Delivery of Methoprene-Tolerant dsRNA to Improve RNAi Efficiency by Modified Liposomes for Pest Control.

The application of RNA interference (RNAi) technology for pest control is environmentally friendly and accurate. However, the efficiency of RNAi is often inconsistent and unreliable, and finding a suitable carrier element is considered critical to success in overcoming biotic and abiotic barriers to reach the target site. The fall armyworm, Spodoptera frugiperda (FAW), which is one of most important global agricultural pests, has recently spread rapidly to other parts of the world. In this study, a method to improve the stability and RNAi efficiency of the dsRNA carrier complex was reported. Methoprene-tolerant gene (Met) was selected as a target, a gene which is critical to the growth and development of FAW. Biomaterials nanoliposomes (LNPs) were modified with polyethylenimine (PEI) to deliver the dsRNA of Met. The synthesized Met3@PEI@LNPs reached a size of 385 nm and were found to load dsRNA effectively. Through stability and protection assays, it was found that LNPs provided reliable protection. In addition, the release curve also demonstrated that LNPs were able to prevent premature release under alkaline condition of the insect midgut but accelerate the release after entering the acidic environment of the target cells. The cell transfection efficiency of the prepared LNPs reached 96.4%. Toxicity tests showed that the use of LNPs could significantly improve the interference efficiency, with 91.7% interference efficiency achieved when the concentration of dsRNA in LNPs was only 25% of that of the control. Successful interference of Met demonstrated it could significantly shorten the larval period and make the larvae pupate earlier, thus achieving the purpose of control. In this study, we have demonstrated the use of nanotechnology to provide a novel RNAi delivery method for pest control.

Gut microbial community supplementation and reduction modulates African armyworm susceptibility to a baculovirus.

Gut microbiota stimulates the immune system and inhibits pathogens, and thus, it is critical for disease prevention. Probiotics represent an effective alternative to antibiotics used for the therapy and prevention of bacterial diseases. Probiotic bacteria are commonly used in vertebrates, although their use in invertebrates is still rare. We manipulated the gut microbiome of the African Armyworm (Spodoptera exempta Walker) using antibiotics and field-collected frass, in an attempt to understand the interactions of the gut microbiome with the nucleopolyhedrovirus, SpexNPV. We found that S. exempta individuals with supplemented gut microbiome were significantly more resistant to SpexNPV, relative to those with a typical laboratory gut microbiome. Illumina MiSeq sequencing revealed the bacterial phyla in the S. exempta gut belonged to 28 different classes. Individuals with an increased abundance of Lactobacillales had a higher probability of surviving viral infection. In contrast, there was an increased abundance of Enterobacteriales and Pseudomonadales in individuals dying from viral infection, corresponding with decreased abundance of these two Orders in surviving caterpillars, suggesting a potential role for them in modulating the interaction between the host and its pathogen. These results have important implications for laboratory studies testing biopesticides.

Partiti-like viruses from African armyworm increase larval and pupal mortality of a novel host: the Egyptian cotton leafworm.

BACKGROUND: The general principle of using microbes from one species to manage a different pest species has a clear precedent in the large-scale release of mosquitoes carrying a Wolbachia bacterium derived from Drosophila flies. New technologies will facilitate the discovery of microbes that can be used in a similar way. Previously, we found three novel partiti-like viruses in the African armyworm (Spodoptera exempta). To investigate further the utility and consistency of host shift of insect viruses as a potential pest management tool, we tested the interaction between the partiti-like viruses and another novel host, the Egyptian cotton leafworm (Spodoptera littoralis). RESULT: We found that all three partiti-like viruses appeared to be harmful to the novel host S. littoralis, by causing increased larval and pupal mortality. No effect was observed on host fecundity, and partiti-like virus infection did not impact host susceptibility when challenged with another pathogen, the baculovirus SpliNPV. Transcriptome analysis of partiti-like virus-infected and noninfected S. littoralis indicated that the viruses could impact host gene-expression profiles of S. littoralis, but they impact different pathways to the two other Spodoptera species through effects on pathways related to immunity (Jak-STAT/Toll and Imd) and reproduction (insulin signaling/insect hormones). CONCLUSION: Taken together with the previous findings in the novel host S. frugiperda, these results indicate a parasitic relationship between the partiti-like viruses and novel insect hosts, suggesting a possible use and novel pest management strategy through the artificial host shift of novel viruses. © 2021 Society of Chemical Industry.

Trans-generational viral transmission and immune priming are dose-dependent.

It is becoming increasingly apparent that trans-generational immune priming (i.e. the transfer of the parental immunological experience to its progeny resulting in offspring protection from pathogens that persist across generations) is a common phenomenon not only in vertebrates, but also invertebrates. Likewise, it is known that covert pathogenic infections may become 'triggered' into an overt infection by various stimuli, including exposure to heterologous infections. Yet, rarely have both phenomena been explored in parallel. Using as a model system the African armyworm Spodoptera exempta, an eruptive agricultural pest and its endemic dsDNA virus (Spodoptera exempta nucleopolyhedrovirus, SpexNPV), the aim of this study was to explore the impact of parental inoculating-dose on trans-generational pathogen transmission and immune priming (in its broadest sense). Larvae were orally challenged with one of five doses of SpexNPV and survivors from these treatments were mated and their offspring monitored for viral mortality. Offspring from parents challenged with low viral doses showed evidence of 'immune priming' (i.e. enhanced survival following SpexNPV challenge); in contrast, offspring from parents challenged with higher viral doses exhibited greater susceptibility to viral challenge. Most offspring larvae died of the virus they were orally challenged with; in contrast, most offspring from parents that had been challenged with the highest doses were killed by the vertically transmitted virus (90%) and not the challenge virus. These results demonstrate that the outcome of a potentially lethal virus challenge is critically dependent on the level of exposure to virus in the parental generation-either increasing resistance at very low parental viral doses (consistent with trans-generational immune priming) or increasing susceptibility at higher parental doses (consistent with virus triggering). We discuss the implications of these findings for understanding both natural epizootics of baculoviruses and for using them as biological control agents.

Novel partiti-like viruses are conditional mutualistic symbionts in their normal lepidopteran host, African armyworm, but parasitic in a novel host, Fall armyworm.

Recent advances in next generation sequencing (NGS) (e.g. metagenomic and transcriptomic sequencing) have facilitated the discovery of a large number of new insect viruses, but the characterization of these viruses is still in its infancy. Here, we report the discovery, using RNA-seq, of three new partiti-like viruses from African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), which are all vertically-transmitted transovarially from mother to offspring with high efficiency. Experimental studies show that the viruses reduce their host's growth rate and reproduction, but enhance their resistance to a nucleopolyhedrovirus (NPV). Via microinjection, these partiti-like viruses were transinfected into a novel host, a newly-invasive crop pest in sub-Saharan Africa (SSA), the Fall armyworm, S. frugiperda. This revealed that in this new host, these viruses appear to be deleterious without any detectable benefit; reducing their new host's reproductive rate and increasing their susceptibility to NPV. Thus, the partiti-like viruses appear to be conditional mutualistic symbionts in their normal host, S. exempta, but parasitic in the novel host, S. frugiperda. Transcriptome analysis of S. exempta and S. frugiperda infected, or not, with the partiti-like viruses indicates that the viruses may regulate pathways related to immunity and reproduction. These findings suggest a possible pest management strategy via the artificial host-shift of novel viruses discovered by NGS.

Discovery and characterization of a novel picorna-like RNA virus in the cotton bollworm Helicoverpa armigera.

We characterize a novel picorna-like virus, named Helicoverpa armigera Nora virus (HaNV), with a genome length of 11,200 nts, the sequence of which was isolated from the lepidopteran host cotton bollworm Helicoverpa armigera, using RNA-Seq. Phylogenetic analysis, using the putative amino acid sequence of the conserved RNA-dependent RNA polymerase (RdRp) domain, indicated that HaNV clustered with Spodoptera exigua Nora virus, Drosophila Nora virus and Nasonia vitripennis virus-3 with a high bootstrap value (100%), which might indicate a new viral family within the order Picornavirales. HaNV was efficiently horizontally transmitted between hosts via contaminated food, and transmission was found to be dose-dependent (up to 100% efficiency with 109 viral copy number/µl). HaNV was also found to be transmitted vertically from parent to offspring, mainly through transovum transmission (virus contamination on the surface of the eggs), but having a lower transmission efficiency (around 43%). Infection distribution within the host was also investigated, with HaNV mainly found in only the gut of both adult moths and larvae (>90%). Moreover, our results showed that HaNV appears not to be an overtly pathogenic virus to its host.

