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.     

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.     

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.     

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.     

Effects of individual Bacillus thuringiensis insecticidal crystal proteins on adult Heliothis virescens (F.) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Bacillus thuringiensis (Bt) is a grampositive, spore forming bacterium, which is principally distinguishedfrom other bacilli by the production of large, insecticidal,protein crystals (Insecticidal Crystal Proteins, or ICPs). Theseproteins are usually thought to act only on the actively feedinglarvae of susceptible species by a mechanism which involvesconsumption and proteolytic processing of the protein followed bybinding to, and lysis of, midgut epithelial cells. However, few authorshave reported Bt toxicity to adult insects. In the followingpaper, we expand on previous reports of toxicity to adult insects andpresent data which demonstrate that: (1) proteolytically activated ICPssignificantly reduce the lifespans of adult Heliothis virescensand Spodoptera exigua at concentrations of 500 g/ml, butnot 167 or 25 g/ml, (2) individual activated ICPs are differentiallytoxic to adult H. virescens and S. exigua, and (3)adult S. exigua are sensitive to Cry1C protoxin at aconcentration of 1 mg/ml.Deceased     

Survival and development of Heliothis virescens (Lepidoptera: Noctuidae) larvae on isogenic tobacco lines with different levels of alkaloids

Levels of pyridine alkaloids were measured in 18 tobacco, Nicotiana tabacum L., entries from three parental isolines ('NC 95', 'SC 58', and 'Coker 139'), grown at Tifton, GA, Florence, SC, and Oxford, NC, in 1991. Levels of alkaloids in bud leaves (first fully unfolded leaf below the apical leaf bud) were negatively correlated to natural infestation ratings of tobacco budworm larvae, Heliothis virescens (F.), 7 wk after transplanting. For artificially infested bud leaves at Oxford, there was a significant negative correlation between levels of total alkaloids and larval weights after 1 wk of feeding. In 1992, four entries from the 'NC 95' isoline were grown at Oxford, and samples for alkaloid analyses were taken every 2 wk at several leaf positions on each plant. During weeks 4, 8, 12, and 16, second instar tobacco budworms were caged on individual, intact leaves inside perforated plastic bags in the field. The survival and development of tobacco budworm larvae after 1 wk were negatively correlated with levels of alkaloids at the various leaf positions. Larvae survived better and grew faster on the bud leaves of each entry where alkaloid levels were lower than they did on leaves further down the stalk where alkaloid levels were higher. More larvae survived on the lower leaves of the low alkaloid lines than on the lower leaves of the high alkaloid lines. Even moderate increases in pyridine alkaloids had negative effects on tobacco budworm survival and development. Nicotine constituted >97% of the pyridine alkaloids in the 'NC95' isoline each year.     

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.     

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.     

Host regulation effects on Heliothis virescens (F.) larvae inducd by teratocytes of Cardiochiles nigriceps Viereck (Lepidoptera,Noctuidae-Hymenoptera,Braconidae)

Teratocytes deriving from the serosal membrane of Cardiochiles nigriceps Viereck, obtained “in vitro” from embryos hatched on a semidefined medium, were injected at different numbers and in different developmental stages of nonparasitized Heliothis virescens (F.) last instar larvae. Host development was affected by teratocyte injections and the responses registered ranged from normal to complete inhibition of pupation, according to the number of teratocytes injected and the developmental stage of the larva at time of injection. Complete pupation failure was observed when teratocytes derived from 4C nigriceps embryos were injected into 1st day 5th instar (new-slender stage) host larvae. Complete pupation occurred when teratocytes from 2 embryos were injected into 3rd or 4th day 5th instars (burrow-digging or day 1 cell formation stage). Intermediate responses, such as the formation of pupal cuticle without ecdysis or with only partial ecdysis, were obtained with intermediate teratocyte numbers, or host developmental stages. All pupae derived from teratocyte injected larvae failed to develop into adults normally obtained from control injected larvae. The larval weight just before pupation was negatively affected only when teratocyte injections were performed on 1st day 5th instar H. virescens larvae. Teratocyte injections altered the hemolymph protein titer to a level similar to that occurring in parasitized larvae. At the same time the ecdysteroid titer was characterized by a late significant increase, which reached values almost 3 times greater than found in normally parasitized larvae, and also surpassed the highest values registered for nonparasitized larvae. Ligation of parasitized larvae between the meso- and metathorax demonstrated that when the prothoracic glands were excluded, there was almost no ecdysteroid production posterior to the ligation. Ligations performed on parasitized larvae to isolate parasitoid eggs before hatching in the last abdominal segments, demonstrated that only virus and venom determined a reduction of the ecdysteroid titer. On the basis of these results the possible role of teratocytes in affecting the biological activity of ecdysteroids is postulated and discussed in a wider context of host-parasitoid physiological interactions.     

