Egt Gene Research Articles

Ecdysteroid UDP-Glucosyltransferase Expression in Beauveria bassiana Increases Its Pathogenicity against Early Instar Silkworm Larvae.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic fungus that can control pests in agriculture and forestry. In this study, encoding ecdysteroid uridine diphosphate glucosyltransferase gene (egt) was successfully screened in B. bassiana on the medium containing 500μg/mL G418 sulfate solution through the protoplast transformation method. This enzyme has the function of 20E (20-hydroxyecdysone) inactivation, thus increasing the mortality of the early instar larvae infected with B. bassiana. In this study, we transformed B. bassiana with the egt gene, which deactivates 20-hydroxyecdysone, a key hormone in insect development. The results showed that transgenic B. bassiana killed more silkworms of the 2nd instar larvae than the wild-type with a shorter LT50 time, which was reduced by approximately 20% (day 1 of the 2nd instar silkworm infection of B. bassiana) and 26.4% (day 2 of the 2nd instar silkworm infection of B. bassiana) compared to the wild-type, and also showed a higher mortality number before molting. The transgenic B. bassiana had a higher coverage of the body surface of silkworms compared to the wild type on the 3rd instar. In summary, improving entomopathogenic fungi using biological methods such as genetic engineering is feasible.

Bombyx mori nucleopolyhedrovirus ptp and egt genes are dispensable for triggering enhanced locomotory activity and climbing behavior in Bombyx mandarina larvae

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors.

Genetic variants in Argentinean isolates of Spodoptera frugiperda Multiple Nucleopolyhedrovirus.

The fall armyworm, Spodoptera frugiperda (JE Smith) is a key pest in the Americas. Control strategies are mainly carried out by use of chemical insecticides and transgenic crops expressing Bacillus thuringiensis toxins. In the last years, resistance of S. frugiperda populations to transgenic corn was reported in different Latin American countries. The baculovirus Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) is a pathogenic agent for the fall armyworm and a potential alternative for its control in integrated pest management strategies. In this work, we analyze some characteristics of two baculovirus isolates collected from maize (SfMNPV-M) and cotton (SfMNPV-C) fields from Argentina. The isolates were compared by restriction enzymes patterns and the analysis reveals the presence of genotypic variants in the SfMNPV-M isolate. We confirmed a deletion by sequencing fragments encompassing egt gene and most part of its contiguous gene (orf A) in a SfMNVP-M genotypic variant. Additionally, we estimated the 50% lethal dose and median survival time of each isolate in bioassays with S. frugiperda larvae.

The potential of novel African isolates of Phthorimaea operculella granulovirus for the control of Tuta absoluta

AbstractPhthorimaea operculella granulovirus (PhopGV) is infectious for larvae of different Gelechiidae insect species, including Tuta absoluta and Phthorimaea operculella. As these are major economic pests in North and sub‐Saharan Africa as well as in the Mediterranean area, the development of locally suitable biocontrol agents is essential. We have studied five isolates of PhopGV from Tunisia (Tns16, Tu1.11 and Tu2.11), Kenya (Ken13) and Yemen (Ym14) for their biological activity and the sequence polymorphism of their granulin and ecdysteroid UDP‐glucosyltransferase (egt) genes and allocated the isolates to two different egt types. Infection experiments with neonate larvae of T. absoluta and P. operculella demonstrated their pathogenicity to both host species. The isolate PhopGV Tu1.11 was the most virulent one for T. absoluta but had a relatively low infectivity to two P. operculella populations originating from Italy and Tunisia.

Heterologous and High Production of Ergothioneine in Escherichia coli.

Ergothioneine (ERG) is a histidine-derived thiol compound suggested to function as an antioxidant and cytoprotectant in humans. Therefore, experimental trials have been conducted applying ERG from mushrooms in dietary supplements and as a cosmetic additive. However, this method of producing ERG is expensive; therefore, alternative methods for ERG supply are required. Five Mycobacterium smegmatis genes, egtABCDE, have been confirmed to be responsible for ERG biosynthesis. This enabled us to develop practical fermentative ERG production by microorganisms. In this study, we carried out heterologous and high-level production of ERG in Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt enzymes and elimination of bottlenecks in the substrate supply, we succeeded in constructing a production system that yielded 24 mg/L (104 μM) secreted ERG.

