The Helicoverpa/Heliothis complex can cause serious damage to agricultural crops. Phenotypic similarity makes it difﬁcult to discriminate between closely related Helicoverpa species. Currently, morphology of the male genitalia complemented with molecular techniques constitutes the best approach for species identiﬁcation. In this work, a broad microscopic examination of adult Helicoverpa zea (Boddie) males (n ¼ 200) captured in central Argentina was carried out in order to provide a detailed description of the val- vae and the phallus. A considerable degree of variability was recorded. Both rounded and sharp valve apices were observed and valvae were not always parallel-sided. Most evident differences were detected concerning the number of cornuti on the phallus. A range of 15–21 cornuti per phallus was recorded, the mode being 18. A signiﬁcant minority of the samples (3.5%) displayed an abnormal genital condition show- ing a constricted phallus lacking cornuti, and pointed valvae. This form was initially attributed to a distinct species, Heliothis stombleri, and later proposed as a synonym of H. zea based on additional morphological observations and molecular studies. Here, a phylogenetic analysis combining mitochondrial (cytochrome oxidase subunit I) and nuclear (elongation factor –1 alpha) genes was performed on these and other Helicoverpa specimens collected in the same geographical region, in order to further verify the taxonomic status of H. stombleri. The tree topology clearly grouped H. stombleri with H. zea, supporting the assump- tion that the former represents, in fact, an anomalous form of the latter. Further experiments are needed to clarify the etiology of this anomaly and its persistence over time. Resumen El complejo Helicoverpa/Heliothis produce graves danos a la agricultura. Las especies del ge´ nero Helicoverpa son difıciles de diferenciar porque poseen un fenotipo similar. Actualmente, su identiﬁcacion se basa en carac- terısticas morfologicas de la genitalia masculina y en el uso de marcadores moleculares. En este trabajo, se examinaron por microscopıa machos adultos de Helicoverpa zea (Boddie; n ¼ 200) capturados en la zona central de Argentina, a ﬁn de detallar la forma y variabilidad de valvas y falo. Las valvas no siempre presentaron lados paralelos, y mostraron apices agudos o redondeados. Las diferencias mas evidentes se reﬁrieron al numero de cornuti del falo (15 a 21, moda ¼ 18). Una minorıa signiﬁcativa de las muestras (3,5%) exhibio una fuerte anomalıa en el aspecto del aparato reproductor: falo sin cornuti, estrechado en su extremo, y valvas terminadas en punta. Esta variante, que habıa sido atribuida inicialmente a una especie distinta, Heliothis stombleri, fue luego considerada como sinonimo de H. zea mediante estudios morfologicos y moleculares. Para veriﬁcar esta conclusion, se llevo a cabo un analisis ﬁlogene´ tico que combino genes mitocondriales (COI) y nucleares (EF-1a) V C The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/3/74/3852624 email@example.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 3 a partir de especımenes de H. stombleri y otras Helicoverpa spp. provenientes de la misma region. La topologıa de los arboles agrupo claramente a H. stombleri con H. zea, permitiendo suponer que, en efecto, ambas formas constituyen la misma especie. Experimentos adicionales son necesarios para clariﬁcar la etiologıa de esta anomalıa, ası como su persistencia en el tiempo. Key words: corn earworm, identiﬁcation, morphology, molecular marker The corn earworm, Helicoverpa zea (Boddie), is an economically impor- As for other insect species, the current classification of the tant agricultural pest distributed across the Americas (Hardwick 1965). Heliothinae is being supported by genetic studies involving molecu- This noctuid is considered a major threat to corn, and occasionally it at- lar markers. Sequencing of conserved regions of the insect genome, tacks other field and horticultural crops, including alfalfa, cotton, to- combined with classical morphological studies, has proven a power- bacco, peanuts, sorghum, and sunflower. In Argentina, H. zea ful tool for resolving taxonomic controversies. The present work completes five generations per year in the north and three to four gener- provides additional details on the morphology of the H. zea male ations in the central region (Margheritis and Rizzo 1965). Adults begin genitalia (both the “normal” and the “H. stombleri forms”), which to emerge from wintering pupae between October and November. The should be useful for routine diagnosis. Furthermore, a phylogenetic moths lay their eggs on the stigmas of carpellate (female) corn flowers analysis comprising nuclear and mitochondrial DNA sequences was (“corn silk”), where the newly-hatched larvae start feeding. As crop performed in order to