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.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

Effects of 20‐hydroxyecdysone and juvenile hormone on octopamine metabolism in females of Drosophila

The effect of exogenous 20‐hydroxyecdysone (20E) and juvenile hormone (JH) on the activities of the tyrosine decarboxylase (TDC), the first enzyme in octopamine (OA) synthesis, has been studied in young females of wild type D. virilis and D. melanogaster under normal and heat stress (38°C) conditions. Flies fed 20E expressed increased TDC activity in both species. JH application decreased TDC activity in both species. A rise in JH and 20E levels did not prevent a TDC response to heat stress, but changed the response intensity. A long‐term increase in JH titre had no effect on the activity of main OA catabolyzing enzyme, arylalkylamine N‐acetyltransferase, in females of both species. A possible mechanism of regulation of OA levels by 20E and JH in Drosophila females is discussed. © 2009 Wiley Periodicals, Inc.     

A VIGS screen identifies immunity in the Arabidopsis Pla‐1 accession to viruses in two different genera of the Geminiviridae

Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus‐resistant genes that derive from alterations in essential host factors. We used a virus‐induced gene silencing (VIGS) vector derived from the geminivirus Cabbage leaf curl virus (CaLCuV) to assess natural variation in virus–host interactions in 190 Arabidopsis accessions. Silencing of CH‐42, encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS, and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS. One accession, Pla‐1, lacked both symptoms and silencing, and was immune to wild‐type infectious clones corresponding to CaLCuV or Beet curly top virus (BCTV), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus (TYLCV). Quantitative trait locus mapping of a Pla‐1 X Col‐0 F₂ population was used to detect a major peak on chromosome 1, which is designated gip‐1 (geminivirus immunity Pla‐1‐1). The recessive nature of resistance to CaLCuV and the lack of obvious candidate genes near the gip‐1 locus suggest that a novel resistance gene(s) confers immunity.     

Host Filamentous Actin Is Associated with <Emphasis Type="Italic">Heliothis armigera</Emphasis> Single Nucleopolyhedrosis Virus (HaSNPV) Nucleocapsid Transport to the Host Nucleus

VP39 is the major capsid protein of Heliothis armigera nucleopolyhedrovirus (HaSNPV), and it might have induced the aggregation of host cellular actin in vitro in our previous study. We demonstrated here that VP39 could interact with host actin in vivo in Helicoverpazea (Hz-AM1 cells) through coimmunoprecipitation assay. With confocal immunofluorescence microscopy, it was confirmed further that the released HaSNPV nucleocapsids/VP39s in the host cytoplasm (0.5 hours after infection) colocalized where the actin aggregated and that the nucleocapsids/VP39s were transported from the host cytoplasm to the nucleus (2 hours after infection). Because cytochalasin D (CD) was used to prevent host global actin from forming filamentous structures, the infection efficiency of the recombinant virus HaSNPV/gfpΔp74, with the gfp gene inserted into HaSNPV p74 gene loci, was decreased to 7.34%, whereas it was 34.7% in normal host cells and 55.7% in the cells whose microtubules had been destroyed by colchicin. Ultramicroscopy assay revealed that HaSNPV nucleocapsids could enter the cytoplasm of CD-treated cells but could not be transported to the nucleus, which resulted in the lower infection efficiency of HaSNPV/gfpΔp74 in CD-treated cells. However, transportation of the nucleocapsids was not inhibited in colchicin-treated cells, demonstrating that the transportation of HaSNPV nucleocapsid from the cytoplasm to the nucleus was associated with actin filaments but not with microtubules, a conclusion that is also strongly supported by evidence from the RNAi interference of host actin during HaSNPV infection.     

Observations of parasitoid behaviour in both no‐choice and choice tests are consistent with proposed ecological host range

The solitary larval endoparasitoid Eadya daenerys Ridenbaugh (Hymenoptera: Braconidae) is a proposed biocontrol agent of Paropsis charybdis Stål (Coleoptera: Chrysomelidae, Chrysomelinae), a pest of eucalypts in New Zealand. Eadya daenerys oviposition behaviour was examined in two assay types during host range testing, with the aim of improving ecological host range prediction. No‐choice sequential and two‐choice behavioural observations were undertaken against nine closely related species of New Zealand non‐target beetle larvae, including a native beetle, introduced weed biocontrol agents, and invasive paropsine beetles. No behavioural measure was significantly different between no‐choice and two‐choice tests. In sequential no‐choice assays the order of first presentation (target–non‐target) had no significant effect on the median number of attacks or the attack rate while on the plant. Beetle species was the most important factor. Parasitoids expressed significantly lower on‐plant attack rates against non‐targets compared to target P. charybdis larvae. The median number of attacks was always higher towards target larvae than towards non‐target larvae, except for the phylogenetically closest related non‐target Trachymela sloanei (Blackburn) (Coleoptera: Chrysomelidae, Chrysomelinae). Most non‐target larvae were disregarded upon contact, which suggests that the infrequent attack behaviour observed by two individual E. daenerys against Allocharis nr. tarsalis larvae in two‐choice tests and the frass of Chrysolina abchasica (Weise) was probably abnormal host selection behaviour. Results indicate that E. daenerys is unlikely to attack non‐target species apart from Eucalyptus‐feeding invasive paropsines (Chrysomelinae). Non‐lethal negative impacts upon less preferred non‐target larvae are possible if E. daenerys does attack them in the field; however, this is likely to be rare.     