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.     

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.     

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.     

Genetic basis of resistance to Cry1Ac and Cry2Aa in Heliothis virescens (Lepidoptera: Noctuidae)

The development of pest resistance to transgenic crop plants producing insecticidal toxins from Bacillus thuringiensis Berliner (Bt) poses a major threat to their sustainable use in agriculture. "Pyramiding" two toxins with different modes of actions in the same plant is now being used to delay the evolution of resistance in the insects, but this strategy could fail if a single gene in a pest confers resistance to both toxins. The CP73 strain of the cotton pest Heliothis virescens (F.) is resistant to both Cry1Ac and Cry2Aa toxins from Bt. We explored the genetic basis of resistance in this strain with a backcross, split-family design. The gene with the largest effect on Cry1Ac resistance in CP73 (BtR-5) maps to linkage group 10 of H. virescens and thus differs from the previously described linkage group 9 BtR-4 resistance found in the YHD2 strain, involving mutation of the gene encoding a 12-domain cadherin-like binding target of the Cry1A toxins. Neither BtR-4 nor BtR-5 seems to confer significant resistance to Cry2Aa. A majority of the linkage groups studied in one backcross family made a small positive contribution to resistance for both toxins. Thus, the Cry2Aa resistance in CP73 is not caused by either of the two major Cry1Ac resistance-conferring genes but instead probably has a quantitative genetic basis.     

Growth and development of Cardiochiles nigriceps viereck (hymenoptera,braconidae) larvae and their synchronization with some changes of the hemolymph composition of their host,Heliothis virescens (F.) (Lepidoptera,Noctuidae)

Larval development of the parasitoid Cardiochiles nigriceps Viereck occurs in the last instar larva of its host, Heliothis virescens (F.). This allows the parasitoid to exploit the nutritional increase in the biosynthetic activity occurring in the host in preparation for metamorphosis. To understand the biochemical basis of this host parasitoid developmental synchrony, we undertook host ligation studies and analyzed host hemolymph for proteins and glycerol esters. Parasitization affected the biochemical profile of the host. The hemolymph protein concentration of parasitized last instar H. virescens larvae increased through time, whereas unparasitized (control) larvae were characterized by a decrease in the protein titer when they reached the prepupal stage. The effect of parasitism on glyceride titers of host hemolymph was not as pronounced as the effect on proteins. Ligation conducted on 5th instar hosts, which were parasitized as 4th instars, affected parasitoid development in a time-dependent way. The percentage of successfully developing C. nigriceps larvae increased with the increase of the time interval between parasitization and ligation. Ligation performed before day 2 of the 5th larval instar of H. virescens completely inhibited parasitoid development. Ligations that disrupted parasitoid developmentwere associated with a low host hernolymph protein concentration. Parasitoid development was successful when hernolymph protein titer was high, as occurred when ligations were performed after day 3 of the 5th host instar in both control and parasitized larvae. Ligations in both situations resulted in a slight increase in glyceride titers. The results suggest that host proteins and/or some factor(s) associated with them may play a role in parasitoid growth and development. © 1993 Wiley-Liss, Inc.     

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.