present supplementary genetic information to phenology progresses, larvae feed on the grains throughout kernel filling corroborate the synonymy between H. zea and H. stombleri. (Navarro et al. 2009). The extent of damaged ears can reach up to 100% in late sowing, even in transgenic hybrids expressing toxins de- Materials and Methods rived from Bacillus thuringiensis (Balbi and Flores 2015). H. zea partially overlaps its geographical range with other pest spe- Insect Sampling cies in the Helicoverpa/Heliothis complex: Heliothis virescens Moth catches were carried out using mercury vapor light traps in (Fabricius; found from USA to central Argentina) and Helicoverpa gelo- January and February 2016 in Marcos Ju arez (central Argentina), topoeon (Dyar; restricted to southern South-America). One of the most near corn and soybean crops. In total, 202 male specimens, which devastating members of the complex, the exotic Helicoverpa armigera had wing markings resembling H. zea, were separated and con- (Hu ¨ bner), has recently become established in Brazil and Argentina served at 20 C for posterior morphological and genetic analyses. (Murua et al. 2016). The regular monitoring of Helicoverpa spp. has thus become imperative in the highly productive South-American tem- Microscopic Examinations perate and subtropical agricultural lands. These polyphagous species The final portion of the abdomen of all individuals was dissected, sometimes co-occur on the same host plants, and due to their similar ex- placed in KOH solution and heated in a water bath for 15 min at ternal morphology, they are difficult to differentiate from each other. increasing temperature below boiling point. Then, the genitalia were Several molecular-based techniques have been developed for species removed and placed in alcohol for 24 h. After cleaning with a fine- identification within Heliothinae (Orui et al. 2000, Behere et al. 2008, bristled brush, genitalia were examined under a Zeiss Stemi DV4 Arneodo et al. 2015, Gilligan et al. 2015, Perera et al. 2015, Nagoshi stereomicroscope (Carl Zeiss GMBH, Hamburg, Germany) at 16 et al. 2016). However, the microscopic observation of adult genitalia magnification. Photographs were taken at 5 zoom with a digital continues to be the most widely used method. The most obvious charac- camera (Canon G10, Canon Inc., Tokyo, Japan). The shape of the ters for the separation of related species are found in the genitalia of valvae was described, and their size assessed using the program Image males. The shape, width and length of the valvae, the presence and Pro Plus (Media Cybernetics, Silver Spring, MD). The presence and number of cornuti in the vesica, the shape and number of coils of the in- number of cornuti in the phallus were determined. The vesica was in- flated vesica, and the presence or absence of lobes at the base of the flated by introducing 70% ethanol, using a syringe with a 0.3 mm di- vesica constitute the key features for species discrimination (Hardwick ameter needle, in order to observe the lobes at its base. 1965, Pogue 2004). A thorough knowledge of these morphological and morphometric traits is essential to avoid misidentification, especially in thecaseof the closelyrelated H. zea and H. armigera. Molecular Characterization During a survey of Heliothinae pests in California, Okumura and Five H. stombleri specimens were subjected to DNA extraction, amplifi- Bauer (1969) recorded the presence of a lepidopteran species resem- cation and sequencing. DNA was extracted by the CTAB method bling H. zea in external appearance, but possessing particular genital (Doyle and Doyle 1990). Amplification of an 812-bp region of the mito- characters. They described it as a new species, which they named chondrial cytochrome oxidase subunit I gene (COI) was performed Heliothis stombleri.Male H. stombleri specimens were reported to with primers H3Fw/H3Rv according to Arneodo et al. (2015).On the have narrower valvae than H. zea, with their apical end sharpened. basis of available Heliothinae sequences, a new primer pair was de- The phallus was apically constricted and lacked cornuti. A subsequent signed to amplify a 587- bp fragment of the nuclear elongation factor – microscopic analysis by Hardwick (1970) concluded that, in fact, H. 1 alpha gene (EF-1a): HelicoEFaFw6 (5 -CGTCAACCAAAATGCC 0 0 0 stombleri was an aberrant form of H. zea. Moreover, he assumed that CTGG-3 ) and HelicoEFaRv6 (5 -GGCGTCACCAGACTTGATGG-3 ). those individuals probably would not succeed in mating. In a more re- For the latter, the following PCR conditions were used: 95 C for cent study, Pogue (2004) re-checked the same above mentioned mor- 5 min, 40 cycles of 95 Cfor 30s, 53 Cfor 30s, and72 Cfor phological attributes, and proposed that H. stombleri and H. zea 45 s; and a final step of 72 C for 5 min. All PCR products