Joel D. Arneodo

Molecular Identification of Helicoverpa armigera (Lepidoptera: Noctuidae: Heliothinae) in Argentina and Development of a Novel PCR-RFLP Method for its Rapid Differentiation From H. zea and H. gelotopoeon

ABSTRACT Helicoverpa armigera (Hu¨ bner) (Lepidoptera: Noctuidae: Heliothinae) is among the most voracious global pests of agriculture. Adults of this species were identified recently in northern Argentina by dissection of male genitalia. In this work, a rapid and simple molecular tool was designed to distinguish H. armigera from the morphologically similar indigenous bollworms Helicoverpa zea (Boddie) and Helicoverpa gelotopoeon (Dyar), regardless of the life stage. Amplification of partial COI gene with a new primer pair, and subsequent digestion with endonuclease HinfI, yielded different RFLP profiles for the three main Helicoverpa pests currently present in South America. The method was validated in Helicoverpa specimens collected across Argentina, whose identity was further corroborated by COI sequencing and phylogenetic analysis. The data reported here constitute the first molecular confirmation of this pest in the country. The survey revealed the occurrence of H. armigera in northern and central Argentina, including the main soybean- and maize-producing area.

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites.

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.

Light microscopy of microsporidian spores and baculovirus occlusion bodies in Epinotia aporema and Helicoverpa gelotopoeon larval tissues, respectively

Entomopathogens help regulate natural insect populations, and their use in pest management is the subject of active research. Classical and molecular techniques are currently available to elucidate disease etiology and pathogenesis. Here, we performed an Azan trichrome stain for light microscopic visualization of pathogens in the larval tissues of two Neotropical lepidopterans. Epinotia aporema larvae exhibiting symptoms of microsporidiosis were fortuitously detected in a rearing facility (IMyZA-INTA, Argentina), while Helicoverpa gelotopoeon specimens were experimentally infected with an Argentinean isolate of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). Caterpillars were fixed in Duboscq-Brasil and paraffin embedded. Microtome sections (5 m-thick) were deparaffinized and stained for observation under a Nikon Eclipse 80i microscope equipped with a Nikon DS-U3 digital camera.

Lignocellulose Degradation by Termites

Host and symbiont enzymes are involved in lignocellulose processing by termites. A brief description of the structure of the main components of the plant cell wall and the most relevant degrading enzymes is presented. This chapter focuses on the dual cellulolytic system in lower and higher termites and provides an update on the current research strategies through culture-dependent and culture-independent “-omic” approaches. Significance for biofuel production and future perspectives are also discussed.

First Report of Chrysodeixis includens Nucleopolyhedrovirus (ChinNPV) Infecting Chrysodeixis includens (Lepidoptera: Noctuidae) in Argentina

Summary Typical baculovirus infection symptoms were observed in Chrysodeixis includens Walker (Lepidoptera: Noctuidae) larvae from a laboratory rearing in Tucumán, Argentina. Scanning electron microscopy revealed the occurrence of polyhedral occlusion bodies displaying a heterogeneous morphology. Amplification, sequencing, and phylogenetic analysis of conserved polyhedrin, lef-8 and lef-9 genes identified the pathogen, for the first time in Argentina, as a variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV). The biocontrol potential of this new isolate is worthy of future research.

Further Description of Helicoverpa zea (Lepidoptera: Noctuidae) Male Genitalia and New Genetic Evidence of Synonymy With Respect to the Anomalous Form, “Heliothis stombleri”

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 = 200) captured in central Argentina was carried out in order to provide a detailed description of the valvae 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 showing 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 assumption 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.