Identification of a polymorphic ryanodine receptor gene from Heliothis virescens (Lepidoptera: noctuidae)

cDNAs encoding the C-terminal 1172 amino acids of a ryanodine receptor (RyR) from the lepidopteran pest Heliothis virescens (Hv-RyR) have been cloned and characterised. Sequence comparisons, organisational studies on corresponding genomic regions and a genetic segregation analysis provide evidence for two polymorphic alleles of the Hv-RyR locus.Comparison of the Hv-RyR C-terminal amino acid sequence with equivalent regions of other RyRs reveals a high level of overall amino acid homology (74% identity with D. melanogaster and between 47.9 and 50.1% with vertebrate isoforms). Homologies are however not uniformly distributed, though regions of high and low similarity are consistent with patterns in other RyR isoforms. The structural similarity of Hv-RyR with other RyRs is also indicated by comparison of hydropathy profiles and other previously described functional domains. Such results are consistent with this region of Hv-RyR containing the Ca(2+) channel itself and being intimately involved in RyR regulation. Potential uses of the cDNAs described in the discovery and development of novel ryanodine like insecticides are discussed.     

Behavioural responses of Heliothis virescens (Lepidoptera: Noctuidae) to stimulation with sugars

Tarsal or antennal stimulation with sugars of adult male or female Heliothis virescens results in proboscis extension. Moths are most responsive to sucrose followed by fructose and glucose. Tarsal response to glucose is higher than that of antennae while stimulation of either appendage with sucrose or fructose is similar. Moths do not respond to ribose, rhamnose and raffinose, constituents of cotton extrafloral nectar. There is also no difference in response between males and females, mated and virgin individuals, with age and during scotophase or photophase.     

Trans-ovum transmission of a cytoplasmic polyhedrosis virus of Heliothis virescens (Lepidoptera: Noctuidae)

Adults of Heliothis virescens infected with a cytoplasmic polyhedrosis virus (CPV) produced healthy offspring when their eggs were surface sterilized with either 15% formaldehyde or 0.2% sodium hypochlorite solution. Larvae from infected parents (1) cultured on a vitamin-deficient medium, (2) exposed to cold treatment (5°C, 24 hr), or (3) as progeny of adults from diapaused infected pupae, produced the same number of infected individuals as larvae reared in the customary way. Field studies indicated that the percent of CPV infection in larvae originating from virus-infected parents was density dependent.     

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Unique intertesticular tissue complex in larvae of Heliothis virescens (F.) (Lepidoptera : Noctuidae)

A unique heart-testis complex has been found in the last instar of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera : Noctuidae). This complex consists of a previously undescribed muscle system, herein designated the cardiotesticular muscle, that attaches the heart to the testes, and a ubiquitous connective tissue sheath that overlays this muscle and surrounding fat body and pericardial cells. These interconnected organs form a sieve-like septum enclosing a sinus that contains clusters of hemocytes and lipid droplets. Lipid droplets appear to be released from the surface of the testes sheath. The cardiotesticular muscle is innervated by neuroendocrine nerves that may be the route by which the brain peptide, testis ecdysiotropin, reaches the testes. The cardiotesticular muscle and products sequestered in the cardiotesticular sinus may be involved in the fusion of the paired testes and maturation of the male reproductive system, because they are present only during this stage of development.     

Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae)

Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) Heliothis virescens strains were compared. The overall pH of gut extracts from YDK and CXC were statistically similar (9.56 and 9.62, respectively), while the pH of extracts from KCBhyb and YHD2-B were significantly more alkaline (9.81 and 10.0, respectively). Gut extracts from YHD2-B and CXC larvae processed Cry1Ac and Cry2Aa protoxin slower than extracts from YDK larvae, suggesting that differences in proteolysis contribute to resistance in these strains. Casein zymogram analysis of gut extracts revealed both qualitative and quantitative differences in caseinolytic activities among all strains, but the overall caseinolytic activity of YHD2-B gut extract was lower. Kinetic microplate assays with a trypsin substrate (l-BApNA) demonstrated that proteinases in YDK gut extract had increased alkaline pH optima compared to resistant strains YHD2-B, CXC and KCBhyb. Gut extracts from YHD2-B had reduced trypsin-like activity, and activity blots indicated that YHD2-B had lost a trypsin-like proteinase activity. In assays with a chymotrypsin substrate (SAAPFpNA), enzymes from all Bt-resistant strains had increased pH optima, especially those from KCBhyb. Activity blots indicated that CXC had lost a chymotrypsin-like proteinase activity. Because serine proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.     

Effects of Serratia marcescens on the F1 generation of laboratory-reared Heliothis virescens (Lepidoptera: Noctuidae)

The effects of the bacterium Serratia marcescens (Bizio) was investigated on the F1 generation of laboratory-reared Heliothis virescens (F.). There was no difference in adult male or female longevity (i.e., parental generation) for individuals inoculated with S. marcescens as larvae (Serratia treatment) and those that were free of the bacterium (control treatment). However, the number of eggs laid and the prevalence of eclosion of eggs from Serratia treatment adults were reduced relative to control treatment adults. A very low number of F1 Serratia treatment eggs exhibited signs of infection, but a higher prevalence of mortality was observed for F1 larvae (n = 2,888) for the Serratia (3.5-4.6%) than for the control (1.1-1.5%) treatment. No S. marcescens was isolated from dead control larvae; whereas, 48 -54% of dead F1 larvae for the Serratia treatment were positive for the bacterium. However, there was no significant difference in larval weights between treatments. There were also no differences in either mortality or weight of F1 male pupae between treatments, but F1 female pupae were significantly smaller and prevalence of mortality was higher for the Serratia treatment. Serratia marcescens was not isolated from any of the control F1 pupae, but 6% of pupal cadavers for the Serratia treatment were positive for the bacterium. No S. marcescens was recovered from the meconia of any of the F1 adults (n = 2,600) regardless of treatment, and there were no differences in adult weights between treatments. Although sublethal effects of S. marcescens were detected, the impact and prevalence of the bacterium were tremendously reduced over the F1 generation in the absence of all but the most basic management strategies.     

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences

Intra- and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton- and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F(S) parameters. In addition, high values of haplotype diversity (?) and low values of nucleotide diversity (π), combined with a high number of low frequency haplotypes and values of θ(π)<θ(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.     

Cross-resistance responses of CrylAc-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein vip3A

One susceptible and three Cry1Ac-resistant strains of tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), were used in laboratory studies to determine the level of cross-resistance between the Bacillus thuringiensis (Berliner) toxins Cry1Ac and Vip3A by using concentration-mortality and leaf tissue experiments. Concentration-mortality data demonstrated that the three Cry1Ac-resistant H. virescens strains, YHD2, KCBhyb, and CxC, were at least 215- to 316-fold resistant to Cry1Ac compared with the susceptible strain, YDK. Results from Vip3A concentration-mortality tests indicated that mortality was similar among all four H. virescens strains. Relative larval growth on Cry1Ac reflected concentration-mortality test results, because YHD2 larval growth was mostly unaffected by the Cry1Ac concentrations tested. Growth ratios for KCBhyb and CXC indicated that they had a more moderate level of resistance to Cry1Ac than did YHD2. Relative larval growth on Vip3A was highly variable at lower concentrations, but it was more consistent on concentrations of Vip3A above 25 microg/ml. Differences in larval growth among strains on Vip3A were not as pronounced as seen in Cry1Ac experiments. Mortality and larval growth also was assessed in leaf tissue bioassays in which YDK, CxC, and KCBhyb neonates were placed onto leaf disks from non-Bt and Bt cotton, Gossypium hirsutum L., for 5 d. Three Bt lines were used in an initial bioassay and consisted of two Vip3A-containing lines, COT203 and COT102, and a Cry1Ac-producing line. Mortality of KCBhyb and CXC was lower than that of YDK larvae in the presence of leaf tissue from the Cry1Ac-producing line. Additionally, increased larval growth and leaf tissue consumption on Cry1Ac-containing leaf disks was observed for KCBhyb and CXC. Mortality and larval weights were similar among strains when larvae were fed leaf tissue of either non-Bt, COT203, or COT102. A subsequent leaf tissue bioassay was conducted that evaluated four cotton lines: non-Bt, Cry1Ab-expressing, Vip3A-expressing, and pyramided-toxin plants that produced both Cry1Ab and Vip3A. Mortality levels were similar among strains when fed non-Bt, Vip3A-expressing, or pyramided-toxin leaf tissues. Mortality was higher for YDK than for KCBhyb or CXC on Cry1Ab-expressing leaf tissues. No differences in larval weights were observed among strains for any genotype tested. Results of these experiments demonstrate that cross-resistance is nonexistent between CrylAc and Vip3A in H. virescens. Thus, the introduction of Vip3A-producing lines could delay Cry1Ac-resistance evolution in H. virescens, if these lines gain a significant share of the market.     