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Characterization of a novel member of genus Iflavirus in Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera, is one of the most important agricultural pests of many economic crops worldwide. Herein, we found a novel single-strand RNA virus by RNA-Seq and Polymerase Chain Reaction (PCR) method in H. armigera named Helicoverpa armigera iflavirus (HaIV), which possessed a genome with 10,017 nucleotides in length and contained a single large open reading frame (ORF) encoding a putative polyprotein of 3021 amino acids with a predicted molecular mass of 344.16kDa and a theoretical isoelectric point (pI) of 6.45. The deduced amino acid sequence showed highest similarity (61.0%) with the protein of Lymantria dispar Iflavirus 1. Phylogenetic analysis with putative RdRp amino acid sequences indicated that the virus clustered with members of the genus Iflavirus. The virus was mainly distributed in the fat body of its host and was found to be capable of both horizontal and vertical transmission. The efficiency of perorally horizontal transmission was dose dependent (100% infection rate with a viral dose of 108copies/µl) while vertical transmission efficiency was found to be relatively low (<28.57%). These results suggest that we have found a novel member of genus Iflavirus in H. armigera.

Structural proteins of Helicoverpa armigera densovirus 2 enhance transcription of viral genes through transactivation.

Herein, we report the identification of putative promoters for the non-structural proteins (NS) and capsid structural proteins (VP) of Helicoverpa armigera densovirus (HaDV2) as well as a potential mechanism for how these promoters might be regulated. For the first time, we report that VP is able to transactivate the VP promoter and, to a lesser degree, the NS promoter in densoviruses. In addition to this, another promoter-like sequence designated P2, when co-transfected with the VP gene, enhanced luciferase activity by approximately 35 times compared to a control. This suggests that there are two promoters for VP in HaDV2 and that the VP of parvoviruses might play a more important role in viral transcription than previously appreciated.

Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells.

BACKGROUND: Densoviruses (DVs) are highly pathogenic to their hosts. However, we previously reported a mutualistic DV (HaDV2). Very little was known about the characteristics of this virus, so herein we undertook a series of experiments to explore the molecular biology of HaDV2 further. RESULTS: Phylogenetic analysis showed that HaDV2 was similar to members of the genus Iteradensovirus. However, compared to current members of the genus Iteradensovirus, the sequence identity of HaDV2 is less than 44% at the nucleotide-level, and lower than 36, 28 and 19% at the amino-acid-level of VP, NS1 and NS2 proteins, respectively. Moreover, NS1 and NS2 proteins from HaDV2 were smaller than those from other iteradensoviruses due to their shorter N-terminal sequences. Two transcripts of about 2.2 kb coding for the NS proteins and the VP proteins were identified by Northern Blot and RACE analysis. Using specific anti-NS1 and anti-NS2 antibodies, Western Blot analysis revealed a 78 kDa and a 48 kDa protein, respectively. Finally, the localization of both NS1 and NS2 proteins within the cell nucleus was determined by using Green Fluorescent Protein (GFP) labelling. CONCLUSION: The genome organization, terminal hairpin structure, transcription and expression strategies as well as the mutualistic relationship with its host, suggested that HaDV2 was a novel member of the genus Iteradensovirus within the subfamily Densovirinae.

Functional opsin retrogene in nocturnal moth.

BACKGROUND: Retrotransposed genes are different to other types of genes as they originate from a processed mRNA and are then inserted back into the genome. For a long time, the contribution of this mechanism to the origin of new genes, and hence to the evolutionary process, has been questioned as retrogenes usually lose their regulatory sequences upon insertion and generally decay into pseudogenes. In recent years, there is growing evidence, notably in mammals, that retrotransposition is an important process driving the origin of new genes, but the evidence in insects remains largely restricted to a few model species. FINDINGS: By sequencing the messenger RNA of three developmental stages (first and fifth instar larvae and adults) of the pest Helicoverpa armigera, we identified a second, intronless, long-wavelength sensitive opsin (that we called LWS2). We then amplified the partial CDS of LWS2 retrogenes from another six noctuid moths, and investigate the phylogenetic distribution of LWS2 in 15 complete Lepidoptera and 1 Trichoptera genomes. Our results suggests that LWS2 evolved within the noctuid. Furthermore, we found that all the LWS2 opsins have an intact ORF, and have an ω-value (ω = 0.08202) relatively higher compared to their paralog LWS1 (ω = 0.02536), suggesting that LWS2 opsins were under relaxed purifying selection. Finally, the LWS2 shows temporal compartmentalization of expression. LWS2 in H. armigera in adult is expressed at a significantly lower level compared to all other opsins in adults; while in the in 1st instar stage larvae, it is expressed at a significantly higher level compared to other opsins. CONCLUSIONS: Together the results of our evolutionary sequence analyses and gene expression data suggest that LWS2 is a functional gene, however, the relatively low level of expression in adults suggests that LWS2 is most likely not involved in mediating the visual process.

