Pathology and ultrastructure of Hz-2V infection in the agonadal female corn earworm,Helicoverpa zea

The pathology and ultrastructure of the reproductive tract of Hz-2V-infected female corn earworm moths, Helicoverpa zea, were studied. The identity of malformed reproductive tissues found in virus-infected moths was determined by examining these tissues in moths that were infected with the virus at different life stages. Malformation of reproductive tissues in the progeny of virus-infected female moths was first observed by 3 days post-pupation (dpp), indicating that virus replication had altered the differentiation of these tissues very early on in their development. The ultrastructure of the grossly malformed agonadal reproductive tissues from insects aged 3-10dpp revealed the absence of the cuticular lining found in the oviducts of normal moths, and the proliferation of epithelial cells in these infected oviduct tissues. In addition, large quantities of virus were found aggregated into a large mass in the lumen of the malformed cervix bursa of 10dpp agonadal female pharate adult moths. Prior to eclosion, the virus in the cervix bursa was observed separated into spherical masses, which are thought to exude through the ductus bursa and collect over the vulva, forming a viral "waxy plug" that is likely to play an important role in virus transmission.     

The sexually transmitted insect virus,Hz-2V

Hz-2V is one of only a very few sexually transmitted viruses currently known in insects. Replication of this insect pathogenic virus results in sterility of infected moths rather than mortality. The sterility of the infected host is a consequence of virus directed malformation of adult reproductive tissues, which in females results in cellular proliferation and hypertrophy of these tissues. Virus replication has additional ramifications in infected females. Infected females produce more mating pheromones and attract more mates than healthy females, ultimately facilitating virus transmission and enhancing viral fitness. The molecular mechanisms used by the virus to manipulate the host to enhance its fitness are yet to be determined. Unraveling the underlying principles of these mechanisms promises to enhance our understanding of insect reproductive physiology, as well as provide molecular tools for use in novel approaches in sterile insect control programs.     

Acquisition, persistence, and species susceptibility of the Hz-2V virus

Hz-2V, formerly called gonad-specific virus, is known to infect the reproductive organs of both males and females of the corn earworm Helicoverpa zea, rendering them agonadal or sterile. The primary mode of transmission is through mating by asymptomatic carrier moths. In this report we show that Hz-2V can be acquired by first instar larvae, through feeding on virus laced diet, although the incidence of agonadal condition was significantly lower. In a laboratory study, the virus appeared to persist for no more than three generations, with the incidence of agonadal progeny decreasing with each generation. Although, Hz-2V has been reported only from H. zea, in our tests when nine species of insects were artificially infected, four of the Noctuid species showed some signs of agonadal condition. Out of the remaining five species, the diamondback moth Plutella xylostella and the German cockroach Blatella germanica, showed no evidence of the virus in progeny of adults that were injected with Hz-2V, even after using the very sensitive PCR based assay.     

Further characterization of the gonad-specific virus of corn earworm, Helicoverpa zea

The gonad-specific virus (GSV) is a DNA virus infecting the reproductive tracts of adults of both sexes of the corn earworm, Helicoverpa zea, causing severe tissue deformities leading to sterility. Atypical occlusion bodies containing large concentrations of virions embedded in a granular matrix were seen in the lumen of the oviduct and the bursa copulatrix of infected females. The virus, transmitted by both sexes, was successfully propagated in vivo and in tissue culture. The GSV genome is about 225 kb in size, with no apparent similarity to the nucleopolyhedrovirus type species, AcMNPV, genomic DNA, as determined by Southern hybridization. PCR amplification of GSV genomic DNA with primers derived from the highly conserved polyhedra gene of several baculoviruses indicated no similarity. GSV at 10(-2) female equivalents (based on virus obtained from the bursa copulatrix and oviducts of one infected female) injected into a newly emerged female and mated to a normal male resulted in >95% agonadal progeny. However, at lower doses, some of the adult progeny looked normal but apparently carried a low level of the virus that could be responsible for sustenance of infection in a given colony, as well as in nature.     