Physiology and glomerular projections of olfactory receptor neurons on the antenna of female Heliothis virescens (Lepidoptera: Noctuidae) responsive to behaviorally relevant odors

The neurophysiology and antennal lobe projections of olfactory receptor neurons housed within short trichoid sensilla of female Heliothis virescens F. (Noctuidae: Lepidoptera) were investigated using a combination of cut-sensillum recording and cobalt-lysine staining techniques. Behaviorally relevant odorants, including intra- and inter-sexual pheromonal compounds, plant and floral volatiles were selected for testing sensillar responses. A total of 184 sensilla were categorized into 25 possible sensillar types based on odor responses and sensitivity. Sensilla exhibited both narrow (responding to few odors) and broad (responding to many odors) response spectra. Sixty-six percent of the sensilla identified were stimulated by conspecific odors; in particular, major components of the male H. virescens hairpencil pheromone (hexadecanyl acetate and octadecanyl acetate) and a minor component of the female sex pheromone, (Z)-9-tetradecenal. Following characterization of the responses, olfactory receptor neurons within individual sensilla were stained with cobalt lysine (N=39) and traced to individual glomeruli in the antennal lobe. Olfactory receptor neurons with specific responses to (Z)-9-tetradecenal, a female H. virescens sex pheromone component, projected to the female-specific central large female glomerulus (cLFG) and other glomeruli. Terminal arborizations from sensillar types containing olfactory receptor neurons sensitive to male hairpencil components and plant volatiles were also localized to distinct glomerular locations. This information provides insight into the representation of behaviorally relevant odorants in the female moth olfactory system. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Prothoracic gland inactivation in Heliothis virescens (F.) (Lepidoptera:Noctuidae) larvae parasitized by Cardiochiles nigriceps Viereck (Hymenoptera:Braconidae)

Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. nigriceps genome, which, in contrast, are undetectable in H. virescens.     

Susceptibilities of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) populations to Cry1Ac insecticidal protein

Susceptibilities of bollworm, Helicoverpa zea (Boddie) and tobacco budworm, Heliothis virescens (F.) to Cry1Ac were measured via a diet-incorporated assay with MPV II at the University of Arkansas during 2002-2004. Lethal concentration-mortality (LC50) estimates of five laboratory, seven laboratory-cross, and 10 field populations of H. virescens varied 12-fold. Pooled susceptibilities of H. virescens across all laboratory and field populations varied five-fold. The LC50 estimates for H. virescens were higher than those reported by previous research before the introduction of transgenic crops. However, the ratio of susceptibility of laboratory and field populations was similar, suggesting no change in overall species susceptibility. Individual LC50 estimates of five laboratory, nine laboratory-cross, and 57 field populations of H. zea varied over 130-fold. Pooled susceptibilities across laboratory and field populations varied widely. Among the field populations, colonies from non-Bacillus thuringiensis (Bt) crops were generally more susceptible than those from Bt crops. Across the Bt crops expressing Cry protein, colonies from Bollgard (Monsanto Company) cotton had lower susceptibility to CrylAc than those from Bt corn and those from non-Bt crops.     

Long-term seasonal abundance patterns of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) in the Texas High Plains

Bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), male adult (moth) activities were monitored between 1982 and 1995 by using sex pheromone traps in the Texas High Plains. Moths were monitored weekly from early March to mid-November near Lubbock and Halfway, two prominent cotton production areas in the Texas High Plains region. Based on trap captures, the bollworm-budworm complex consisted of approximately 98% bollworms and approximately 2% tobacco budworms. Seasonal activity patterns varied between location for bollworm but not for tobacco budworm. The 14-yr average (+/- SE) bollworm moth abundance (moths per trap per week) at Lubbock was significantly higher (226.5 +/- 10.4) compared with that at Halfway (153.7 +/- 8.1). Correlation analyses showed a significant positive relationship between moth abundance and average weekly temperatures, whereas a significant negative relationship was observed between moth abundance and average weekly wind velocity for both species. Analyses also showed a positive correlation between moth abundance and cumulative degree-days (> 0.0 degrees C) from 1 January. A strong positive relationship was observed between moth abundance and weekly average precipitation for both species. Average weekly abundances were positively correlated between adjacent months during most of the active cotton fruiting season (June-September). However, the relationship between populations that contributed to the overwintering generation and the following spring populations varied between species and study sites. Nevertheless, data from this study indicated that late-season moth catches could be indicative of the dynamics of the early-season moth catches the following year in the High Plains. The mean population abundance curve based on 14-yr averages showed two bollworm population peaks at Lubbock, but only one peak at Halfway. Separate degree-day-based models were developed to describe long-term seasonal abundance patterns of bollworm moths for the Lubbock and Halfway sites.     

The role of heliothine hairpencil compounds in female Heliothis virescens (Lepidoptera: Noctuidae) behavior and mate acceptance

Studies on numerous insect species suggest that male-produced sex pheromones play a role in attracting females; as aphrodisiacs, making females more quiescent; or as a means of inhibiting competing males. Male heliothine moths display abdominal hairpencils during courtship, but the specific effects of the odors released on female behavior have not yet been elucidated. This study investigates the role of male hairpencil compounds in female Heliothis virescens mating behavior. Female H. virescens were exposed to filter paper loaded with hairpencil extracts of male H. virescens, Heliothis subflexa and Helicoverpa zea, and observed for behavioral responses to odors. Single synthetic compounds found in the H. virescens hairpencil blend were also tested. In mating assays between single male and female H. virescens it was found that: (i) antennectomized females mated less frequently than sham-operated females; (ii) females mated less frequently with males whose hairpencils had been surgically removed; (iii) females mated with males with ablated hairpencils if a filter paper loaded with one male equivalent of H. virescens hairpencil extract was presented simultaneously; and (iv) this effect was species-specific, as presentation of H. subflexa or H. zea hairpencil extracts did not restore mate acceptance. This study suggests that odors released by male hairpencils are important in mate acceptance by female H. virescens, and may play a role in mate choice and species isolation.     

Effects of Heliothis virescens ascovirus (HvAV-3e) on a novel host, Crocidolomia pavonana (Lepidoptera: Crambidae)

Ascoviruses are double-stranded DNA viruses which cause fatal disease in lepidopteran host larvae. They induce a unique pathology, causing cleavage of host cells into virion-containing vesicles. With the single exception of Diadromus pulchellus ascovirus, all ascoviruses have been exclusively reported from the Noctuidae. To investigate whether Heliothis virescens AV (HvAV-3e) has a broader host range at the family level, larvae of Crocidolomia pavonana F. (Lepidoptera: Crambidae), a major pest of brassica crops in tropical and sub-tropical regions of the Old World and Australasia, were inoculated with HvAV-3e. Larvae were readily infected by the ascovirus and feeding, growth and survival were significantly affected. However, the milky white discolouration of the haemolymph which is characteristic of ascovirus infection in noctuid hosts was not apparent. In further contrast to infected noctuid host larvae that do not develop to the pupal stage, a significant proportion of infected C. pavonana larvae pupated but all were killed at this stage. Thus, C. pavonana appears to be a semi-permissive host of the ascovirus, the presence of such hosts in the field might be an explanation for the conundrum for the ascovirus-noctuid-wasp relationship, helping explain the persistence of the ascovirus.     

Effect of temperature on the fitness of a Vip3A resistant population of Heliothis virescens (Lepidoptera: Noctuidae)

Microbial insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) have become increasingly important for pest management. In addition to crystal (Cry) insecticidal protein toxins formed during sporulation, vegetative insecticidal protein (Vip) toxins can be produced during the vegetative phase. Resistance to Cry toxins has been reported in laboratory- and field-selected populations of various Lepidoptera species and several studies have identified fitness costs associated with Cry toxin resistance. Here, fitness costs are examined in the first insect population to be reported with resistance to a Vip toxin, a laboratory-selected Vip3A-resistant subpopulation of the tobacco budworm, Heliothis virescens (L.) (Vip-Sel). The Vip-Sel population showed reduced survival to adult eclosion compared with an unselected subpopulation at all test temperatures, including the culture temperature (25 degrees C). Vip3A resistance was also associated with reduced egg viability and mating success and a lower intrinsic rate of population increase (r(m)) at temperatures below (20 degrees C) and above (30 degrees C) the optimal laboratory culture temperature. The latter findings agree with previous studies, that fitness costs associated with resistance are usually greater under stressful conditions. Such data can help predict the impact of fitness costs on the rate of development of resistance in the field and in the development of resistance management strategies that more fully exploit fitness costs.     

Overwintering of Heliothis virescens (F.) and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in cotton fields of northeast Mississippi

Following the 1995-2001 crop seasons, population densities of pupae of tobacco budworm, Heliothis virescens (F.), and corn earworm, Helicoverpa zea (Boddie), overwintering in cotton fields, Gossypium hirsutum L., in Monroe County, MS, were estimated by digging a total of 43,542 m-row. Densities of pupae varied greatly among years with the highest densities occurring in years with low rainfall in the late summer and early fall. The sex ratio of pupae was variable and significantly male-biased in some years. The total area-wide, tobacco budworm population emerging from all overwintering sites was estimated from catches in pheromone traps whose absolute efficiency is known. After the introduction of Bacillus thuringiensis Berliner (Bt)-transgenic cotton in 1996 but before a several-fold increase in use of herbicide resistant (HR)-transgenic cotton cultivars in 2001, local cotton fields accounted for <2% of the total overwintered tobacco budworm population. In the pretransgenic cotton production era, it is estimated cotton fields typically accounted for <10% of the overwintered tobacco budworm population. Driving surveys of the tillage status of cotton fields and pheromone trapping to determine the timing of adult emergence in the spring indicate that--until the 2000 crop season--postseason tillage destroyed most tobacco budworm and corn earworm pupae overwintering in cotton fields. Adoption of HR-transgenic cultivars has quintupled cotton field area planted no-till, but the contribution of cotton fields to overwintered tobacco budworm populations has remained low. Preemptive tillage of cotton fields to kill overwintering tobacco budworm pupae would not appreciably suppress in-season population densities of this pest. However, the absence of such tillage might increase the rate at which the tobacco budworm develops resistance to insecticides and counteradaptation to antibiotic host plant resistance traits including the Bt-transgenic trait.