The fall armyworm (FAW), Spodoptera frugiperda Smith & Abbot (Lepidoptera: Noctuidae), is the main pest of maize (corn) and some other crops in Latin America (Hernández-Mendoza et al. 2008), causing yield reductions and economic losses (Sparks 1986; Casmuz et al. 2010). Chemical control is the practice most often used to control this insect pest; however, this method has been in efficient due to incorrect and indiscriminate use, thus causing acute and chronic poisoning to farm workers, and inducing development of resistance, elimination of native natural enemies, and pollution of soil (Tinoco & Halperin 1998; Gómez-Valderrama et al. 2010). An alternative to the use of insecticides is to control this pest using native natural enemies (Rios-Velasco et al. 2011), which has great advantages, such as not having harmful effects on human health and environment. Also, natural enemies often are specific, some have high search capabilities, and the great majority can be handled easily and released in the field.

The parasitoids of FAW in the families Ichneumonidae, Braconidae, Eulophidae (order Hymenoptera) and Tachinidae (order Diptera) have been inventoried in the Mexican states Coahuila, Michoacán, Jalisco, Sinaloa, Nayarit, Veracruz, Colima, Yucatán, among others (Molina-Ochoa et al. 2004; Delfín-González et al. 2007; Rios-Velasco et al. 2011; Estrada-Virgen et al. 2013). Also, entomopathogenic bacteria, viruses, nematodes, and fungi have been reported (Molina-Ochoa et al. 2003; Hajek et al. 2007; Rios-Velasco et al. 2011). However, the fauna of natural enemies of FAW larvae in Chihuahua State has not been reported. Therefore, the main objectives of this study were to identify natural enemies of S. frugiperda and to estimate their level of parasitism in 5 localities of Chihuahua, Mexico.

Materials and Methods

All instars of S. frugiperda were collected from infested maize (corn) fields from Chihuahua, Mexico, in Aug and Sep 2014 (Table 1). Habitats surrounding maize fields were other crops as apple (Malus pumila Mill.; Rosales: Rosaceae) and soybean (Phaseolus vulgaris L.; Fabales: Fabaceae); a mix of noncrop vegetation predominated by sagebrush (Artemisia sp. L.; Asterales: Asteraceae) and grasses (Poales: Poaceae); and a forest-species complex composed primarily of hardwood/coniferous woodland species such as juniper Juniperus spp. (Pinales: Cupressaceae) and oak Quercus spp. (Fagales: Fagaceae). All corn fields had been without chemical pesticide application. Larvae were placed in 1 oz (29.6 mL) plastic cups (Grupo Convermex, S.A. de C.V. Puebla, Puebla, Mexico) with artificial diet (Southland Products, Inc., Village, Arkansas, USA). Collected larvae were transported to the Centro de Investigación en Alimentación y Desarrollo, A.C., (CIAD, Campus Cuauhtémoc, Chihuahua, Mexico) and held at 26 ± 2 °C, a 12:12 h photoperiod, and > 70% RH. Plastic containers were examined daily until the emergence of parasitoids, until the presence of mycosis, nematodes, or viral infections, or until FAW larvae reached adulthood.

The parasitoids obtained were identified using a stereoscope (Leica G26, Barrington, New Jersey, USA) and compound microscope (Carl Zeiss, Jena, Germany), and the keys published by Townes & Townes (1966), Cave (1993), Wharton et al. (1997), and Triplehorn & Johnson (2005). Confirmations of identifications were made by Dra. Juana Maria Coronado Blanco (Universidad Autónoma de Tamaulipas, Tamaulipas, México). Parasitoids were kept in 70% ethanol for preservation. Percentage parasitism was calculated based on the total number of FAW larvae that were positive for parasitoids or entomopathogens divided by the total number of larvae collected × 100 (Pair et al. 1986; Rios-Velasco et al. 2011).

Table 1.

Geographical location of maize fields in the state of Chihuahua, Mexico, where Spodoptera frugiperda larvae were collected in 2014.

The predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae) was observed consuming FAW larvae in corn plots. Specimens were collected and placed in 1 oz (29.6 mL) plastic cups with 70% ethanol and identified using taxonomic and pictorial keys (University of Florida 2010; Rider 2012).