Comparison of a South African and Canadian Isolate of the Nucleopolyhedrosis Virus Infecting the InsectTrichoplusia ni

The nucleotide and amino acid sequence of the ecdysteroid UDP-glucosyltransferase (egt), polyhedrin (pol), inhibitor of apoptosis (iap2 and iap3), late expression factor (lef) 9 and vp1054 genes of a South African isolate of Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) was compared to a Canadian isolate of TnSNPV. Nucleotide and amino acid sequence identity for the compared genes ranged from 89%to 98%and 95%to 99%, respectively. The deduced amino acid sequences of the TnSNPV egt, pol, iap3, lef-9 and vp1054 genes were used to infer phylogenetic trees and these were compared to the tree inferred from combined data sets consisting of the amino acid sequences of polyhedrin/granulin, lef-8 and lef-9 genes of 48 completely sequenced baculoviruses. The topologies of trees for the baculovirus core genes pol, lef-9 and vp1054 were better resolved than that of the auxiliary genes iap2, iap3 and egt when compared to the tree inferred from complete genome sequences. Sequence and phylogenetic analysis confirm...

Parasitic Manipulation of Host Behaviour: Baculovirus SeMNPV EGT Facilitates Tree-Top Disease in Spodoptera exigua Larvae by Extending the Time to Death.

Many parasites enhance their dispersal and transmission by manipulating host behaviour. One intriguing example concerns baculoviruses that induce hyperactivity and tree-top disease (i.e., climbing to elevated positions prior to death) in their caterpillar hosts. Little is known about the underlying mechanisms of such parasite-induced behavioural changes. Here, we studied the role of the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in tree-top disease in S. exigua larvae. Larvae infected with a mutant virus lacking the egt gene exhibited a shorter time to death and died before the induction of tree-top disease. Moreover, deletion of either the open reading frame or the ATG start codon of the egt gene prevented tree-top disease, indicating that the EGT protein is involved in this process. We hypothesize that SeMNPV EGT facilitates tree-top disease in S. exigua larvae by prolonging the larval time to death. Additionally, we discuss the role of egt in baculovirus-induced tree-top disease.

Genetic and biological characterisation of a novel Plutella xylostella granulovirus, PlxyGV-SA

Plutella xylostella granulovirus (PlxyGV) has been isolated from insect populations in many countries and is considered a potential biopesticide for sustainable control of P. xylostella (L.) (Lepidoptera: Plutellidae). Several PlxyGV isolates have been genetically characterised, and the full genome sequence of PlxyGV-Japan is available for comparison with novel isolates. A South African PlxyGV was recently recovered from an overcrowded laboratory P. xylostella colony and identified as a genetically distinct isolate by sequencing of the granulin gene and restriction endonuclease (REN) analysis of genomic DNA. In this report, PlxyGV-SA was further characterised by PCR amplification and sequencing of egt, lef-8 and lef-9 genes, and several amino acid substitutions were observed. The PstI REN profile of PlxyGV-SA was different from that of PlxyGV-Japan in terms of band size and number, thereby confirming its novel genetic identity. Surface dose bioassays showed that PlxyGV-SA is pathogenic to neonate but not late instar larvae at the same and higher virus doses, indicating that a biopesticide should be targeted at early larval stages in the field.

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 Lymantriadispar 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 Autographacalifornica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusiani and Spodopteraexigua, 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.

The isolation and genetic characterisation of a South African strain of Phthorimaea operculella granulovirus, PhopGV-SA

The Phthorimaea operculella granulovirus (PhopGV) is considered a promising biopesticide that can be incorporated into integrated pest management programmes for sustainable control of the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), a major pest of solanaceous crops in sub-tropical and tropical regions worldwide. Several PhopGV isolates recovered from geographically different insect populations have been genetically characterised, and the full genome of the Tunisian PhopGV-1346 isolate has been sequenced, providing a reference strain for comparison of novel isolates. Here we report the identification and genetic characterisation of a South African PhopGV isolate recovered from a P. operculella colony held under laboratory conditions. Transmission electron microscopy examination of purified occlusion bodies together with analysis of granulin and late expression factor-8 (lef-8) gene sequences confirmed the identity of the virus as PhopGV. The sequenced ecdysteroid UDP-glucosyltransferase (egt) gene was 1353nt in length, placing PhopGV-SA in egt group II. Finally, a phylogenetic analysis using a range of egt sequences grouped PhopGV-SA together with the Kenyan, Ecuadorian, Indonesian and Colombian isolates. The results are discussed with reference to the possible origin of PhopGV-SA, and provide a platform for future studies involving virulence evaluation against geographically different P. operculella populations with a view to biopesticide development.

Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates

False codling moth, Thaumatotibia leucotreta (Meyrick) is a serious pest of economic importance to the South African fruit industry. As part of sustainable efforts to control this pest, biological control options that involve the application of baculovirus-based biopesticides such as Cryptogran and Cryptex (both formulated with a South African isolate of Cryptophlebia leucotreta granulovirus, CrleGV-SA) are popularly used by farmers. In order to safeguard the integrity of these biopesticides as well as protect against any future development of resistance in the host, we conducted a study to bioprospect for additional CrleGV isolates as alternatives to existing ones. Using overcrowding as an induction method for latent infection, we recovered five new CrleGV isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGV-SA Nels). Single restriction endonuclease (REN) analysis of viral genomic DNA extracted from purified occlusion bodies showed that isolates differed in their DNA profiles. Partial sequencing of granulin and egt genes from the different isolates and multiple alignments of nucleotide sequences revealed the presence of single nucleotide polymorphisms (SNPs), some of which resulted in amino acid substitutions in the protein sequence. Based on these findings as well as comparisons with other documented CrleGV isolates, we propose two phylogenetic groups for CrleGV-SA isolates recovered in this study.

Development of a viral biopesticide for the control of the Guatemala potato tuber moth Tecia solanivora

The Guatemala potato tuber moth Tecia solanivora (Povolny) (Lep. Gelechiidae) is an invasive species from Mesoamerica that has considerably extended its distribution area in recent decades. While this species is considered to be a major potato pest in Venezuela, Colombia, and Ecuador, currently no specific control methods are available for farmers. To address this issue we developed a biopesticide formulation to be used in integrated pest management of T. solanivora, following three steps. First, search for entomopathogenic viruses were carried out through extensive bioprospections in 12 countries worldwide. As a result, new Phthorimaea operculella granulovirus (PhopGV) isolates were found in T. solanivora and five other gelechid species. Second, twenty PhopGV isolates, including both previously known and newly found isolates, were genetically and/or biologically characterized in order to choose the best candidate for a biopesticide formulation. Sequence data were obtained for the ecdysteroid UDP-glucosyltransferase (egt) gene, a single copy gene known to play a role in pathogenicity. Three different sizes (1086, 1305 and 1353bp) of egt were found among the virus isolates analyzed. Unexpectedly, no obvious correlation between egt size and pathogenicity was found. Bioassays on T. solanivora neonates showed a maximum of a 14-fold difference in pathogenicity among the eight PhopGV isolates tested. The most pathogenic PhopGV isolate, JLZ9f, had a medium lethal concentration (LC50) of 10 viral occlusion bodies per square mm of consumed tuber skin. Third, we tested biopesticide dust formulations by mixing a dry carrier (calcium carbonate) with different adjuvants (magnesium chloride or an optical brightener or soya lecithin) and different specific amounts of JLZ9f. During laboratory experiments, satisfactory control of the pest (>98% larva mortality compared to untreated control) was achieved with a formulation containing 10 macerated JLZ9f-dead T. solanivora larvae per kg of calcium carbonate mixed with 50mL/kg of soya lecithin. The final product provides an interesting alternative to chemical pesticides for Andean farmers affected by this potato pest.

Concentration- and time-response characteristics of plaque isolates of Agrotis ipsilon multiple nucleopolyhedrovirus derived from a field isolate

Plaque isolates derived from the Illinois field isolate of Agrotis ipsilon multiple nucleopolyhedrovirus are distinguished by the presence or absence of a small deletion in the baculovirus egt (ecdysteroid UDP-glucosyltransferase) coding sequence. In concentration–response and time-response bioassays with both plaque and field isolates, plaque isolates with a mutated egt gene were less pathogenic against A. ipsilon larvae than other isolates, but killed larvae faster. Mixed infections with isolates representing the two different egt genotypes caused the same level of mortality as the field isolate and a plaque isolate with a wild-type egt gene.

Experimental mixtures of Phthorimaea operculella granulovirus isolates provide high biological efficacy on both Phthorimaea operculella and Tecia solanivora (Lepidoptera: Gelechiidae)

The Guatemalan potato moth Tecia solanivora (Povolny) recently invaded part of South America, colonizing zones where Phthorimaea operculella (Zeller), another potato moth species belonging to the same group, was previously established. T. solanivora is now the major insect pest of potato in this area encompassing Venezuela, Colombia and Ecuador. P. operculella granulovirus (PhopGV) (Betabaculovirus) is a biocontrol agent to be considered for the simultaneous management of these two potato pests, instead of classical chemical insecticides. In a previous work, five PhopGV isolates were isolated in Colombia from T. solanivora and were tested against larvae of the same species showing variable efficacies. Infections with mixtures of different genotypes of Baculoviruses had been carried out in a wide range of species and several showed interesting results. In the present study, the effect of sequential passages of PhopGV in P. operculella and T. solanivora larvae was analyzed through biological assays. Three different mixtures containing a Peruvian PhopGV isolate (Peru) adapted to P. operculella and a Colombian PhopGV isolate (VG003) adapted to T. solanivora were tested. A preliminary analysis of the correlation between the genotypic marker egt gene and the level of pathogenicity after a variable number of replication cycles was made. Mixtures of virus isolates showed a higher efficacy in both hosts compared to individual PhopGV isolates. This higher pathogenicity was maintained through passages. In P. operculella the mixtures were between 2.8 and 23.6-fold (from 7.15OB/mm2 to 0.10OB/mm2) more pathogenic than isolate Peru applied alone. In T. solanivora they were between 2.3 and 4.9-fold (from 12.29OB/mm2 to 1.25OB/mm2) more pathogenic than isolate VG003 alone. Viral biopesticide containing a mixture of selected genotypes active against each hosts seemed suitable for the development of a biopesticide aimed to simultaneously control P. operculella and T. solanivora.