Viability of Claviceps africana spores ingested by adult corn earworm moths, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

A study was conducted in College Station, TX, to determine the viability of Claviceps africana spores in the digestive tract of adult corn earworm moths, Helicoverpa zea (Boddie). Both sexes were exposed to ergot-infected sorghum panicles for 30 min, and spores were recovered from excreta of the moths at 24-, 48-, and 72-h intervals after feeding. Recovered spores were quantified, and viability was determined by the germination rate of macroconidia. Nearly a 100-fold greater concentration of spores was recovered from female excreta at the three time intervals compared with male excreta. Concentration of spores in female and male excreta was greatest at 24 h, with a significant reduction at the later time intervals. Spore germination rates for both sexes were greater at 24 h, with survival being significantly reduced at the 72-h interval. Spores in female excreta survived longer than those from male excreta. Spore survival over time was significantly reduced in male excreta. Spore concentration and survival were greater from female excreta, which is key, because egg-laying activities on sorghum panicles intensify during flowering, and this source of ergot spores could contribute to the spread of the disease. This study demonstrates that corn earworm moths can internally carry viable ergot spores for several days and can act as primary dispersal agents for the fungus. This is important because contaminated moths migrating from areas in Mexico and southern Texas where ergot is endemic could transmit and spread the disease to other sorghum growing regions of the United States.     

In vivo dose-response of insects to Hz-2V infection

Background

Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus.

Results

We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells.

Conclusions

When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.     

Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures

The serial passaging of baculoviruses in cell lines numerous times can result in a variety of mutations or defective viral populations becoming predominant in the cultures. The generation of these mutants during cell culture passage, also known as "the passage effect," can seriously hinder the use of in vitro methods for large-scale production of baculoviruses for use as biopesticides. In an effort to develop a large-scale in vitro method of producing Helicoverpa armigera singly enveloped nucleopolyhedrovirus (HaSNPV), it was essential to determine whether or not the passage effect was evident when this virus is serially passaged in cell cultures. An isolate of HaSNPV was serially passaged in Helicoverpa zea cell cultures up to 10 times. The production of occlusion bodies decreased with increasing passage number and there was evidence of defective viruses becoming predominant in cultures after 5 passages. The number of virions present within cross sections of passage 3 occlusion bodies was 1.5 times higher than those from passage 10 occlusion bodies when quantified using electron microscopy. A laboratory bioassay showed that potencies of passage 3 isolates against H. armigera larvae were 8 times higher than potencies of passage 10 isolates. This study indicated that changes typical of the passage effect were evident when HaSNPV was serially passaged in H. zea cell cultures up to 10 times.     

Horizontal and vertical transmission of wild-type and recombinant Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus

Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts. Here, we report on the horizontal and vertical transmission dynamics of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV-WT) and a genetically modified variant (HaSNPV-AaIT) with enhanced speed of action through the expression of an insect-selective scorpion toxin (AaIT). In caged field plots, horizontal transmission of both HaSNPV variants was greatest when inoculated 3rd instar larvae were used as infectors, transmission was intermediate with 2nd instar infectors and lowest with 1st instar infectors. Transmission was greater at a higher density of infectors (1 per plant) than at a lower density (1 per 4 plants); however, the transmission coefficient (number of new infections per initial infector) was lower at the higher density of infectors than at the lower density. HaSNPV-AaIT exhibited a significantly lower rate of transmission than HaSNPV-WT in the field cages. This was also the case in open field experiments. In the laboratory, the vertical transmission of HaSNPV-AaIT from infected females to offspring of 16.7+/-2.1% was significantly lower than that of HaSNPV-WT (30.9+/-2.9%). Likewise, in the field, vertical transmission of HaSNPV-AaIT (8.4+/-1.1%) was significantly lower than that of HaSNPV-WT (12.6+/-2.0%). The results indicate that the recombinant virus will be transmitted at lower rates in H. armigera populations than the wild-type virus. This may potentially affect negatively its long-term efficacy as compared to wild-type virus, but contributing positively to its biosafety.     