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

Naturally 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.     

Biological Comparison of Two Genotypes of Helicoverpa armigera Single-Nucleocapsid Nucleopolyhedrovirus

Baculovirus isolates from the same host species often show a considerable degree of variation on phenotypes. The completely sequenced genotypes C1 and G4 of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) were compared. Bioassay studies suggested that nearly double of HaSNPV G4 virus was required compared with HaSNPV C1 to achieve a similar LD₅₀, and at the LD₉₀ level the insect-killing speed for HaSNPV C1 was quicker than that of HaSNPV G4. The budded virus (BV) production of HaSNPV C1 was nearly two- to threefold higher at 24 and 48 h post-infection (p.i.) than that of HaSNPV G4. However, the kinetics of polyhedral inclusion body (PIB) formation in HzAM1 cells was similar in both the genotypes, which implied that the insect-killing speed was not influenced by PIB formation, but by the kinetics of BV production. The results suggested that the HaSNPV C1 isolate was a better choice than HaSNPV G4 virus for controlling H. armigera.     

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus‐induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense‐orientated target gene sequence of 100‐200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV‐based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E‐knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector‐mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees.     

In vitro effects of juvenile hormone analog on wing disc morphogenesis under ecdysteroid treatment in the female-wingless bagworm moth Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, lack wings completely, whereas male adults of this species have functional wings. We previously found that ecdysteroid induces apoptotic events in the female wing rudiment of E. variegata in vitro, whereas the male wing discs cultured with 20-hydroxyecdysone (20E) underwent apolysis and then cell differentiation. To investigate whether juvenile hormone (JH) in involved in sex-specific cellular response to ecdysteroid during wing development between sexes of E. variegata, we tested the effects of juvenile hormone analog (JHA), methoprene, and 20E on wing disc morphogenesis between sexes in vitro. Using transmission electron microscopy (TEM), we found that both higher concentration of JHA (5 μg/ml) and 20E (1 μg/ml) addition induced cell death (apoptosis) in the male wing discs but not induced cell death in the female wing rudiments in vitro in E. variegata. These culture experiments clearly detected the differential responses of wing discs to JHA under ecdysteroid treatment between sexes. We propose two important hypotheses: (1) JH is not significantly involved in the suppression of the female wing rudiment morphogenesis under 20E treatment, (2) female wing rudiment has lost the ability for cell proliferation in response to the stimulus of 20E.     

Contrasting impacts of a novel specialist vector on multihost viral pathogen epidemiology in wild and managed bees

Typically, pathogens infect multiple host species. Such multihost pathogens can show considerable variation in their degree of infection and transmission specificity, which has important implications for potential disease emergence. Transmission of multihost pathogens can be driven by key host species and changes in such transmission networks can lead to disease emergence. We study two viruses that show contrasting patterns of prevalence and specificity in managed honeybees and wild bumblebees, black queen cell virus (BQCV) and slow bee paralysis virus (SBPV), in the context of the novel transmission route provided by the virus‐vectoring Varroa destructor. Our key result is that viral communities and RNA virus genetic variation are structured by location, not host species or V. destructor presence. Interspecific transmission is pervasive with the same viral variants circulating between pollinator hosts in each location; yet, we found virus‐specific host differences in prevalence and viral load. Importantly, V. destructor presence increases the prevalence in honeybees and, indirectly, in wild bumblebees, but in contrast to its impact on deformed wing virus (DWV), BQCV and SBPV viral loads are not increased by Varroa presence, and do not show genetic evidence of recent emergence. Effective control of Varroa in managed honeybee colonies is necessary to mitigate further disease emergence, and alleviate disease pressure on our vital wild bee populations. More generally, our results highlight the over‐riding importance of geographical location to the epidemiological outcome despite the complexity of multihost‐parasite interactions.     

Host‐associated genomic differentiation in congeneric butterflies: now you see it,now you do not

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host‐associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host‐adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host‐adapted traits, resulting in incipient reproductive isolation among host‐associated ecotypes. We show here that Euphydryas aurinia had much weaker host‐associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host‐adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation.     

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.