Development of a Real-Time qPCR Assay for Quantification of Covert Baculovirus Infections in a Major African Crop Pest.

Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR) procedure using a 5' nuclease hydrolysis (TaqMan) probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV). The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics.

Transgenerational effects modulate density-dependent prophylactic resistance to viral infection in a lepidopteran pest.

There is an increasing appreciation of the importance of transgenerational effects on offspring fitness, including in relation to immune function and disease resistance. Here, we assess the impact of parental rearing density on offspring resistance to viral challenge in an insect species expressing density-dependent prophylaxis (DDP); i.e. the adaptive increase in resistance or tolerance to pathogen infection in response to crowding. We quantified survival rates in larvae of the cotton leafworm (Spodoptera littoralis) from either gregarious- or solitary-reared parents following challenge with the baculovirus S. littoralis nucleopolyhedrovirus. Larvae from both the parental and offspring generations exhibited DDP, with gregarious-reared larvae having higher survival rates post-challenge than solitary-reared larvae. Within each of these categories, however, survival following infection was lower in those larvae from gregarious-reared parents than those from solitary-reared, consistent with a transgenerational cost of DDP immune upregulation. This observation demonstrates that crowding influences lepidopteran disease resistance over multiple generations, with potential implications for the dynamics of host-pathogen interactions.

Densovirus is a mutualistic symbiont of a global crop pest (Helicoverpa armigera) and protects against a baculovirus and Bt biopesticide.

Mutualistic associations between symbiotic bacteria and their hosts are common within insect systems. However, viruses are often considered as pathogens even though some have been reported to be beneficial to their hosts. Herein, we report a novel densovirus, Helicoverpa armigera densovirus-1 (HaDNV-1) that appears to be beneficial to its host. HaDNV-1 was found to be widespread in wild populations of H. armigera adults (>67% prevalence between 2008 and 2012). In wild larval populations, there was a clear negative interaction between HaDNV-1 and H. armigera nucleopolyhedrovirus (HaNPV), a baculovirus that is widely used as a biopesticide. Laboratory bioassays revealed that larvae hosting HaDNV-1 had significantly enhanced resistance to HaNPV (and lower viral loads), and that resistance to Bacillus thuringiensis (Bt) toxin was also higher at low doses. Laboratory assays indicated that the virus was mainly distributed in the fat body, and could be both horizontally- and vertically-transmitted, though the former occurred only at large challenge doses. Densovirus-positive individuals developed more quickly and had higher fecundity than uninfected insects. We found no evidence for a negative effect of HaDNV-1 infection on H. armigera fitness-related traits, strongly suggesting a mutualistic interaction between the cotton bollworm and its densovirus.

Locusts increase carbohydrate consumption to protect against a fungal biopesticide.

There is growing evidence to suggest that hosts can alter their dietary intake to recoup the specific resources involved in mounting effective resistance against parasites and pathogens. We examined macronutrient ingestion and disease-resistance in the Australian plague locust (Chortoicetes terminifera), challenged with a fungal pathogen (Metarhizium acridum) under dietary regimes varying in their relative amounts of protein and digestible carbohydrate. Dietary protein influenced constitutive immune function to a greater extent than did carbohydrate, indicating higher protein costs of mounting an immune defence than carbohydrate or overall energy costs. However, it appears that increased immune function, as a result of greater protein ingestion, was not sufficient to protect locusts from fungal disease. We found that locusts restricted to diets high in protein (P) and low in carbohydrate (C) were more likely to die of a fungal infection than those restricted to diets with a low P:C ratio. We hypothesise that the fungus is more efficient at exploiting protein in the insect's haemolymph than the host is at producing immune effectors, tipping the balance in favour of the pathogen on high-protein diets. When allowed free-choice, survivors of a fungus-challenge chose a less-protein-rich diet than those succumbing to infection and those not challenged with fungus locusts. These results are contrary to previous studies on caterpillars in the genus Spodoptera challenged with bacterial and baculoviral pathogens, indicating that nutrient ingestion and pathogen resistance may be a complex interaction specific to different host species and disease agents.