FAW larvae with mycosis were incubated in humid chambers, and the possible entomopathogenic fungi were isolated and purified in an artificial medium of potato dextrose agar (PDA) for Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) and V8-Agar for Metarhizium rileyi (Farl.) Kepler, S.A. Rehner & Humber comb. nov. (= Nomuraea rileyi [Farl.] Samson; Hypocreales: Clavicipitaceae) (Kepler et al. 2014). The purified entomopathogenic fungi were identified according to their microscopic and macroscopic characteristics using taxonomic keys in Barnett & Hunter (1998), Dugan (2006), and Watanabe (2010).

Larvae infected with nematodes were observed with a stereoscope (Leica G26, Barrington, New Jersey, USA) and compound microscope (Carl Zeiss, Jena, Germany) for identification of the nematodes with the taxonomic keys in Nickle (1972) and Nguyen & Smart (1996). However, identification to species level was not possible.

Larvae with symptoms of viral infection, such as white or dark color and fragility of the integument when dead, were obtained (Nicholls-Estrada 2008). To verify that virus was the cause of death, a sample was mounted on a slide and stained with 0.4% Giemsa stain (Sigma Aldrich, St. Louis, Missouri, USA) and/or Naphthol Blue Black (Sigma Aldrich, St. Louis, Missouri, USA) to observe the occlusion bodies (OBs) that showed polyhedric and granular characteristics of the nucleopolyhedrovirus (NPV) and granulovirus (GV) genera, respectively, for their identification. Also, a pathogenicity test was performed in which 100 μL of the OBs suspension was mixed with 100 μL of a solution containing 4% sucrose and blue food dye at 1%. In accordance with the droplet feeding method described by Hughes & Wood (1981), a suspension of 2 μL droplets was administered orally to a group of 2nd instars that previously had been subjected to a 24 h starvation period. After FAW larvae ingested the viral suspension, which was evidenced by a blue color inside the body, these larvae were transferred to plastic cups with artificial diet, held under controlled conditions (26 ± 2 °C, 12:12 h photoperiod, and > 70% RH), and checked daily for symptoms of viral infection, such as loss of appetite and mobility, color change, or fragility and rupture of the integument (Caballero et al. 2001; Vasquez et al. 2002). Infected FAW larvae were placed in 15 mL polypropylene tubes with sterile distilled water and stored at -70 °C.

Results and Discussion

In total, 5,870 FAW larvae were collected from 9 locations with 1 or 2 sampling dates per location. From these samples, 473 larvae were found to be parasitized, but there was no emergence from 133 larvae, in which only the immature stages of the parasitoids were observed. Adult parasitoids emerged from 340 FAW larvae. These parasitoids represent species of Hymenoptera (Ichneumonidae, Braconidae, and Eulophidae) and Diptera (Tachinidae) (Table 2). Molina-Ochoa et al. (2004) found FAW larvae parasitized by specimens belonging to the families Braconidae (Aleoides, Chelonus, Cotesia, Glyptapanteles, Homolobus, and Meteorus genera), Ichneumonidae (Campoletis, Eiphosoma, Ophion, and Pristomerus genera), and Eulophidae (Aprostocetus, Euplectrus, and Horismenus) in Michoacán, Jalisco, Sinaloa, Nayarit, Veracruz, and Colima, Mexico, in various crops. In the present study, we found only 5 of the 13 genera reported by these authors. Parasitoid species found in this study were Meteorus arizonensis Muesebeck, Campoletis sonorensis (Cameron), Campoletis flavicincta (Ashmead), Pristomerus sp., Chelonus insularis (Cresson), Euplectrus platyhypenae Howard, Lespesia sp. Archytas marmoratus (Townsend), and other unknown tachinids of which twelve morphospecies were identified (Table 3). These results were similar to those reported by Delfín-González et al. (2007), who found Lespesia archippivora (Riley), A. marmoratus, and E. platyhypenae in the state of Yucatán, Mexico. Murúa et al. (2009) reported Campoletis grioti (Blanchard), Ch. insularis, A. marmoratus, and E. platyhypenae, as in this study. Additionally, they found Archytas incertus (Giglio-Tos), Ophion sp., and Incamyia chilensis (Aldrich). Further Jourdie et al. (2008) found Cotesia marginiventris (Cresson), Chelonus cautus Cresson, Meteorus laphygmae Viereck, Pristomerus spinator (F.), and Eiphosoma vitticolle Cresson. The data obtained suggest the existence of a great diversity of parasitoids species and parasitism levels, depending on geographic region, though all sites share many of the same species. The regional differences in parasitoid species and levels of parasitism may be caused largely by environmental differences, besides weather, adjacent crops, and alternate hosts. Also, extent of sampling, population density of the host, the level of adaptation of natural enemies, and population growth in both host and parasitoids, among other factors, will affect determination of natural enemies.

