew052-B47], [@iew052-B49], [@iew052-B23], [@iew052-B3], [@iew052-B20]). [@iew052-B6] reported the use of two partial mitochondrial DNA genes \[cytochrome oxidase subunit I (COI), cytochrome b (Cyt b)\] as markers to differentiate *H. armigera*, *H. punctigera*, *H. assulta*, and *H. zea*. [@iew052-B3] designed a rapid and simple molecular tool to distinguish *H. armigera* from *H. zea* and *H. gelotopoeon*. The method was validated using *Helicoverpa* specimens collected across Argentina, and their identity was further corroborated by COI sequence and phylogenetic analysis.

*Helicoverpa armigera* seems to be well adapted to the climate of agricultural regions of South America and, according to [@iew052-B20] it is spreading rapidly throughout Argentina, Bolivia, Brazil, Paraguay, and Uruguay, and most recently to Puerto Rico. However, no studies have been performed to investigate if *H. armigera* detected in these non-Brazilian locations were due to natural spread of Brazilian populations, or if they represented separate incursion events.

It is important to mention that *H. armigera* has developed resistance to insecticides and Cry proteins ([@iew052-B16], [@iew052-B2], [@iew052-B24], [@iew052-B27], [@iew052-B17], [@iew052-B25], [@iew052-B8], [@iew052-B48]). Therefore, it is necessary to know how the Heliothinae complex is distributed in legumes of economic importance in NOA, considering the difficulty of identifying these species.

The objective of this study was to determine population fluctuations of the Heliothinae complex in soybean and chickpea crops using male moths collected in pheromone traps. The distribution of *H. gelotopoeon* in Tucumán province and an update of the geographical distribution of *H. armigera* in Argentina are also provided herein.

Materials and Methods
=====================

### Identification of the Heliothinae Complex

Larvae were collected in chickpea and soybean fields in Tucumán and Santiago del Estero provinces. In chickpea, collections took place on September 12, 2013 in Viclos (27°10′38″S, 64°52′42″W). In soybean, larvae were collected on January 31 and February 26, 2014 in La Cocha (27°45′55″S, 65°30′6″W), on February 7, 2014 in Jiménez (26°52′51″S, 64°42′3″W), and on February 15, 2015 in San Agustín (26°50′21″S, 64°51′32″W). Samples were collected during vegetative and reproductive stages in both crops. One hectare was monitored in each locality. In each hectare, 10--15 plants of soybean or chickpea were sampled at each of ten locations. Larvae were collected using a sheet method as described by [@iew052-B12].

The collected larvae were taken to the laboratory and reared on artificial diet, which included dry bean flour, wheat germ (Grandiet, Buenos Aires, Argentina), brewer's yeast (Calsa, Tucumán, Argentina), vitamin C, sorbic acid (Anedra, Buenos Aires, Argentina), vitamin supplement, amino acids (Ruminal, Buenos Aires, Argentina), and methylparaben (Todo Droga, Córdoba, Argentina) ([@iew052-B31]), in growth chambers under controlled conditions \[27 ± 2°C, 70--75% relative humidity, 14:10 (L:D) h cycles\] until adult emergence. This diet has been used to rear other polyphagous lepidopteran species such as *Spodoptera frugiperda* (J. E. Smith), *Diatraea saccharalis* (Fabricius), *Rachiplusia nu* (Guenée), and *Chrysodeixis includens* (Walker) (Lepidoptera: Noctuidae) ([@iew052-B31], [@iew052-B30]; [@iew052-B40], [@iew052-B5]). Only male moths were examined using genitalic observation to confirm the species according to [@iew052-B50] and [@iew052-B38]. The females collected were not considered for this study, which might have affected the accuracy of data regarding species composition in the Heliothinae complex, mainly in species with unequal sex ratios. Some insects of each of these populations were deposited as voucher specimens in the collection of Sección Zoología Agrícola, at Estación Experimental Agroindustrial Obispo Colombres, Tucumán province, Argentina.

### Population Fluctuations

Male moths were sampled in chickpea and soybean crops from 2013 to 2015 in Tucumán province. Traps were placed in chickpea crops from August to October 2013 in Viclos, and from July to December 2014 in San Agustín. In soybean, traps were placed from January to June 2014 and from January to May 2015 in San Agustín. Yellow traps (Unitrap, ChemTica Internacional S.A., Heredia, Costa Rica) were used and installed according to manufacturer's recommendations. The lures were changed every 20 or 30 d and the insects were collected every 7 or 10 d. Traps were baited with *H. armigera* and *H. gelotopoeon* pheromones (ChemTica Internacional S.A., Heredia, Costa Rica). Growth stages of chickpea and soybean crops were determined based on the description by [@iew052-B45] and [@iew052-B13], respectively.

The data obtained were tested for normality using the Shapiro--Wilk test. The data regarding *H. gelotopoeon* and *H. armigera* adults obtained from each trap that did not show normal distribution or homogeneity of variance were subjected to a square root transformation \[√(*X* + 0.5)\]. The transformed data were analyzed using a *t*-test to detect differences between the adults of both species collected on both crops from different years. All the analyses were conducted using the software [@iew052-B19].

### *Geographical Distribution of H.* *armigera*

Pheromone and light traps were used to update the distribution of this noctuid pest in Tucumán province. As described previously, males were collected using pheromone traps in San Agustín (Cruz Alta). A light trap (A todo campo, Buenos Aires, Argentina) was installed in Las Talitas (26°47′S, 65°11′W) from September to November 2014 and only male moths were selected for further identification. Moreover, larvae of this species were caught directly from sunflower, and spiny plumeless thistle (*Carduus acanthoides* L.; Asterales: Asteraceae).

To report the geographical distribution of *H. armigera* in other regions of Argentina, the database of Sistema Nacional Argentino de Vigilancia y Monitoreo de Plagas (SINAVIMO) of the Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA) was utilized.

Results
=======

### Identification of Heliothinae complex

A total of 880 larvae were collected from 2013 to 2015 on chickpea and soybean crops and 416 male moths were obtained and identified. Species identification used adult males only, because using male genitalic characters are more reliable than using females ([@iew052-B37]). Female obtained were 394 and remainder of larvae died by unknown reasons. The species found were determined as *H. gelotopoeon*, *H. armigera*, *H. zea*, and *C. virescens.* Regardless of province, county, crop, and year, the predominant species was *H. gelotopoeon* ([Table 1](#iew052-T1){ref-type="table"}). Table 1.Larvae of the Heliothinae complex (*Helicoverpa* spp. and *Chloridea virescens*) collected in Viclos, La Cocha, San Agustín and Jiménez counties in chickpea and soybean crops used to identify the adults of each speciesProvinceCountyCropDateLarvae collected (no.)Males obtained and identified[^a^](#iew052-TF1){ref-type="table-fn"} (no.)*H. gelotopoeonH. armigeraH. zeaC. virescens*TucumánViclosChickpeaSeptember 12 20132509694101La CochaSoybeanJanuary 31 20142009489410February 26 20141005453100San AgustínSoybeanJanuary 15 20151809595000Santiago del EsteroJiménezSoybeanFebruary 15 20141507771231[^2]

### Population Fluctuations

Population fluctuations of *H. gelotopoeon* and *H. armigera* males in chickpea and soybean crops are shown in [Fig. 1](#iew052-F1){ref-type="fig"}. Considering the total number of moths obtained (*H. gelotopoeon*, *n* = 440; *H. armigera*, *n* = 53), *H. gelotopoeon* was the most abundant species in both crops, in all years sampled, and the difference in number of moths between the two crops was statistically significant (*t* = −6.15; df = 89; *P* \< 0.0001). In chickpea ([Fig. 1a and b](#iew052-F1){ref-type="fig"}), the first species that appeared was the native, *H. gelotopoeon*, and it was present, in general, during the entire crop cycle in 2013 and 2014. The largest collections of *H. gelotopoeon* and *H. armigera* were from October to November and from September to October in 2013, respectively, and adults were only collected in some weeks from September to November in 2014. Fig. 1.Population fluctuations of *H. gelotopoeon* and *H. armigera* adults, in chickpea and soybean crops, from 2013 to 2015, in Tucumán province, Argentina. Chickpea crop: (A) Viclos county; (B) San Agustín county. Soybean crop: (C, D) San Agustín county.

In soybeans ([Fig. 1c and d](#iew052-F1){ref-type="fig"}), the largest collection of *H. gelotopoeon* was recorded in February in both years and, in general, this pest was present during all months in 2015. Male *H. armigera* individuals were only recorded in 2014 from mid-February to April. In 2014, male *H. gelotopoeon* specimens were observed before *H. armigera*.

Geographical Distribution of *H.* *armigera*
============================================

Considering our collections, *H. armigera* was found in two counties (Las Talitas and San Agustín) of Tucumán province and one county (Jiménez) of Santiago del Estero province, and these were new records for both provinces. Larvae of this species were recorded on soybean, chickpea, sunflower, and spiny plumeless thistle. This is the first report of *H. armigera* in sunflower and spiny plumeless thistle in Argentina ([Table 2](#iew052-T2){ref-type="table"} and [Fig. 2](#iew052-F2){ref-type="fig"}). Fig. 2.Geographical distribution of *H. armigera* in different provinces of Argentina. Table 2.Geographical distribution of *H. armigera* in ArgentinaProvinceSampling siteElevation (masl)Stage collectedCollection/host plantMonth/yearBuenos Aires1 Pergamino[^a^](#iew052-TF2){ref-type="table-fn"}56AdultsPheromone trap/soybean cropsJuly 2014Chaco2 Nueve de Julio[^b^](#iew052-TF3){ref-type="table-fn"}50AdultsCotton and soybeanJuly 20143 Las Breñas[^b^](#iew052-TF3){ref-type="table-fn"}105AdultsPheromone trapOctober 2014 to January 2015Córdoba4 Río Segundo[^a^](#iew052-TF2){ref-type="table-fn"}335AdultsPheromone trapsJune 20145 Marcos Juárez[^a^](#iew052-TF2){ref-type="table-fn"}112AdultsLight trap near cereals and oleaginous cropsNovember 2014Entre Ríos6 Paraná[^a^](#iew052-TF2){ref-type="table-fn"}77AdultsPheromone traps near to soybean and corn cropsJuly 2014Santiago del Estero7 Jiménez[^a^](#iew052-TF2){ref-type="table-fn"}1,030LarvaeSoybeanJanuary 20148 Ojo de Agua[^a^](#iew052-TF2){ref-type="table-fn"}600AdultsPheromone traps on soybean cropsFebruary 20159 La Paloma[^a^](#iew052-TF2){ref-type="table-fn"}606AdultsPheromone traps on soybean cropsFebruary 201510 Quimili[^a^](#iew052-TF2){ref-type="table-fn"}137AdultsPheromone trapsJune 201511 El Puna[^a^](#iew052-TF2){ref-type="table-fn"}130AdultsPheromone trapsJune 201512 El Puyehue[^a^](#iew052-TF2){ref-type="table-fn"}191AdultsPheromone trapsJune 201513 Otumpa[^a^](#iew052-TF2){ref-type="table-fn"}155AdultsPheromone trapsJune 201514 Rapelli[^b^](#iew052-TF3){ref-type="table-fn"}395LarvaeChickpeaOctober 2014 to January 2015Santa Fé15 General Obligado[^a^](#iew052-TF2){ref-type="table-fn"}21AdultsCottonMay 2014Salta16 Cerrillos[^a^](#iew052-TF2){ref-type="table-fn"}1,217AdultsChickpeaNovember 2014Tucumán17 La Cocha397Adults[^c^](#iew052-TF4){ref-type="table-fn"}ChickpeaNovember 2013LarvaeSoybeanFebruary 201418 Viclos345Adults[^c^](#iew052-TF4){ref-type="table-fn"} and larvaeChickpeaSeptember 2013LarvaeSpiny plumeless thistleSeptember 201419 Las Talitas591AdultsLight trap near to sugarcane crops and lemon tree fieldsOctober 201420 San Agustín1,190AdultsPheromone traps on soybean cropsJanuary--June 2014LarvaeSunflowerJanuary 2015[^3][^4][^5]

Taking into account SINAVIMO database, the reports of [@iew052-B3], and our own collections, *H. armigera* was recorded in eight provinces and 20 counties in Argentina ([Table 2](#iew052-T2){ref-type="table"} and [Fig. 2](#iew052-F2){ref-type="fig"}).

Discussion
==========

In Tucumán province, Argentina, *H. gelotopoeon*, the native species, was the most abundant and predominant Heliothinae in chickpea and soybean crops. Our results showed that *H. gelotopoeon* and *H. armigera* had a seasonal distribution in both crops during all years studied in Tucumán province, being more prevalent in September and October on chickpea and during February and March on soybean.

The influence of crops on the abundance of *H. armigera* was reported by [@iew052-B26]. The authors found that alfalfa and corn always had positive effects, whereas sorghum presented a negative effect on the light-trap catch in the second and third generations.

Considering crop phenology, [@iew052-B1] observed that the appearance of *H. armigera* always coincided with the initiation of flowering of chickpea, irrespective of the fact of how early or late the crop started flowering. In our study, an increase in population of both species was detected from September to October, coinciding with the beginning of the podding stage (R3) of chickpea. In soybean, *H. armigera* was not abundant and the first adults were detected at R2, by the end of February, whereas *H. gelotopoeon* adults were found from V3, in the beginning of January, in both years.

The results in this study indicate that *H. armigera* is widely distributed and the species has been adapted to elevations ranging from 20 to 1,200 masl in Argentina. [@iew052-B52], [@iew052-B41], and [@iew052-B14] reported that *H. armigera* adults are highly mobile. They can move many hundreds of kilometers between regions and extensively between fields within regions. *Helicoverpa armigera* seems to be well adapted to the climate of agricultural regions of South America. According to [@iew052-B20], this insect species has spread rapidly throughout Argentina, Bolivia, Brazil, Paraguay, Uruguay, and most recently to Puerto Rico. However, no studies have been conducted to investigate whether *H. armigera* detected in these non-Brazilian locations were due to natural spread of Brazilian populations or they represented separate incursion events. In addition, *H. armigera* has been detected in Brazil in October 2008 ([@iew052-B46]), meaning that the presence of this species in other countries could be due to late detection more than to fast spreading. Also, the detection in Brazil does not mean that this country has been the entrance of this insect species to Latin American countries. *Helicoverpa armigera* could have invaded South America through other countries where its identification might have been neglected.

So far, in Argentina, larvae of *H. armigera* have been found on chickpea ([@iew052-B3], [@iew052-B44]), soybean, cotton ([@iew052-B44]), sunflower and spiny plumeless thistle. Studies performed in Brazil indicate a widespread distribution of *H. armigera* throughout Midwest and Northeast Brazil in a variety of crops, particularly dicotyledons, beans (*Phaseolus vulgaris* L. Fabales: Fabaceae), soybean, and cotton, and to a lesser extent on monocotyledons, e.g., millet (*Pennisetum glaucum* L. Poales: Poaceae), sorghum, and corn ([@iew052-B23]).

Considering the importance of *H. gelotopoeon* in the region, few studies on the bioecology, crop damage, chemical control, natural enemies, host plants, oviposition preference or monitoring resistance to insecticides and *Bt* cultivars, have been performed. Our study is the first to provide information about its seasonal distribution.

Despite the low frequency of *H. armigera*, the results from the current study show that this pest is widely distributed in Argentina. Because of that, it is important to intensify the monitoring and correctly identify the different species of the Heliothinae complex using taxonomic methods or molecular techniques.

The sustainable management of this species poses a significant challenge for Argentinean entomology in the coming years, especially considering its polyphagous feeding habit, great dispersal ability, and numerous reports of resistance to insecticides and *Bt* crops ([@iew052-B4], [@iew052-B24], [@iew052-B17], [@iew052-B25], [@iew052-B32], [@iew052-B8]).

It is necessary to address different studies, as mentioned, in order to contribute to future studies regarding management for *H. gelotopoeon* and *H. armigera* in Argentina.

We thank, Lucas Fadda and David González, from EEAOC, for their assistance in the collection of material and excellent technical support in the laboratory; Javier Carreras Baldres (SrySig, EEAOC) for his assistance in the preparation of the map; SENASA NOA-Sur for providing database of *H. armigera* pheromone traps; Lic. E. Willink for his constructive comments on an earlier draft of the manuscript; Dr. Fernando Navarro \[CONICET, Instituto Superior de Entomología Dr. Abraham Willink (INSUE), Facultad de Ciencias Naturales, Universidad Nacional de Tucumán\] for the taxonomic identification of the Heliothinae complex adults, for his unconditional support, and for sharing memorable moments.

Funding
=======

This study was supported by EEAOC, CONICET, Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT no. G535/26), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Project no. 308947/2014-2).

[^1]: Subject Editor: Xinzhi Ni

[^2]: ^a^Only male moths obtained from larvae collected were examined using genitalia to confirm the species. Females obtained were not considered for this study.

[^3]: Database of SINAVIMO.

[^4]: Data reported by [@iew052-B3].

[^5]: Data used to report the first time of *H. armigera* collection in Argentina ([@iew052-B29]).
