Juvenile hormone analogs greatly increase the production of a nucleopolyhedrovirus

The commercial production of baculovirus insecticides is limited by the need to produce the virus in living insects. The influence of juvenile hormone analogs (JHA) on the growth and survival of Spodoptera exigua larvae placed on treated diet in the fifth instar was examined. Weight increases observed in methoprene- and fenoxycarb-treated larvae were over three-fold greater than that of control insects, whereas other compounds resulted in lower weight gains (pyriproxyfen) or highly variable responses (hydroprene). Approximately 90% and 70% of fenoxycarb and methoprene-treated larvae, respectively, molted to a supernumerary sixth instar and attained a final weight at 8–10 days post-treatment that was approximately double the maximum weight observed in control larvae. Inoculation of fenoxycarb and methoprene-treated sixth instars with a nucleopolyhedrovirus (SeMNPV) resulted in 2.4- or 2.9-fold increases in final weights, compared to control larvae inoculated in the fifth instar. The total yield of SeMNPV occlusion bodies (OBs) per larva was 2.7- and 2.9-fold greater in fenoxycarb- and methoprene-treated larvae, respectively, compared to fifth instar controls. A significant but small increase in the yield of OBs/mg larval weight was observed in fenoxycarb-treated insects but not in the methoprene treatment. The LC₅₀ value of OBs harvested from fenoxycarb-treated insects was slightly higher than that of OBs from control insects, whereas no such difference was observed in OBs from methoprene-treated insects. We conclude that appropriate use of JHA technology is likely to provide considerable benefits for the mass production of baculoviruses.     

Baculovirus infection triggers a positive phototactic response in caterpillars to induce ‘tree-top’ disease

Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions (‘tree-top’ disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.     

Adaptation of the Se301 insect cell line to suspension culture. Effect of turbulence on growth and on production of nucleopolyhedrovius (SeMNPV)

As chemical pesticides are being banned as control agents for agricultural pests, the use of the highly specific, safe to non-target organisms baculoviruses has been proposed. These viruses can be produced either in vivo or in vitro. In vitro production requires appropriated host insect cell lines with the ability for growing as freely-suspended cells. In this work, the Spodoptera exigua Se301 cell line was used to produce the commercially available S. exigua nucleopolyhedrovirus (SeMNPV) in suspension. Se301 cells showed to be very sensitive to the hydrodynamic shear rates developed in bioreactors. A process of progressive adaptation to freely-suspended cultures using protective additives against shear stress and disaggregant was proposed. The best combinations were polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) with the disaggregant dextran sulfate (DS). Both static and freely-suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Production of occluded baculovirus (OB) increased with the multiplicity of infection (MOI > 0.1).     

Greenhouse Evaluation of Dose– and Time–Mortality Relationships of Two Nucleopolyhedroviruses for the Control of Beet Armyworm, Spodoptera exigua, on Chrysanthemum

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV) against synchronized populations of S. exigua larvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exigua target stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exigua were controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigua larvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigua larvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.     

Interactions between an ectoparasitoid and a nucleopolyhedrovirus when simultaneously attacking Spodoptera exigua (Lepidoptera: Noctuidae)

Insect pathogenic viruses and parasitoids represent distinct biological entities that exploit a shared host resource and have similar effects in suppressing host populations. This study explores the interactions between the ectoparasitoid Euplectrus plathypenae (Hymenoptera: Eulophidae) and the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in larvae of S. exigua (Lepidoptera: Noctuidae). Parasitoid progeny failed to complete development in hosts that had been infected prior to parasitism. However, infection of S. exigua fourth instars at 48 h post‐parasitism had no significant effects on the survival of parasitoid progeny. Larval and pupal development times of E. plathypenae that survived on virus‐infected S. exigua did not differ significantly from that of parasitoids on healthy hosts. Virus‐induced mortality and the production of occlusion bodies were very similar in parasitized and non‐parasitized S. exigua. The virus was genetically stable over three passages in parasitized and unparasitized hosts. These results suggest that applications of SeMNPV‐based insecticides are unlikely to disrupt pest control exerted by the parasitoid E. plathypenae in biological pest control programs as long as virus applications are timed not to coincide with parasitoid releases.     

