A host-plant specialist, Helicoverpa assulta, is more tolerant to capsaicin from Capsicum annuum than other noctuid species

Plant secondary compounds not only play an important role in plant defense, but have been a driving force for host adaptation by herbivores. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), an alkaloid found in the fruit of Capsicum spp. (Solanaceae), is responsible for the pungency of hot pepper fruits and is unique to the genus. The oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), is a specialist herbivore feeding on solanaceous plants including Capsicum annuum, and is one of a very few insect herbivores worldwide capable of feeding on hot pepper fruits. To determine whether this is due in part to an increased physiological tolerance of capsaicin, we compared H. assulta with another specialist on Solanaceae, Heliothis subflexa, and four generalist species, Spodoptera frugiperda, Heliothis virescens, Helicoverpa armigera, and Helicoverpa zea, all belonging to the family Noctuidae. When larvae were fed capsaicin-spiked artificial diet for the entire larval period, larval mortality increased in H. subflexa and H. zea but decreased in H. assulta. Larval growth decreased on the capsaicin-spiked diet in four of the species, was unaffected in H. armigera and increased in H. assulta. Food consumption and utilization experiments showed that capsaicin decreased relative consumption rate (RCR), relative growth rate (RGR) and approximate digestibility (AD) in H. zea, and increased AD and the efficiency of conversion of ingested food (ECI) in H. armigera; whereas it did not significantly change any of these nutritional indices in H. assulta. The acute toxicity of capsaicin measured by injection into early fifth instar larvae was less in H. assulta than in H. armigera and H. zea. Injection of high concentrations produced abdominal paralysis and self-cannibalism. Injection of sub-lethal doses of capsaicin resulted in reduced pupal weights in H. armigera and H. zea, but not in H. assulta. The results indicate that H. assulta is more tolerant to capsaicin than the other insects tested, suggesting that this has facilitated expansion of its host range within Solanaceae to Capsicum after introduction of the latter to the Old World about 500 years ago. The increased larval survival and growth due to chronic dietary exposure to capsaicin suggests further adaptation of H. assulta to that compound, the mechanisms of which remain to be investigated.     

Discovery of novel trimethylalkanes in the internal hydrocarbons of developing pupae of Heliothis virescens and Helicoverpa zea

Novel trimethyl-branched alkanes which eluted with the monomethylalkanes were identified in the internal lipids of Helicoverpa zea but were not present in Heliothis virescens. Their structures were unique in that the first methyl branch occurred on carbon 2 and the 2nd and 3rd methyl branch points were separated by a single methylene. Novel trimethylalkanes identified from their chemical ionization and electron impact mass spectra were 2,18,20-trimethyltetratriacontane, 2,18,20-trimethylhexatriacontane, and 2,24,26-trimethyldotetracontane. Previous reports did not find these trimethylalkanes in the cuticular surface lipids of larvae, pupae or adults of either species. The internal pupal hydrocarbons of H. virescens and H. zea amounted to 123 μg and 304 μg per pupa, respectively. They consisted of n-alkanes (8 and 4%, respectively) and methyl-branched alkanes (88 and 94%, respectively). The n-alkanes ranged in chain length from approximately 21 to 35 carbons and the methyl-branched alkanes from approximately 26 to 55 carbons vs. methyl-branched alkanes from 28 to 37 carbons previously reported for hydrocarbons from the pupal cuticular surface. The major n-alkane was heptacosane (3.3 and 1.2%, respectively, in H. virescens and H. zea). The major methyl-branched alkanes in H. virescens were methylhentriacontane (15%), methyltritriacontane (12%) and dimethyltritriacontane (10%), and in H. zea were methylnonacosane (17%), dimethylnonacosane (9%) and methylhentriacontane (20%). Except for the novel trimethylalkanes, the methylalkane branch points were predominantly on odd-numbered carbons as has been reported for these and other species.     