were pu- should be treated as synonyms. In agreement with this, a later work rified using ADN Puri-Prep-GP kit (INBIO, Tandil, Argentina) and using molecular markers revealed high similarity between H. stom- both strands sequenced in an automated genetic analyzer ABI bleri and H. zea haplotypes (Nagoshi et al. 2016). PRISM 3500 XL (Applied Biosystems, Foster City, CA) at Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/3/74/3852624 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 3 3 Fig. 1. Helicoverpa zea male genitalia. (A) Valvae and phallus with 17 cornuti. (B) Valvae and phallus with 15 cornuti. (C) Valvae and phallus with 21 cornuti. (D) Vesica inﬂated. Arrows and arrowheads show the cornuti and the lobes at the base of the vesica, respectively. CICVyA-INTA (Hurlingham, Argentina). Unlike COI, EF-1a se- corresponded to the “H. stombleri form”. The remaining two were quences from local specimens of “normal” H. zea and the two identified as H. armigera.In H. zea, the length of valvae ranged other Heliothinae reported in the study area were not available at from 4.7 to 5.5 mm. A certain degree of variability was observed in GenBank. DNAs extracted from H. zea, H. armigera and H. gelo- the shape of the valve apex. Some samples had a rounded apex and topoeon during a recent survey in central Argentina (Arneodo others had a sharper one. In some moths, valvae were not parallel- et al. 2015) were used as templates to amplify this gene. A phylo- sided and the internal side was more curved (Fig. 1A). Notorious dif- genetic analysis of COI and EF-1a sequences was conducted on ferences were observed concerning the number of cornuti on the newly-obtained and reference sequences retrieved from public vesica. Most of the samples (>70%) exhibited 18 cornuti, but this databases. The noctuid Spodoptera exigua served as outgroup. value fluctuated from 15 to 21 (Fig. 1A–C). When the vesica was in- Maximum likelihood trees were constructed with 1,000 bootstrap flated, three lobes or diverticula were observed at the base, with the replicates using MEGA7 software (Kumar et al. 2016). central smallest (Fig. 1D). On the other hand, H. stombleri specimens displayed a distinct genital morphology. Valvae were similar in length to H. zea but Results with a tapering apex (Fig. 2A). The phallus presented a more or less Morphological Features clear constriction near the apical end and had no cornuti (Fig. 2B One hundred ninety three out of the 202 male moths dissected were and C). The inflated vesica did not evert. However, three small lobes “normal” H. zea specimens, whereas seven individuals appeared close to the constriction (Fig. 2C). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/3/74/3852624 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 3 Fig. 2. Heliothis stombleri male genitalia. (A) Valvae. (B) Vesica uninﬂated. (C) Vesica inﬂated. Arrows and arrowhead show the apical constriction and lobes, respectively. Fig. 3. Maximum likelihood phylogenetic tree based on COI sequences of H. stombleri (highlighted with a black circle) and related Heliothinae occurring in central Argentina. Numbers at the nodes indicate bootstrap support (1,000 replicates). Spodoptera exigua was chosen as outgroup. GenBank accession numbers are provided next to the species names. The scale bar shows the number of nucleotide substitutions per site. Genetic Analyses H. armigera (KY623665) and H. gelotopoeon (KY623666) were PCR amplifications of partial COI and EF-1a genes yielded frag- also submitted. ments of the expected sizes, as observed by agarose gel electrophore- Overall, and regardless of the gene considered, sequence analyses sis (data not shown). The resulting sequences were edited and clearly clustered together H. stombleri and H. zea, whereas the other aligned (Clustal W). Both the mitochondrial and nuclear sequences Helicoverpa spp. formed separate clades. Indeed, the mitochondrial obtained from the five H. stombleri specimens analyzed were identi- COI phylogeny revealed three well-supported groups enclosing the cal among them. Representative H. stombleri COI and EF-1a se- different species, one of which grouped H. zea and H. stombleri quences are available at GenBank under accession numbers with a bootstrap support of 100 (Fig. 3). Phylogenetic analysis of KY623667 and KY623663, respectively. EF-1a sequences corre- nuclear EF-1a sequences showed the same pattern. Again, all nodes sponding to local male specimens of “normal” H. zea (KY623664), had high bootstrap values (>70%; Fig. 4). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/3/74/3852624 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 3 5 Fig. 4. Maximum likelihood phylogenetic tree based on EF-1a sequences of H. stombleri (highlighted with a black circle) and related Heliothinae. Newly reported sequences obtained in this work are marked with a white circle. Numbers at the nodes indicate bootstrap support (1,000 replicates). Spodoptera exigua was cho- sen as outgroup. GenBank accession numbers are provided next to the species names. The scale bar shows the number of nucleotide substitutions per site. Discussion populations, also supports the affirmation that H. stombleri and H. zea constitute the same species. In fact, phylogenies of mito- In his pioneer work, Hardwick (1965) described and illustrated the chondrial (maternal lineage) and nuclear (inherited from all ances- genitalia of Heliothinae from different continents. The article in- tors) markers were congruent in placing H. stombleri within the cluded a series of figures focusing on the most useful characters to H. zea cluster. Crosses between Heliothinae spp. have been re- discriminate one species from another. This study still serves as an ported (Laster et al. 1988), and these can yield individuals with identification guide for world-wide researchers dealing with genital malformations (Zhao et al. 2005). According to the nucleo- Heliothinae pests. However, variability is found in some cases, and tide sequences provided in this paper, however, the possibility that species determination should be based on many characters consid- H. stombleri could be the result of hybridization between species ered as a whole. seems unlikely. The present paper analyzed and documented the variability of Hardwick (1970) reported an incidence of 3.6%, 5.6% and male genitalia in H. zea individuals captured in central 15.2% of the “H. stombleri form” among H. zea captures from Argentina. Emphasis was placed on the description of the valvae Argentina, Hawaii (USA) and Brazil, respectively. The percentage de- and on the number of cornuti in the phallus. With respect to the tected during a sampling in Texas (USA) was 9.1% (Pogue 2004). latter, a non-negligible proportion of moths have been shown to The ratio of H. stombleri to the total H. zea obtained in this study (7 possess more or fewer (15–21) cornuti than the mode (18). This out of 200, i.e., 3.5%) was similar to that obtained more than four must be kept in mind when trying to differentiate H. zea from H. decades ago in the Argentinian sample (Hardwick 1970). It has to be armigera, which has a lower number of cornuti (Pogue 2004). noted that, at this time, only 28 specimens were analyzed. H. stom- Nevertheless, the supplementary diagnostic character of three bleri individuals were considered by Hardwick (1970) as “mutant lobes at the base of the inflated vesica (vs. only one in the case of phenotypes”, doubtfully fertile in any degree and not even capable of H. armigera) was observed in all H. zea samples. This fact rein- copulating. He suggested that the abnormal genitalia would be sub- forces the necessity of evaluating the morphological traits in a jected to genetic control, and not to physiological or environmental comprehensive manner. factors. The accumulated data, including those from the present Some moths displayed the unusual genitalia originally attrib- work, lead to the inference that the regular and persistent occurrence uted to a different species, H. stombleri (Okumura and Bauer of the “H. stombleri form” may represent a polymorphism. What is 1969). On the basis of morphology, Hardwick (1970) and then the origin of such anomaly, and how these forms are maintained at a Pogue (2004) concluded that H. stombleri was an abnormal form relatively stable frequency is worthy of future research. of H. zea. This question was taken up again by Nagoshi et al. (2016), who partially sequenced COI and Z-linked triosephos- phate isomerase (Tpi) genes from “normal” H. zea males and ex- Acknowledgments ternally similar Helicoverpa specimens with aberrant genital structures, collected in Florida (USA). Their results were in line We are grateful to the JIS reviewers who contributed valuable remarks that with the previous morphological deductions. The evidence pre- improved our manuscript. This work was funded by Instituto Nacional de sented here from additional genomic regions and different insect TecnologuˆUa Agropecuaria (Argentina). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/3/74/3852624 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 3 Murua , M. G., L. E. Cazado, A. Casmuz, M. I. Herrero, M. E. Villagran, A. References Cited Vera, D. R. Sosa-Gomez , and G. Gastaminza. 2016. Species from the Arneodo, J. D., E. I. Balbi, F. M. Flores, and A. Sciocco-Cap. 2015. Molecular Heliothinae complex (Lepidoptera: Noctuidae) in Tucum an, Argentina, an identiﬁcation of Helicoverpa armigera (Lepidoptera: Noctuidae: update of geographical distribution of Helicoverpa armigera. J. 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Published: May 25, 2017
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