The overall parasitism level by natural enemies observed in this study, including both parasitoids and entomopathogens, was 18.2% (Table 3). In contrast, Estrada-Virgen et al. (2013) reported a parasitism level of 29.7% in the state of Nayarit, which was substantially greater than in this study. The most commonly encountered species of parasitoids were M. arizonensis and C. flavicincta, parasitizing 3.0% and 1.3% FAW larvae, respectively. These results are different from what was reported by Molina-Ochoa et al. (2003), who indicated that Ch. insularis was the most common and widely distributed parasitoid species occurring in Central America and North America. The tachinid flies recovered in this study, Lespesia sp. and A. marmoratus, showed parasitism rates of only 0.1% and 0.02%, respectively. However, a parasitism incidence of 0.56% by other unknown species of tachinids was found (Table 3).

Thirty-four specimens of the predator P. maculiventris were found consuming FAW larvae in corn fields. This species has been recognized as a polyphagous predator associated with several orders of insects including Lepidoptera (De Clercq & Degheele 1992; Zanuncio et al. 2008).

Table 2.

Incidence of natural enemies of Spodoptera frugiperda larvae in Chihuahua, Mexico, in 2014.

Table 3.

Natural enemy taxa of Spodoptera frugiperda larvae found in maize fields from Chihuahua, Mexico in 2014.

The incidence of entomopathogenous fungi, viruses, and nematodes in this study was 10.1%, which was higher than the 3.8% found by Wyckhuys & O'Neil (2006) in Honduran subsistence maize. Five hundred forty-two (9.2%) FAW larvae showed fungal mycosis, of which 504 (8.6%) were infected by M. rileyi and 38 (0.65%) by B. bassiana. The high incidence of fungal infections in Bachiniva County (Table 2) may possibly be due to the significant level of precipitation (166.8 mm) and cool weather (18.5 °C and 18.8 °C for Aug and Sep 2014, respectively) that occurred in the days just prior to the sampling date (Unifrut 2014). Growth and germination of fungi are influenced strongly by environmental conditions, mainly by temperature and RH (Ignoffo & García 1985; Vimala-Devi et al. 1996; Rios-Velasco et al. 2010).

Forty-nine isolates of nucleopolyhedrovirus (Baculoviridae) were obtained from FAW larvae. Each infected larva was considered to be an isolate, and 0.8% of the larvae were infected with this entomopathogen. Similar results were obtained in Colombia by Gómez-Valderrama et al. (2010), who collected 2,140 larvae of which only 3 larvae were infected with nucleopolyhedrovirus, which corresponded to an occurrence rate of 0.14%. This result is also similar to that reported by Valicente & Barreto (1999), who collected 14,000 larvae, which produced 21 nucleopolyhedrovirus isolates, corresponding to an occurrence rate of 0.15%.

Among the collected larvae, 0.07% showed infection by nematodes. Molina-Ochoa et al. (2003) asserted that entomopathogenic nematodes have been shown to be agents with high potential for the control of the FAW. Four hundred eighty-three FAW larvae (8.2%) died from unknown causes, and the remaining larvae (4,319) reached adulthood (Table 2).

A great diversity of natural enemies of the FAW occurs in the state of Chihuahua, which is similar to other locations in Mexico and Central America, and may have potential to regulate population of this pest. Properly managed, this diversity may represent a viable alternative for implementation in integrated FAW management programs.