Role of the exoprotease InA in the pathogenicity of Bacillus thuringiensis in pupae of Hyalophora cecropia

Two geographical biotypes of Nomuraea rileyi (from Ecuador and the United States) were topically bioassayed against seven lepidopteran species, i.e., Anticarsia gemmatalis, Heliothis zea, Heliothis virescens, Heliothis subflexa, Pseudoplusia includens, Spodoptera exigua, and Trichoplusia ni. There was an average difference of 1.7-fold in mortality in how cultures of the same insect species from different sources responded to topical applications of either biotype of N. rileyi. Regression equations and LC₅₀ values were obtained for each insect species and fungal biotype combination. Larvae of S. exigua were equally susceptible to both biotypes of N. rileyi. Although larvae of A. gemmatalis were moderately susceptible to the Ecuadoran biotype, they were relatively nonsusceptible to the Mississippian biotype. Species of Heliothis (H. zea, H. virescens, and H. subflexa) were about equally susceptible to the Mississippian biotype. Larvae of H. subflexa and H. virescens, however, were significantly less susceptible than H. zea to the Ecuadoran biotype. When the integumental barrier was breached via intrahemocoelic injections, larvae of H. virescens were as susceptible as H. zea larvae to blastospores of either biotype of N. rileyi.     

The surface hydrocarbons of larval Heliothis virescens and Helicoverpa zea

Third instar larvae of Heliothis virescens and Helicoverpa zea could be distinguished based on the hydrocarbons of their surface lipids. Hydrocarbons were the major components of the surface lipids and a distinctive capillary gas chromatographic profile could be obtained from a hexane extract of the surface lipids of a single larva. Analysis of hexane extracts of the surface lipids by capillary gas chromatography-mass spectrometry (CGC-MS) showed several obvious differences between the two species: (1) in their gas chromatographic profiles; (2) in the presence of a major alkene, hentriacontene, only in H. zea; (3) in H. virescens the CGC-MS peak with a KI of approximately 2860 was 2-methyloctacosane, but in H. zea it was a mixture of 4-methyloctacosane plus 9,13- and 8,12-dimethyloctacosanes; and (4) in the methyl branch positions of dimethyl-branched alkanes with carbon backbones of C₃₁, C₃₃, C₃₅, C₄₅, C₄₇, C₄₉ and C₅₁. The methyl branch points of H. virescens dimethylalkanes were separated by seven or nine methylenes, while in H. zea the methyl branch points of the dimethylalkanes were separated by three and sometimes five methylenes.     

THE GENETIC ARCHITECTURE OF A COMPLEX ECOLOGICAL TRAIT: HOST PLANT USE IN THE SPECIALIST MOTH,HELIOTHIS SUBFLEXA

We used genetic mapping to examine the genetic architecture of differences in host plant use between two species of noctuid moths, Heliothis subflexa, a specialist on Physalis spp., and its close relative, the broad generalist H. virescens. We introgressed H. subflexa chromosomes into the H. virescens background and analyzed 1462 backcross insects. The effects of H. subflexa‐origin chromosomes were small when measured as the percent variation explained in backcross populations (0.2–5%), but were larger when considered in relation to the interspecific difference explained (1.5–165%). Most significant chromosomes had effects on more than one trait, and their effects varied between years, sexes, and genetic backgrounds. Different chromosomes could produce similar phenotypes, suggesting that the same trait might be controlled by different chromosomes in different backcross populations. It appears that many loci of small effect contribute to the use of Physalis by H. subflexa. We hypothesize that behavioral changes may have paved the way for physiological adaptation to Physalis by the generalist ancestor of H. subflexa and H. virescens.     

Mitochondrial DNA analysis of field populations of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) and of its relationship to <Emphasis Type="Italic">H. zea</Emphasis>

Background  

Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.     