Baculovirus‐induced tree‐top disease: how extended is the role of egt as a gene for the extended phenotype?

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP‐glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree‐top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree‐top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree‐top disease. Here, we studied tree‐top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree‐top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting‐dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree‐top disease in lepidopteran larvae. The conclusion that egt is a ‘gene for an extended phenotype’ is therefore not generally applicable for all baculovirus–host interactions. We hypothesize that in some baculovirus–host systems (including LdMNPV in L. dispar), an effect of egt on tree‐top disease can be observed through indirect effects of egt on moulting‐related climbing behaviour.     

Relative activity of baculoviruses of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.) (Lepidoptera: Plutellidae)

The relative activities of the granulovirus (PxGV) and the nucleopolyhedrovirus (PxMNPV) of the diamondback moth, Plutella xylostella (L.), against this insect were evaluated in the laboratory. Similar numbers of occlusion bodies (OBs) of PxGV and PxMNPV were required to kill P. xylostella. However, cadavers of insects infected with PxGV contained more OBs per unit weight than did cadavers infected with PxMNPV. Less cadaver material was thus required to kill insects with PxGV. Larvae that were killed by PxGV survived approximately 2 d longer than did those killed by PxMNPV. When P. xylostella larvae were fed both viruses together, mortality was higher than that caused by either virus alone. The virulence of PxMNPV produced in P.␣xylostella did not differ from that of PxMNPV produced in the beet armyworm, Spodoptera exigua (Hübner). S. exigua was less susceptible to PxMNPV than to its homologous nucleopolyhedrovirus, SeMNPV. Use of trade names does not imply endorsements by USDA-ARS of products named nor criticism of similar ones not mentioned.     

Validation of a Comprehensive Process-Based Model for the Biological Control of Beet Armyworm,Spodoptera exigua,with Baculoviruses in Greenhouses

This paper describes the validation and sensitivity analysis of a process-based simulation model (BACSIM) for the control of beet armyworm, Spodoptera exigua, with baculoviruses. Model predictions are compared to results of independent greenhouse experiments in which second, third, or fourth instar larvae of S. exigua in chrysanthemum plots are treated with different concentrations of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and S. exigua MNPV (SeMNPV), two viruses with distinct differences in infectivity and mean time to kill. BACSIM provides robust predictions for the control of S. exigua populations in greenhouse chrysanthemum with both AcMNPV and SeMNPV. Mortality levels caused by AcMNPV and SeMNPV were generally predicted within a 25% margin of error compared to the observed values. None of the deviations was higher than 40%. All values of simulated foliage consumption, caused by S. exigua populations treated with AcMNPV or SeMNPV applications, fell within 95% confidence intervals of measurements. Simulated time to kill was, in general, lower than the measurements. This discrepancy may be caused by the behavior of S. exigua larvae which feed on the underside of chrysanthemum leaves where they are protected from polyhedra. This suggests that the larval foraging behavior may play an important role in the efficacy of baculovirus applications and should be further studied experimentally. This validated model can be used for the pretrial evaluation of the efficacy of genetically modified baculoviruses as biological control agents and for the optimization of spraying regimes in chrysanthemum cultivation.     

Contribution of a parasitoid species to multiplication and transmission of a multiple nucleopolyhedrovirus in caterpillars

Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Microplitis pallidipes are both used as biocontrol agents of the beet armyworm (Spodoptera exigua). However, it has not been determined how beet armyworms respond when these agents interact. Here, we studied the effects of M. pallidipes on virus multiplication and transmission using quantitative detection of SeMNPV. Our results indicated that parasitoids promoted virus multiplication in caterpillars (10⁵ copies per caterpillar) and that it was more advantageous when the M. pallidipes oviposited one day prior to infection with NPV. Interestingly, SeMNPV was transmitted by M. pallidipes in four ways. Transmission efficiency was higher for parasitoids whose body surfaces were contaminated with NPV, and for parasitoids ovipositing on NPV-infected caterpillars, than for those emerging from NPV-infected caterpillars, or feeding on mixtures of honey, water and NPV. Our study reveals that parasitoids do affect the proliferation and transmission of NPV in caterpillars and suggests that M. pallidipes could be used to strengthen the effectiveness of SeMNPV as a biocontrol agent.     