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein ( hsp) 23.7 promoter, with a neomycin-resistance gene ( neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene ( gfp) under the control of the B. mori actin 3 ( A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.

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.

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

AbstractNaturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.

Dose dependency of time to death in single and mixed infections with a wildtype and egt deletion strain of Helicoverpa armigera nucleopolyhedrovirus

Recombinant insect nucleopolyhedroviruses lacking the egt gene generally kill their hosts faster than wild-type strains, but the response of insects to mixtures of virus genotypes is less well known. Here, we compared the survival time, lethal dose and occlusion body yield in third instar larvae of Helicoverpa armigera (Hübner) after challenge with wild-type H. armigera SNPV (HaSNPV-wt), a strain with a deletion of the egt gene, HaSNPV-LM2, and a 1:1 mixture of these two virus strains. A range of doses was used to determine whether the total number of OBs influenced the response to challenge with a mixture of virus strains versus single strains. At high virus doses, HaSNPV-LM2 killed H. armigera larvae significantly faster (ca. 20 h) than HaSNPV-wt, but at low doses, there was no significant difference in survival time between the viruses. The survival time after challenge with mixed virus inoculum was significantly different from and intermediate between that of the single viruses at high doses, and not different from that of the single viruses at low doses. No differences in lethal dose were found between single and mixed infections or between virus genotypes. The number of occlusion bodies produced per larva increased with time to death and decreased with virus dose, but no significant differences among virus types were found.

Mixed-genotype infections of Trichoplusia ni larvae with Autographa californica multicapsid nucleopolyhedrovirus: Speed of action and persistence of a recombinant in serial passage

Fast-acting recombinant baculoviruses have potential for improved insect pest suppression. However, the ecological impact of using such viruses must be given careful consideration. One strategy for mitigating risks might be simultaneous release of a wild-type baculovirus, so as to facilitate rapid displacement of the recombinant baculovirus by a wild-type. However, at what ratio must the two baculoviruses be released? An optimum release ratio must ensure both fast action, and the eventual competitive displacement of the recombinant virus and fixation of the wild-type baculovirus in the insect population. Here we challenged Trichoplusia ni larvae with different ratios of wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a derived recombinant, vEGTDEL, which has the endogenous egt gene (coding for ecdysteroid UDP-glucosyltransferase) deleted. Time to death increased with the proportion wild-type virus in the inoculum mixture, although a 1:10 ratio (wild-type: recombinant) resulted in equally rapid insecticidal action as vEGTDEL alone. Five serial passages of three different occlusion body (OB) mixtures of the two viruses were also performed. OBs from 10 larval cadavers were pooled and used to initiate the following passage. Although the wild-type baculovirus was maintained over five passages, it did not go to fixation in most replicates of the serial passage experiment (SPE), and there was no good evidence for selection against the recombinant. Long-term maintenance of a recombinant in serial passage suggests an ecosystem safety risk. We conclude that for assessing ecological impact of recombinant viruses, SPEs in single and multiple larvae are relevant because of potential modulating effects at the between-host level.

Mixed infections and the competitive fitness of faster-acting genetically modified viruses.

Faster-acting recombinant baculoviruses have shown potential for improved suppression of insect pests, but their ecological impact on target and nontarget hosts and naturally occurring pathogens needs to be assessed. Previous studies have focused on the fitness of recombinants at the between-hosts level. However, the population structure of the transmission stages will also be decided by within-host selection. Here we have experimentally quantified the within-host competitive fitness of a fast-acting recombinant Autographa californica multicapsid nucleopolyhedrovirus missing the endogenous egt gene (vEGTDEL), by means of direct competition in single- and serial-passage experiments with its parental virus. Quantitative real-time PCR was employed to determine the ratio of these two viruses in passaged mixtures. We found that vEGTDEL had reduced within-host fitness: per passage the ratio of wild type to vEGTDEL was on average enhanced by a factor of 1.53 (single passage) and 1.68 (serial passage). There is also frequency-dependence: the higher the frequency of vEGTDEL, the stronger the selection against it is. Additionally, the virus ratio is a predictor of time to host death and virus yield. Our results show that egt is important to within-host fitness and allow for a more complete assessment of the ecological impact of recombinant baculovirus release.