Susceptibility of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) to Vip3A insecticidal protein expressed in VipCot™ cotton

Susceptibility of laboratory and field colonies of Helicoverpa zea (Boddie) and Heliothis virescens F. to Vip3A insecticidal protein was studied in diet incorporation and diet overlay assays from 2004 to 2008. Responses of field populations were compared to paired responses of University of Arkansas laboratory susceptible H. zea (LabZA) and H. virescens (LabVR) colonies. After 7 d of exposure, observations were made on number of dead larvae (M) and the number of larvae alive but remaining as first instars (L1). Regression estimates using M (LC₅₀) and M plus L1 (MIC₅₀) data were developed for laboratory and field populations. Susceptibility of laboratory and field populations exposed to Vip3A varied among different batches of protein used over the study period. Within the same batch of Vip3A protein, susceptibilities of laboratory colonies of both species (LabZA and LabVR) were similar. Field colonies were significantly more susceptible to Vip3A than the respective reference colonies of both species. Within field populations, susceptibility to Vip3A varied up to 75-fold in H. zea and 132-fold in H. virescens in LC₅₀ estimates. Variabilities in MIC₅₀s were up to 59- and 11-fold for H. zea and H. virescens, respectively.     

Resistance to insecticides in Heliothine Lepidoptera: a global view

The status of resistance to organophosphate, carbamate, cyclodiene and pyrethroid insecticides in the heliothine Lepidoptera is reviewed. In particular, resistance in the tobacco budworm, Heliothis virescens, and the corn earworm, Helicoverpa zea, from the New World, and the cotton bollworm, Helicoverpa armigera, from the Old World, are considered in detail. Particular emphasis has been placed on resistance to the most widely used of these insecticide groups, the pyrethroids. In each case, the incidence and current status of resistance are considered before a detailed view of the mechanisms of resistance is given. Controversial issues regarding the nature of mechanisms of resistance to pyrethroid insecticides are discussed. The implications for resistance management are considered.     

Populations of Predaceous Natural Enemies Developing on Insect-Resistant and Susceptible Tomato in North Carolina

Naturally occurring populations of immature and adultGeocoris punctipes,adultColeomegilla maculataand immature coccinellids were monitored on field-grown tomato lines susceptible and resistant toManduca sextaandHelicoverpa zea. Helicoverpa zeaandHeliothis virescenseggs and small larvae that serve as prey for these predators also were monitored. MoreH. zeaandH. virescenseggs and small larvae were found on resistant than on susceptible plant lines. However, similar populations of largeH. zeaandH. virescenslarvae were found on resistant and susceptible plants. The number of adultGeocoris punctipes,adultColeomegilla maculataand immature coccinellids on resistant plants was always as high or higher than the number on susceptible plants. The data demonstrate no incompatibility of host-plant resistance with biological control provided by these predaceous insects, but indicate that the number ofG. punctipesand coccinellids required to provide effective biological control may develop too late in the season to be of practical value. Large populations of stilt bugs (Jalysus wickhami,Hemiptera: Berytidae) and spiders were observed to occur earlier in the growing season than eitherG. punctipesor coccinellids and may be a significant source of mortality forH. zeaeggs and small larvae.     

Behavioural and chemical mechanisms of plant‐mediated deterrence and attraction among frugivorous insects

1. Herbivory often induces systemic plant responses that affect the host choice of subsequent herbivores, either deterring or attracting them, with implications for the performance of both herbivore and host plant. Combining measures of herbivore movement and consumption can efficiently provide insights into the induced plant responses that are most important for determining choice behaviour. 2. The preferences of two frugivorous stink bug species, Nezara viridula and Euschistus servus between cotton plants left undamaged or damaged by Helicoverpa zea and Heliothis virescens larvae were investigated. A novel consumer movement model was used to investigate if attraction rates or leaving rates determined preferences. Stink bug consumption rates were measured using salivary sheath flanges. Finally, the systemic induction of selected phenolic‐based and terpenoid secondary metabolites were measured from heliothine herbivory on developing cotton bolls, to investigate if they explained stink bug feeding responses. 3. Heliothine herbivory did not affect the N. viridula feeding preference. However, we found opposing effects of H. zea and H. virescens herbivory on the behaviour of E. servus. Avoidance of H. zea‐damaged plants is not obviously related to phenolic or terpenoid induction in cotton bolls; whereas a preference for H. virescens‐damaged plants may be related to reductions in chlorogenic acid in boll carpel walls. 4. The present results highlight the inferential power of measuring both consumer movement and consumption in preference experiments and combining behavioural responses with phytochemical responses. Furthermore, while plant‐mediated interactions among herbivorous insects are well studied, interactions among frugivorous species specifically have been poorly documented.     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

Responses of male Helicoverpa zea to single pulses of sex pheromone and behavioural antagonist

Male Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) flying in a pheromone plume respond to the loss of pheromone when they fly into a large pocket of clean air by going into crosswind casting flight in a mean of 0.48 s; 0.62 s after re‐contacting pheromone presented as a single pulse, they surge upwind in a kind of narrow zigzagging flight. After 0.36 s of surging, they lapse into casting flight once again in the clean air following the pulse. The addition of a known behavioural antagonist (Z)‐11‐hexadecenyl acetate (Z11–16:Ac), to the pheromone significantly increases the mean latency of the response to a single pulse to 0.85 s. No other aspects of the surge were significantly changed by the presence of antagonist in the single pulse of pheromone. Thus, unlike males of the related species, Heliothis virescens, which show significant changes in track and course angles when antagonist is present in single pulses, only an increased latency of response to a filament containing antagonist occurred in H. zea males. The increased latency could act cumulatively when the male is exposed rapidly and repeatedly to filaments in a natural plume and explain the profound arrestment effect of the antagonist in such plumes. The latencies to casting and surging in response to a pulse of pheromone blend are longer than those of the smaller species, H. virescens, and may be due to size‐related differences in manoeuverability of H. zea vs. H. virescens.     

The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components

Primary alcohols varying in chain length from C₁₃ to C₁₆, and in number, position, and geometric configuration of double bonds, were applied in dimethyl sulfoxide to the surface of the female sex pheromone glands of Heliothis subflexa (Gn.) and Hydraecia micacea (Esper). Capillary gas chromatographic analysis of extracts of the treated glands indicated that the alcohols were converted to the corresponding aldehydes by H. subflexa females and to the acetates by H. micacea females. Conversions of the alcohols showed no preferences for molecular weight, number, position, or geometry of the double bonds in either species. Application of the acetates of the primary alcohols to the gland surface of H. subflexa females resulted in the production of both the corresponding alcohols and aldehydes, while neither alcohols nor aldheydes were produced when acetates were applied to the glands of H. micacea. In addition, application of the acetates to the gland surface of Heliothis virescens (F.) resulted in the production of both the corresponding alcohols and aldehydes. However, no evidence was found to indicate that acetates are ever produced by the pheromone gland of females of H. virescens.     

Microsatellite variation in Helicoverpa gelotopoeon (Lepidoptera: Noctuidae) populations from Argentina

1. Helicoverpa gelotopoeon (Dyar) (Lepidoptera: Noctuidae) is a polyphagous pest that has been reported causing damage in many agricultural crops such as soybean, chickpea and cotton. This species, together with Helicoverpa zea (Boddie), Helicoverpa armigera (Hübner) and Chloridea virescens (Fabricius), belongs to the Heliothinae complex of economic importance in Northwestern Argentina.
2. Many species in the Heliothinae subfamily have developed resistance to insecticides and insecticidal proteins from Bacillus thuringiensis (Bt). Managing resistance to these control tactics in H. gelotopoeon is of high importance and dependant on knowledge of population genetics of this species.
3. The objective of this study was to evaluate the genetic variability and structure of H. gelotopoeon populations from different host plants and geographic regions of Argentina. Towards this goal, six microsatellite loci were screened across 196 individuals.
4. The results obtained revealed high genetic diversity and lack of clear structure among the populations of H. gelotopoeon studied. This genetic homogeneity between populations from different geographical regions and hosts could be attributed to the long-range migratory capacity of Heliothinae and the generalist behaviour of H. gelotopoeon.