Competition between wild-type and a marked recombinant baculovirus (Spodoptera exigua nucleopolyhedrovirus) with enhanced speed of action in insect larvae

Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6-12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findings suggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems.     

Phenotypic characteristics and relative proportions of three genotypic variants isolated from a nucleopolyhedrovirus of Spodoptera exigua

US2A, US2D, and US2F are three in vivocloned genotypic variants from the wild-type strain of a Spodoptera exiguanucleopolyhedrovirus (SeMNPV) isolated in Florida (USA) and is the active component of the commercial bioinsecticide Spod-X^®. These variants were compared in terms of pathogenicity (LD₅₀), speed of kill (expressed as mean time to death) and viral progeny productivity (OBs/larva). LD₅₀values were similar for the three cloned genotypes. The mean time to death value for US2D (113.7 h) was significantly higher than those of US2A (31.7 h) and US2F (27.8 h). Virus yield was determined for L₄larvae infected with the estimated LD₉₉. US2D infected larvae attained higher weight than those infected with US2A and US2F, and produced a higher OB yield than larvae infected with US2A or US2F. An outstanding feature of US2F, in contrast to US2A and US2D, was its inability to disrupt the teguments of NPV-killed larvae. To study the relative proportion of the three genotypic variants throughout successive passages, S. exigualarvae were originally infected with a viral inoculum containing a 1:1:1 mixture of the three genotypes. After three successive passages, US2D was no longer detected in either of the three replicate experiments performed, while US2A was the predominant genotype in all of them, and US2F remained at similar proportions throughout the three passages. The influence of the phenotypic characteristics of the three variants on their relative proportions in mixed infections is discussed.     

Efficacy of Spodoptera exigua multiple nucleopolyhedrovirus as a biological insecticide for beet armyworm control in greenhouses of southern Spain

Chemical control measures targeted at Spodoptera exigua in greenhouse sweet pepper crops in Spain have resulted in pest resistance to virtually all commercially available insecticidal products. A multicapsid nucleopolyhedrovirus (SeMNPV), isolated from diseased S. exigua in Spain, was produced in laboratory reared larvae, tested for insecticidal activity in a laboratory bioassay, and was then applied in eleven commercial greenhouses planted with sweet pepper. Virus occlusion bodies (OBs) were applied on two occasions, at an interval of ～7 days, at a rate of 5×10⁸ OBs/L of spray in a volume of ～600 L/ha, depending on crop phenology and greenhouse area. The percentage of plants showing recent (<48 h old) feeding damage fell dramatically in greenhouses with high infestations of S. exigua; the same pattern was observed, although less dramatically, in greenhouses with low infestations. Average mortality of larvae collected from treated plants at 4 days after each application, and reared in the laboratory until death, was high (70–89%) and was not significantly affected by the degree of crop infestation. In a separate trial, the rate of acquisition of infection was examined in larvae that fed on plants treated with 1×10⁸ or 5×10⁸ OBs/L of spray. Of the 27 and 60% of larvae, respectively, that acquired infection in the 48 h period after spraying, about half became infected in the first 6 h post-application, irrespective of application rate. Acquisition of infection proceeded more slowly during the night-time compared to the daytime period, underlining the advantages of early morning applications of the virus. We conclude that the Spanish SeMNPV isolate merits registration as a biological insecticide for use in greenhouse crops in this region.     

Nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes affected haemolymph titre of 20‐hydroxyecdysone in Spodoptera exigua larvae