Receptor-mediated endocytosis of storage proteins by the fat body of Helicoverpa zea

The selective uptake of storage proteins by the fat body of the corn earworm Helicoverpa zea is mediated by a membrane-bound receptor protein. In this study, the major storage proteins of this insect species, arylphorin and very high density lipoprotein, were directly labeled with colloidal gold-particles of different size. After the fat body had been incubated with the labeled storage proteins, the distribution of these proteins was examined by electron microscopy. Both storage proteins were found at the extracellular side of coated pits and within coated vesicles. Moreover, fusion products of several coated vesicles such as endosomes and multivesicular bodies contained both proteins in their lumen. Ultimately, the proteins accumulated in electron-dense storage granules. Equal numbers of either storage protein were present in each organelle, supporting the notion that a single receptor mediates the uptake of both proteins. In contrast, only small numbers of gold-labeled immunoglobulin G molecules were found in the organelles, indicating that the protein uptake is specific for storage proteins. The results show that storage protein uptake in this lepidoteran species occurs in a process of receptor-mediated endocytosis that is similar to the well-established uptake of specific proteins into mammalian tissues.     

Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo

A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.     

Mode of transmission of nuclear-polyhedrosis virus to progeny of adult Heliothis zea

Late second-instar Heliothis armigera larvae were infected with a granulosis and a nuclear polyhedrosis virus, and all the externally visible symptoms for each virus are described. The effects of the virus infections on the feeding habits of the insects are also described, and it was found that a granulosis infection can prolong the larval period by up to 100%. The larvae continue feeding during this prolonged larval period, and can reach almost double the size and mass of normal larvae.It was further found that each of the viruses displays a distinct set of symptoms which could indicate beyond any doubt which of the two viruses induced death in the host.     

Yield response of dual-toxin Bt cotton to Helicoverpa zea infestations

Field cage experiments were conducted to determine the impact of bollworms, Helicoverpa zea (Boddie), on yields of Bollgard II and Widestrike cotton, Gossypium hirsutum L. One-day-old bollworm larvae were infested in white flowers of Bollgard II and in white flowers and terminals of Widestrike cotton. The infestation levels included 0, 50, and 100% of white flowers for each type of cotton. Terminal infestations included one or two larvae per terminal on Widestrike cotton. Larvae were placed in flowers of Bollgard II cotton each day for 1 to 4 wk during the first 4 wk of flowering during 2003, 2004, and 2005 seasons and in the flowers or terminals of Widestrike cotton each day for 1 to 3 wk. Averaged across years and durations of infestation, yields of Bollgard II cotton were significantly reduced compared with noninfested Bollgard II cotton when 100% of white flowers were infested. For Widestrike cotton, there was a reduction in yield when 100% of white flowers were infested in 2005, but not in 2006. There was a significant relationship for cumulative numbers of white flowers infested on seedcotton yield of Bollgard II during one of the 3 yr of the experiment. The regression equation during that year had a slope of -0.77. No significant relationships were observed for cumulative numbers of white flowers infested on yields of Widestrike cotton. Results of the current experiment suggest bollworms will rarely cause yield losses of Bollgard II and Widestrike cotton. Future research will need to focus on developing specific thresholds for bollworms on Bollgard II and Widestrike cotton.     

Fine-scale resolution of closely spaced pheromone and antagonist filaments by flying male Helicoverpa zea

The limits of a male moth's ability to resolve closely spaced odor filaments have been investigated. Male Helicoverpa zea normally respond to their conspecific sex pheromone blend by exhibiting an upwind flight, which culminates in source contact by at least 50% of the bioassayed individuals. When loaded onto the same filter paper source containing this hitherto attractive pheromone blend, or onto a separate filter paper and co-emitted from the same pipette source with pheromone, (Z)-11-hexadecenyl acetate severely reduced upwind flight and source contact by male H. zea. A similar level of upwind flight inhibition was recorded when the antagonist (Z)-11-hexadecenyl acetate was emitted from its own point source placed 1 mm upwind of the pheromone point source, both plumes being simultaneously emitted in a continuous mode to form a confluent strand. However, (Z)-11-hexadecenyl acetate was less effective in reducing upwind flight and source contact when it was isolated and pulsed from its own source, placed 1 mm either upwind, downwind or cross-wind of a pipette source from which pheromone was simultaneously being pulsed, such that both filaments were separated in time by 0.001–0. 003 s. These results suggest that male H. zea are able to distinguish between odor sources separated by as little as 1 mm in space and 0.001 s in time. Accepted: 31 March 1999     

Cold hardiness of Helicoverpa zea (Lepidoptera: Noctuidae) pupae

An insect's cold hardiness affects its potential to overwinter and outbreak in different geographic regions. In this study, we characterized the response of Helicoverpa zea (Boddie) pupae to low temperatures by using controlled laboratory measurements of supercooling point (SCP), lower lethal temperature (LT(50)), and lower lethal time (LLTime). The impact of diapause, acclimation, and sex on the cold hardiness of the pupae also were evaluated. Sex did not significantly affect the SCP, LT(50), or LLTime. However, the mean SCP of diapausing pupae (-19.3°C) was significantly lower than nondiapausing pupae (-16.4°C). Acclimation of nondiapausing pupae to constant temperatures from 10 to 20°C before supercooling also produced a significantly lower SCP than nondiapausing pupae held at 25°C. The LT(50)s of nondiapausing and diapausing were not significantly different, but confirmed that H. zea pupae are chill-intolerant because these lethal temperatures are warmer than the corresponding mean SCPs. Diapausing pupae survived longer than nondiapausing pupae at the same, constant, cold temperatures, a finding consistent with the SCP results. Both of these results suggest enhanced cold hardiness in diapausing pupae. When laboratory results were compared with field temperatures and observed distributions of H. zea in the contiguous United States, the laboratory results corroborated what is currently perceived to be the northern overwintering limit of H. zea; approximately the 40(th) parallel. Moreover, our research showed that areas north of this limit are lethal to overwintering pupae not because of low temperature extremes, but rather the length of time spent at near-zero temperatures.     

PBAN stimulation of pheromone biosynthesis by inducing calcium influx in pheromone glands of Helicoverpa zea

Isolated pheromone glands of Helicoverpa zea were utilized to investigate the physiological action of pheromone biosynthesis activating neuropeptide (PBAN) with regard to the role of calcium ions in stimulating pheromone biosynthesis under various incubation conditions. Incubation of glands with 1 microM or 1 nM PBAN produced a significant amount of pheromone after a 5 min incubation period and reached maximum pheromone production after 30 min. Glands incubated with PBAN for 1 min, and then without PBAN for 30 min, produced pheromone whether or not extracellular calcium was present during the first 1 min. The presence of lanthanum as a calcium channel blocker did not affect pheromone production if present during the first 1 min of incubation with PBAN. However, if calcium was absent or lanthanum ion was present during the 30 min of incubation, no pheromone was produced. A maximum amount of pheromone was reached when glands were incubated for 1 min with PBAN and for 10 min without PBAN, and repeated three times. The present results indicate that a time interval exists between PBAN binding to a receptor and opening of extracellular calcium channels. Calcium influx into the cytosol from extracellular stores is required for PBAN to stimulate pheromone production. This could be achieved by PBAN either binding periodically to the receptor or the plasma membrane calcium channel could remain activated for a period of time after the initial activation.     

Per os transmission of iridescent virus of Heliothis zea (Lepidoptera: Noctuidae)

In per os transmission of iridescent virus (IV), the first signs of infection are small iridescent patches in the prolegs, clypeus, labrum, and intersegmental membranes. Per os exposure of neonatal larvae to IV produced 18–40% infection. Per os exposure of 5- and 9-day-old larvae produced 11–47% and 10–30% infection, respectively. A few larvae with a patent infection pupated; however, none reached the adult stage. Infected larvae remained in the larval stage up to 89 days, compared to 12–21 days for normal larvae. Transovum or transovarian transmission was not detected. Examination of fat body, silk glands, and muscles of infected larvae by electron microscopy confirmed the presence of numerous intracytoplasmic virus particles. The mean particle diameter of hexagonal profiles within viral paracrystals was 118±3.5 nm.     

Frequent perinatal transmission of feline immunodeficiency virus by chronically infected cats.

Vertical transmission of feline immunodeficiency virus (FIV) was studied in cats infected with either of two FIV clinical isolates (FIV-B-2542 or FIV-AB-2771) prior to breeding and conception. Queens infected 4 to 30 months (mean = 14 months) prior to conception transmitted FIV to 59 of 83 (71%) kittens; 50.6% were virus positive on the day of birth. To examine potential routes of FIV transmission from mother to offspring, kittens were delivered via either vaginal or cesarean birth and nursed by either their virus-infected natural mothers or uninfected surrogate mothers. Comparison of FIV infection rates at birth with those at 6 months of age in kittens delivered by cesarean and surrogate raised demonstrated that late in utero transmission occurred in approximately 20% of kittens. Comparison of kittens nursed by FIV mothers with those by uninfected surrogate mothers demonstrated a 13.5% increase in infection rate of kittens exposed to milk-borne virus. Isolation of virus from 40% of maternal vaginal wash samples and the slightly greater infection rate in vaginally versus cesarean-delivered surrogate-nursed kittens suggested that intrapartum transmission may occur. In addition, we found that low maternal CD4 count (<200 cells per microl), longer duration of maternal infection (>15 months), and maternal symptoms of clinical immunodeficiency correlated with increased rates of mother-to-kitten FIV transmission, paralleling observations in human immunodeficiency virus-infected women. We conclude that FIV infection provides a model in which to explore aspects of human immunodeficiency virus vertical transmission and intervention difficult to address in human patients.     

Sequestration of host plant carotenoids in the larval tissues of Helicoverpa zea

To determine the cause of the unique yellow coloration in mandibular glands of soybean-fed Helicoverpa zea larvae, the accumulation of carotenoids in various tissues of last instar larvae fed soybean, cotton and tomato foliage was quantified. Five carotenoids were detected in the foliage of all host plants but at significantly different concentrations. Xanthophylls rather than carotenes were most likely to accumulate in larval tissues. Carotenoids accumulated at different rates and some were significantly affected by larval diet. Highest levels of carotenoid accumulation, notably lutein, were detected in the testes, followed by midgut epithelium, fat body and integument. The midgut epithelium contained the greatest and the testes the least diversity of carotenoid types. Low levels of lutein were detected in both labial and mandibular glands. Tomato foliage had the highest carotenoid content and caterpillar tissues fed these leaves often had the highest amounts of carotenoid. However, the accumulation of carotenoids did not protect larvae from antibiotic effects of tomato foliage because these caterpillars had the highest mortality and slowest growth rates of all the three host plants. Transport and absorption of lipid and oxidative stress may be some reasons for differential carotenoid accumulation.     

Damage to lima beans by Helicoverpa zea larvae treated as different instars with a nucleopolyhedrovirus

Larvae of the corn earworm,Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), were caged assecond, third, or fourth instars on lima bean,Phaseolus lunatus L., plants in the fieldwith or without treatment of the plants withlethal concentrations of the H. zeanucleopolyhedrovirus. The virus treatmentprevented over 90% of damage to pods and beansif larvae were second or third instars whenplaced in the cages. Damage to pods and beanswas reduced by 73 and 86%, respectively, ifthe larvae were fourth instars when placed onthe plants. When insects survived to the endof the test (which they did only on controlplants), numbers of pods or beans damaged byeach surviving insect was not affected by thestadium in which the larva was when it wasplaced on the plant. This result indicates thatmost damage was done by fourth or laterinstars. If treatments are to effectivelyprevent damage to lima bean, they should thusbe applied before the insects reach the fourthinstar.