     

Immunological Detection of Hymenopteran Parasitism inHelicoverpa zeaandHeliothis virescens

We have developed a monoclonal antibody that recognizes the larval and adult stages ofMicroplitis croceipes(Cresson) (Hymenoptera: Braconidae) andCotesia marginiventris(Cresson) (Hymenoptera: Braconidae), two principal endoparasitoids of the polyphagous pestsHelicoverpa zea(Boddie) (Lepidoptera: Noctuidae) andHeliothis virescens(F.) (Lepidoptera: Noctuidae). The antibody has been incorporated into an indirect enzyme-linked immunosorbent assay that can be used to distinguish parasitized from unparasitized pests. The antibody cross-reacts with several different hymenopteran parasitoids, but not with any of the noctuid pests we have assayed. An immunoassay based on a broadly reactive antibody such as this one enables comprehensive detection of hymenopteran parasitism with a single antibody reagent.     

Effects of cuticle source and concentration on germination of conidia of two isolates of Nomuraea rileyi

The effects of cuticle from larvae of Trichoplusia ni, Heliothis zea and H. virescens on rate and extent of germination of conidia of a Mississippian isolate (MS) and an Ecuadoran (EC) isolate of Nomuraea rileyi were studied. Solid substrates generally stimulated more germination than submerged substrates. There was little or no effect of cuticle source (H. zea or H. virescens) on germination of either the EC isolate or the MS isolate cultured on a solid substrate, however, differences in patterns of germination were obtained in submerged substrates. Addition of cuticle of H. zea or H. virescens generally increased the germination time for the MS isolate. Germination time for the EC isolate was significantly increased when H. virescens cuticle was used.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by U.S. Department of Agriculture.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

Identification of mariner‐like elements belonging to the cecropia subfamily in two closely related Helicoverpa species

Abstract Mariner transposons are widespread in eukaryote genomes and have been used as transposon vectors in insect transgenesis. We examined two closely related Helicoverpa species, the cotton bollworm Helicoverpa armigera and corn earworm Helicoverpa zea, for the presence of mariner‐like elements (MLEs). Multiple copies of two distinct MLEs, Hamar1 and Hamar2, were isolated in H. armigera, and a MLE showing a high degree of conservation to Hamar1 was detected in H. zea and was named Hzmar1. These MLEs belong to the cecropia subfamily, containing indels in the transposase coding region. Sequence analysis indicated the earlier invasion of Hamar1 and relatively recent activity of Hamar2.     

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.     

Population structure and gene flow in the global pest,Helicoverpa armigera

Helicoverpa armigera is a major agricultural pest that is distributed across Europe, Asia, Africa and Australasia. This species is hypothesized to have spread to the Americas 1.5 million years ago, founding a population that is at present, a distinct species, Helicoverpa zea. In 2013, H. armigera was confirmed to have re‐entered South America via Brazil and subsequently spread. The source of the recent incursion is unknown and population structure in H. armigera is poorly resolved, but a basic understanding would highlight potential biosecurity failures and determine the recent evolutionary history of region‐specific lineages. Here, we integrate several end points derived from high‐throughput sequencing to assess gene flow in H. armigera and H. zea from populations across six continents. We first assemble mitochondrial genomes to demonstrate the phylogenetic relationship of H. armigera with other Heliothine species and the lack of distinction between populations. We subsequently use de novo genotyping‐by‐sequencing and whole‐genome sequences aligned to bacterial artificial chromosomes, to assess levels of admixture. Primarily, we find that Brazilian H. armigera are derived from diverse source populations, with strong signals of gene flow from European populations, as well as prevalent signals of Asian and African ancestry. We also demonstrate a potential field‐caught hybrid between H. armigera and H. zea, and are able to provide genomic support for the presence of the H. armigera conferta subspecies in Australasia. While structure among the bulk of populations remains unresolved, we present distinctions that are pertinent to future investigations as well as to the biosecurity threat posed by H. armigera.