Male-killing Wolbachia and mitochondrial selective sweep in a migratory African insect.

BACKGROUND: Numerous recent studies have shown that resident symbiotic microorganisms of insects play a fundamental role in host ecology and evolution. The lepidopteran pest, African armyworm (Spodoptera exempta), is a highly migratory and destructive species found throughout sub-Saharan Africa, that can experience eruptive outbreaks within the space of a single generation, making predicting population dynamics and pest control forecasting extremely difficult. Three strains of Wolbachia have recently been identified infecting this species in populations sampled from Tanzania. In this study, we examined the interaction between Wolbachia pipiensis infections and the co-inherited marker, mtDNA, within populations of armyworm, as a means to investigate the population biology and evolutionary history of Wolbachia and its host. RESULTS: A Wolbachia-infected isofemale line was established in the laboratory. Phenotypic studies confirmed the strain wExe1 as a male-killer. Partial sequencing of the mitochondrial COI gene from 164 individual field-collected armyworm of known infection status revealed 17 different haplotypes. There was a strong association between Wolbachia infection status and mtDNA haplotype, with a single dominant haplotype, haplo1 (90.2% prevalence), harbouring the endosymbiont. All three Wolbachia strains were associated with this haplotype. This indicates that Wolbachia may be driving a selective sweep on armyworm haplotype diversity. Despite very strong biological and molecular evidence that the samples represent a single species (including from nuclear 28S gene markers), the 17 haplotypes did not fall into a monophyletic clade within the Spodoptera genus; with six haplotypes (2 each from 3 geographically separate populations) differing by >11% in their nucleotide sequence to the other eleven. CONCLUSIONS: This study suggests that three strains of Wolbachia may be driving a selective sweep on armyworm haplotype diversity, and that based on COI sequence data, S. exempta is not a monophyletic group within the Spodoptera genus. This has clear implications for the use of mtDNA as neutral genetic markers in insects, and also demonstrates the impact of Wolbachia infections on host evolutionary genetics.

Wolbachia in a major African crop pest increases susceptibility to viral disease rather than protects.

Wolbachia are common vertically transmitted endosymbiotic bacteria found in < 70% of insect species. They have generated considerable recent interest due to the capacity of some strains to protect their insect hosts against viruses and the potential for this to reduce vector competence of a range of human diseases, including dengue. In contrast, here we provide data from field populations of a major crop pest, African armyworm (Spodoptera exempta), which show that the prevalence and intensity of infection with a nucleopolydrovirus (SpexNPV) is positively associated with infection with three strains of Wolbachia. We also use laboratory bioassays to demonstrate that infection with one of these strains, a male-killer, increases host mortality due to SpexNPV by 6-14 times. These findings suggest that rather than protecting their lepidopteran host from viral infection, Wolbachia instead make them more susceptible. This finding potentially has implications for the biological control of other insect crop pests.

Complete sequence, analysis and organization of the Orgyia leucostigma nucleopolyhedrovirus genome.

The complete genome of the Orgyia leucostigma nucleopolyhedrovirus (OrleNPV) isolated from the whitemarked tussock moth (Orgyia leucostigma, Lymantridae: Lepidoptera) was sequenced, analyzed, and compared to other baculovirus genomes. The size of the OrleNPV genome was 156,179 base pairs (bp) and had a G+C content of 39%. The genome encoded 135 putative open reading frames (ORFs), which occupied 79% of the entire genome sequence. Three inhibitor of apoptosis (ORFs 16, 43 and 63), and five baculovirus repeated ORFs (bro-a through bro-e) were interspersed in the OrleNPV genome. In addition to six direct repeat (drs), a common feature shared among most baculoviruses, OrleNPV genome contained three homologous regions (hrs) that are located in the latter half of the genome. The presence of an F-protein homologue and the results from phylogenetic analyses placed OrleNPV in the genus Alphabaculovirus, group II. Overall, OrleNPV appears to be most closely related to group II alphabaculoviruses Ectropis obliqua (EcobNPV), Apocheima cinerarium (ApciNPV), Euproctis pseudoconspersa (EupsNPV), and Clanis bilineata (ClbiNPV).