This study examined the physiological effects of joint and separate parasitism and infection by the endoparasitoid Microplitis pallidipes Szépligeti and the nucleopolyhedrovirus (NPV), respectively, on haemolymph 20‐hydroxyecdysone (20‐E) titre in Spodoptera exigua (Hübner) larvae. The results indicated that in parasitized larvae, virus‐infected larvae (5.7 × 10³ and 5.7 × 10⁵ OB/ml) and parasitized larvae infected with virus at 5.7 × 10⁵ OB/ml, compared to healthy larvae, the 20‐E all declined during the first 3 days but began to increase from day 4 after treatment, while in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), the 20‐E declined during the first 4 days but began to increase on day 5 after treatment. Meanwhile, compared to parasitized larvae, the 20‐E declined during the first 4 days but significantly increased on day 5 in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), while significantly increased during the first 2 days but began to decrease from day 3 after treatment in jointly parasitized and infected larvae (5.7 × 10⁵ OB/ml). Finally, in larvae that were both parasitized and virus infected (5.7 × 10³ OB/ml), compared to just virus‐infected larvae (5.7 × 10³ OB/ml), the 20‐E was lower on days 3 and 4 but higher on other days after treatment; in larvae that were both parasitized and virus infected (5.7 × 10⁵ OB/ml), compared to just virus‐infected larvae (5.7 × 10⁵ OB/ml), the 20‐E was significantly higher at the first 2 days but lower from day 3 after treatment. Our results revealed that 2nd instar larval M. pallidipes in host bodies may release 20‐E into the haemolymph of S. exigua larvae and that NPV infection may stimulate S. exigua to release more 20‐E during its third to fourth instar larval moulting. We found that this stimulatory effect was greater with higher virus concentrations.     

Adverse ffects of covert iridovirus infection on life history and demographic parameters of Aedes aegypti

Abstract Sublethal viral infections can cause changes in the body size and demography of insect vectors, with important consequences for population dynamics and the probability that individual mosquitoes will transmit disease. This study examined the effects of covert (sublethal) infection by Invertebrate iridescent virus 6 (IIV‐6) on the demography of female Aedes aegypti and the relationship between key life history parameters in covertly infected female insects compared with healthy (control) insects or non‐infected mosquitoes that had survived exposure to virus inoculum without becoming infected. Of the female mosquitoes that emerged following exposure to virus inoculum and were offered blood meals, 29% (43/150) proved positive for covert IIV‐6 infection. The net reproductive rate (R₀) of covertly infected females was 50% lower for infected females compared to control mosquitoes, whereas non‐infected exposed females had an R₀ approximately 15% lower than that of controls. Reproduction caused a significant decrease of about 13 days in mosquito longevity compared to females that did not reproduce (P < 0.001). Infected females lived 5–8 days less than non‐infected exposed females or controls, respectively (P = 0.028). Infected females and non‐infected exposed females both had significantly shorter wings than control insects (P < 0.001). There was a significant positive correlation between wing length and longevity in covertly infected female mosquitoes but not in control or non‐infected exposed mosquitoes. Longer lived females produced more eggs in all treatments. There were no significant correlations between body size and fecundity or the production of offspring. There was also no correlation between fecundity and fertility, suggesting that sperm inactivation was a more likely cause of decreased fertility in older mosquitoes than sperm depletion. We conclude that covert infection by iridescent virus is likely to reduce the vectorial capacity of this mosquito.     

Patterns of covert infection by invertebrate pathogens: iridescent viruses of blackflies

Recently, it has been recognized that blackfly populations may host two forms of infection by iridescent viruses (IVs); a covert (inapparent, nonlethal) form which was common in springtime populations in the River Ystwyth, Wales, and a patent (obvious, lethal) form which was rare. This study aimed to investigate the changes in frequency of the two types of infection in blackfly populations over the reproductive period of the flies, April-September 1992. Blackfly larvae sampled from three different sites along the river were bioassayed for the presence of covert IV infection. Of 870 larvae assayed, 17 were found to be infected. All the infected larvae appeared to be Simnulium variegatum, the dominant species during the sampling period. IV infections were common in the spring (17–37% depending on site) but appeared absent in the S. variegatum population for most of the summer months, reappearing again in the autumn (0–20% infected). These fluctuations were concurrent with biotic and abiotic factors: elevated levels of covert infection occurred at low population densities, high water flow rates, low temperatures (and presumably slower growth rates), although it is not clear if any cause-and-effect relationship exists. Patent infections occurred immediately after the peak of covert infection in the spring, and again in the autumn. Virus characterization of isolates from covertly infected larvae showed that three distinct groups of isolates were present in the blackfly population. Isolates from the springtime populations were mostly variants of an isolate found in patently infected blackfly larvae in the 1970s (Aberystwyth IV). Isolates from the autumn populations were mostly variants of an isolate from a patently infected larva found in September the previous year. A third group comprised a single novel isolate which was detected in a covertly infected larva. The mechanisms by which IVs persist in blackfly populations remain unknown, although the role of alternative hosts is a possibility which needs to be studied.     

Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen.