Translator Disclaimer
23 September 2021 Re-Examination of Morphological Variations in the Female Internal Genitalia of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) for Identification and Pest Management
Takayuki Amano, Masashi Nomura
Author Affiliations +

Two of the most serious agricultural pests, Helicoverpa armigera (Hübner) and Helicoverpa zea (Boddie) (both Lepidoptera: Noctuidae), are similar in their appearances. To distinguish them, morphological characters of female internal genitalia were re-examined. Specimens were collected from 7 regions in 17 countries. All specimens were identified in advance by molecular methods. Significant differences were observed in the length of the bursa copulatrix, ductus bursa, and pigmented area on the base of appendix bursa. Helicoverpa armigera had dense spicules on the luminal surface of the appendix bursa which were absent on the corresponding surface in H. zea. Additionally, differences of length on these parts in female genitalia will be helpful for identification. We are confident these morphological characters will advance resolution of noctuid speciation by taxonomists to properly identify these species based on distinguishing features of the female genitalia.

The genus Helicoverpa (Lepidoptera: Noctuidae) contains several important agricultural pests, especially, Helicoverpa armigera (Hübner) and Helicoverpa zea (Boddie) are recognized as 2 of the most serious pest species within the genus. Both species severely damage a wide range of important crops (Fitt 1989; Cunningham & Zalucki 2014). In the past, both species were geographically isolated from each other. However, the invasion of H. armigera into South America has changed this situation. The first report of H. armigera in the New World came from Brazil in 2013 (Czepak et al. 2013), and thereafter, distribution of this species rapidly spread in South and Central America (SENAVE 2013; Tay et al. 2013; Murúa et al. 2014; NAPPO 2014; Gilligan et al. 2015, 2019; Castiglioni et al. 2016).

Rapid and proper identification of noctuids is critical for pest detection, monitoring, and timely management. However, similarity in appearance of these 2 species makes it difficult to distinguish them using external morphological characters. Recently, several molecular-based methods have been developed to distinguish these 2 species (Behere et al. 2008; Gilligan et al. 2015; Perera et al. 2015; Zink et al. 2017; Amano & Nomura 2020). Molecular-based identification has several advantages: the applicability for any life stage, the efficiency for multiple and bulk samples, and the uniformity of techniques. However, these methods usually need expensive equipment and reagents, and sometimes can be more time consuming than morphological identification by experienced entomologists. Therefore, it is necessary to use molecular and morphological approaches to speed up the proper identification and diagnosis of these species. In terms of morphological identification, comprehensive keys on the genus Helicoverpa based on male and female internal genitalia were assembled by Hardwick (1965). Detailed morphological differences in male genitalia were provided by Pogue (2004). Morphological features in female genitalia were investigated by Yoshida and Takahashi (2005). Helicoverpa armigera samples used in their study were restricted to specimens from Japan (Yoshida & Takahashi 2005). In more recent studies, Perini et al. (2019) provided a morphological key to identification of Helicoverpa species based on male prothoracic leg characters, but these characters are not applicable to female moths.

In this study, we re-examined the morphological characters of female internal genitalia to identify H. zea and H. armigera using specimens collected from 7 regions and 17 countries. All samples were identified using molecular-based methods as a cross-check.

Materials and Methods


Pinned dry specimens of adult individuals were obtained from the Japanese Plant Protection Station, Yokohama, Kanagawa, Japan (Table 1), and identified by macroscopic features and molecular-based methods.


Multiplex Polymerase Chain Reaction (PCR) was employed as a molecular-based identification method with a specific primer set provided by Perera et al. (2015). DNA extraction and PCR assay were performed with the same procedure described in Amano and Nomura (2020).


The abdomen was removed from each dry specimen and treated with 10% KOH at 80 °C for 30 min, then the genitalia was dissected. Observation of genitalia was carried out under a stereomicroscope system SZX 16 (Olympus, Tokyo, Japan). The length of bursa copulatrix, ductus bursa, and pigmented area on the base of appendix bursa (Fig. 1) were measured under the stereomicroscope with an ocular micrometer UOCMC 10/100XY (Olympus, Tokyo, Japan). In this study, the length of bursa copulatrix was defined as the length between the apex of ostium bursa and apex of appendix bursa, and bursa copulatrix was straightened for measurement using tweezers without stretching. The length of ductus bursa and pigmented area were measured according to Yoshida and Takahashi (2005).

The texture of the appendix bursa was observed under the stereomicroscope system. To confirm the dense spicules on the luminal surface, slides were examined using a biological microscope system BX 53 (Olympus, Tokyo, Japan). A small piece of tissue was cut off from the appendix bursa of H. armigera with a scalpel, and the slice was mounted on the slide with Hoyer's Medium (Anderson 1954) prepared in our laboratory.

Table 1.

Details of specimens used in this study.



The lengths of bursa copulatrix, ductus bursa, and pigmented area were significantly longer in H. zea than in H. armigera. Overlap was not found (Table 2). The mean (± SD) measurements of bursa copulatrix, ductus bursa, and pigmented area were, respectively, 11.8 ± 0.6 mm, 1.7 ± 0.14 mm, and 2.0 ± 0.17 mm in H. zea, whereas the measurements were 8.7 ± 0.5 mm, 1.0 ± 0.12 mm, and 1.1 ± 0.18 mm in H. armigera (Table 2).

Figure 1B, E shows the outward appearances of the appendix bursa. A punctation-like texture was observed in all specimens of H. armigera but was absent in H. zea (Fig. 1B, E). Figure 1C shows the luminal surface texture on the appendix bursa of H. armigera. It indicates that dense spicules on the luminal surface look like punctations under stereomicroscopic observation (Fig. 1B, C).


The bursa copulatrix was significantly longer in H. zea than in H. armigera. This result seems to be consistent with the difference in the male aedeagus because the bursa copulatrix receives the aedeagus and inflated vesica (sensu Pogue 2004; the ‘extension’ of Mallet 2018) during copulation (Callahan 1958; Mallet 2018), and the inflated vesica also is longer in H. zea than in H. armigera (Pogue 2004).

As in the previous study of Yoshida and Takahashi (2005), lengths of both ductus bursa and pigmented area also were significantly longer in H. zea than in H. armigera. However, these 2 characters are difficult to use because the observed length of the ductus bursa depends on the observation angle, and measuring the pigmented area sometimes is difficult due to its curved shape. Moreover, overlaps were observed in both lengths of the ductus bursa and in the pigmented areas of the 2 species in the previous study (Yoshida & Takahashi 2005).

In this study, we immersed abdomens of specimens in 10% KOH at high temperature to dissect genitalia from dried specimens. Use of hot or boiling caustic is a common procedure in preparation of slide mounts of small insects such as mealybugs or aphids (Williams & de Willink 1992; Blackman & Eastop 2000). If the specimens are treated under the same procedure, it appears that these specimens are usable for morphological comparison. On the other hand, Hardwick (1950) mentioned that the use of cold caustic generally gave more uniform results than short immersion in hot or boiling caustic. Thus, the influence of artifacts from hot caustics on the genitalia should be clarified by further studies.

Hardwick (1965) described the difference in the luminal surface texture of appendix bursa. In the same way, dense spicules on the luminal surface were observed in H. armigera but were absent in H. zea. Although it needs high magnification to observe the spicules, this feature is observed easily under relatively low magnification as a punctated texture.

When there are only a few samples to be identified, morphological identification is faster and easier than the molecular-based method. After 30 min of KOH treatment and dissection, careful observation of the appendix bursa is performed. If dense spicules are absent on the appendix bursa, the specimen is identified as H. zea. If dense spicules are observed on the appendix bursa, the specimen is identified as H. armigera. Moreover, measurement of the bursa copulatrix also can be helpful for identification.

Fig. 1.

(A) Female genitalia of Helicoverpa armigera. (B) Texture on appendix bursa of H. armigera. (C) Luminal surface on appendix bursa of H. armigera. (D) Female genitalia of Helicoverpa zea. (E) Texture on appendix bursa of H. zea. (ab) appendix bursa; (bc) bursa copulatrix; (cb) corpus bursa; (db) ductus bursa; (ob) ostium bursa; (pa) pigmented area.


Table 2.

Comparison of the characteristics between Helicoverpa armigera and Helicoverpa zea.


We believe that the morphological characters re-examined in the female internal genitalia could be useful to distinguish H. armigera from H. zea. We are confident that findings of this study will contribute toward the detection, monitoring, and management of these invasive pests.


We would like to express our gratitude to entomology specialists of Yokohama Plant Protection Station, Yokohama, Kanagawa, Japan, for providing specimens used in this study. Also, we appreciate the review of an earlier version of this paper by Muhammad Haseeb, Center for Biological Control, College of Agriculture and Food Sciences, Florida A&M University, Tallahassee, Florida, USA.

References Cited


Amano T, Nomura M. 2020. A diagnostic loop-mediated isothermal amplification method to distinguish Helicoverpa armigera (Lepidoptera: Noctuidae) from other related species in the New World. Journal of Insect Science 20: 10. Google Scholar


Anderson LE. 1954. Hoyer's solution as a rapid permanent mounting medium for bryophytes. The Bryologist 57: 242–244. Google Scholar


Behere GT, Tay WT, Russell DA, Batterham P. 2008. Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae). Bulletin of Entomological Research 98: 599–603. Google Scholar


Blackman RL, Eastop VF. 2000. Aphids on the World's Crops: An Identification and Information Guide, Second edition. John Wiley & Sons Ltd., Hoboken, New Jersey, USA. Google Scholar


Callahan PS. 1958. Serial morphology as a technique for determination of reproductive patterns in the corn earworm, Heliothis zea (Boddie). Annals of the Entomological Society of America 51: 413–428. Google Scholar


Castiglioni E, Perini CR, Chiaravalle W, Arnemann JA, Ugalde G, Guedes JVC. 2016. Primer registro de ocurrencia de Helicoverpa armigera (Hübner, 1808) (Lepidoptera: Noctuidae) en soja, en Uruguay. Agrociencia - Sitio en Reparación 20: 31–35. Google Scholar


Cunningham JP, Zalucki MP. 2014. Understanding Heliothine (Lepidoptera: Heliothinae) pests: what is a host plant? Journal of Economic Entomology 107: 881–896. Google Scholar


Czepak C, Albernaz KC, Vivan LM, Guimarães HO, Carvalhais T. 2013. Primeiro registro de ocorrência de Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) no Brasil. Pesquisa Agropecuária Tropical 43: 110–113. Google Scholar


Fitt GP. 1989. The ecology of Heliothis species in relation to agroecosystems. Annual Review of Entomology 34: 17–52. Google Scholar


Gilligan TM, Goldstein PZ, Timm AE, Farris R, Ledezma L, Cunningham AP. 2019. Identification of Heliothine (Lepidoptera: Noctuidae) larvae intercepted at U.S. ports of entry from the New World. Journal of Economic Entomology 112: 603–615. Google Scholar


Gilligan TM, Tembrock LR, Farris RE, Barr NB, van der Straten MJ, van de Vossenberg BTLH, Metz-Verschure E. 2015. A multiplex real-time PCR assay to diagnose and separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World. PLoS ONE 10: e0142912. journal.pone.0142912 Google Scholar


Hardwick DF. 1950. Preparation of slide mounts of lepidopterous genitalia. The Canadian Entomologist 82: 231–235. Google Scholar


Hardwick DF. 1965. The Corn Earworm Complex. Memoirs of the Entomological Society of Canada 97: 5–274. Google Scholar


Mallet J. 2018. Invasive insect hybridizes with local pests. Proceedings of the National Academy of Sciences 115: 4819–4821. Google Scholar


Murúa MG, Scalora FS, Navarro FR, Cazado LE, Casmuz A, Villagrán ME, Lobos E, Gastaminza G. 2014. First record of Helicoverpa armigera (Lepidoptera: Noctuidae) in Argentina. Florida Entomologist 97: 854–856. Google Scholar


NAPPO – North American Plant Protection Organization. 2014. Detection of Old World bollworm (Helicoverpa armigera) in Puerto Rico. Phytosanitary Alert System, Raleigh, North Carolina, USA. (last accessed 1 Jun 2021). Google Scholar


Perera OP, Allen KC, Jain D, Purcell M, Little NS, Luttrell RG. 2015. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1. Journal of Insect Science 15: 155. Google Scholar


Perini CR, Angulo AO, Olivares TS, Arnemann JA, Guedes VC. 2019. New morphological key using male prothoracic leg characters to identify Helicoverpa (Lepidoptera: Noctuidae) species. Australian Journal of Crop Science 13: 1561–1565. Google Scholar


Pogue MG. 2004. A new synonym of Helicoverpa zea (Boddie) and differentiation of adult males of H. zea and H. armigera (Hübner) (Lepidoptera: Noctuidae: Heliothinae). Annals of the Entomological Society of America 97: 1222–1226. Google Scholar


SENAVE. 2013. Senave en alerta tras ingreso de peligrosa plaga agrícola – Edicion Impresa – ABC Color. Azeta SA, Yegros, Asunción, Paraguay. (last accessed 1 Jun 2021). Google Scholar


Tay WT, Soria MF, Walsh T, Thomazoni D, Silvie P, Behere GT, Anderson C, Downes S. 2013. A brave new world for an Old World pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil. PLoS ONE 8: e80134. Google Scholar


Williams DJ, de Willink MG. 1992. Mealybugs of Central and South America. CAB International, Wallingford, United Kingdom. Google Scholar


Yoshida Y, Takahashi G. 2005. Difference in morphological characteristics between Helicoverpa zea and Helicoverpa armigera. Research Bulletin of the Plant Protection Service Japan 41: 47–51. Google Scholar


Zink FA, Tembrock LR, Timm AE, Farris RE, Perera OP, Gilligan TM. 2017. A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples. PLoS ONE 12: e0178704. journal.pone.0178704 Google Scholar
Takayuki Amano and Masashi Nomura "Re-Examination of Morphological Variations in the Female Internal Genitalia of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) for Identification and Pest Management," Florida Entomologist 104(3), 218-221, (23 September 2021).
Published: 23 September 2021

Back to Top