Kaat Cappelle

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract  RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.

A depauperate immune repertoire precedes evolution of sociality in bees

BackgroundSociality has many rewards, but can also be dangerous, as high population density and low genetic diversity, common in social insects, is ideal for parasite transmission. Despite this risk, honeybees and other sequenced social insects have far fewer canonical immune genes relative to solitary insects. Social protection from infection, including behavioral responses, may explain this depauperate immune repertoire. Here, based on full genome sequences, we describe the immune repertoire of two ecologically and commercially important bumblebee species that diverged approximately 18 million years ago, the North American Bombus impatiens and European Bombus terrestris.ResultsWe find that the immune systems of these bumblebees, two species of honeybee, and a solitary leafcutting bee, are strikingly similar. Transcriptional assays confirm the expression of many of these genes in an immunological context and more strongly in young queens than males, affirming Bateman’s principle of greater investment in female immunity. We find evidence of positive selection in genes encoding antiviral responses, components of the Toll and JAK/STAT pathways, and serine protease inhibitors in both social and solitary bees. Finally, we detect many genes across pathways that differ in selection between bumblebees and honeybees, or between the social and solitary clades.ConclusionsThe similarity in immune complement across a gradient of sociality suggests that a reduced immune repertoire predates the evolution of sociality in bees. The differences in selection on immune genes likely reflect divergent pressures exerted by parasites across social contexts.

High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects

Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA.

Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris

To date, there are no validated internal reference genes for the normalization of RT-qPCR data from virus infection experiments with pollinating insects. In this study we evaluated the stability of five candidate internal reference genes: elongation factor-1-alpha (ELF1α), peptidylprolyl isomerase A (PPIA), 60S ribosomal protein L23 (RPL23), TATA-binding protein (TBP) and polyubiquitin (UBI), in relation to Israeli acute paralysis virus (IAPV) infection of Bombus terrestris. We investigated the stability of these genes: in whole bodies and individual body parts, as well as in whole bodies collected at different time intervals after infection with IAPV. Our data identified PPIA as the single, most-optimal internal reference gene and the combination of PPAI-RPL23-UBI as a fully-sufficient multiple internal reference genes set for IAPV infection experiments in B. terrestris.

Differential transcriptome analysis of the common shrimp Crangon crangon: Special focus on the nuclear receptors and RNAi-related genes

The decapod Crangon crangon is one of the most valuable European fisheries commodities. Despite its economic importance, little sequence data is available for this shrimp species. In this paper, we report the transcriptome sequencing for five different stages of C. crangon (early embryo, late embryo, larva, female adults and male adults) and the annotation and stage-specific expression analysis of nuclear receptors (NRs) and RNA interference (RNAi)-related genes. The NRs are transcription factors that play an essential role in growth, development, cell differentiation, molting/metamorphosis and reproduction, while the RNAi-related genes are very important for internal gene expression regulation and in antiviral defense. We discovered a NR in the female C. crangon which is either a very rapidly evolved homolog of HR10, or a novel NR altogether. This new NR could act as a biological marker for sex determination as it is not expressed in male adults. Most RNAi-related genes were present in C. crangon, proving that the requirements for successful RNAi is present in this decapod shrimp. RNAi-based applications in Crangon such as its use in functional genomics or as antiviral therapeutics could become very important in the near future.

Persistent RNA virus infection of lepidopteran cell lines : interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.

A model species for agricultural pest genomics: The genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Israeli Acute Paralysis Virus Infection Leads to an Enhanced RNA Interference Response and Not Its Suppression in the Bumblebee Bombus terrestris

RNA interference (RNAi) is the primary antiviral defense system in insects and its importance for pollinator health is indisputable. In this work, we examined the effect of Israeli acute paralysis virus (IAPV) infection on the RNAi process in the bumblebee, Bombus terrestris, and whether the presence of possible functional viral suppressors could alter the potency of the host’s immune response. For this, a two-fold approach was used. Through a functional RNAi assay, we observed an enhancement of the RNAi system after IAPV infection instead of its suppression, despite only minimal upregulation of the genes involved in RNAi. Besides, the presence of the proposed suppressor 1A and the predicted OrfX protein in IAPV could not be confirmed using high definition mass spectrometry. In parallel, when bumblebees were infected with cricket paralysis virus (CrPV), known to encode a suppressor of RNAi, no increase in RNAi efficiency was seen. For both viruses, pre-infection with the one virus lead to a decreased replication of the other virus, indicating a major effect of competition. These results are compelling in the context of Dicistroviridae in multi-virus/multi-host networks as the effect of a viral infection on the RNAi machinery may influence subsequent virus infections.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Background The Colorado potato beetle, Leptinotarsa decemlineata Say, is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to not only rapidly adapt to a broad range of solanaceaeous plants and variable climates during its global invasion, but, most notably, to rapidly evolve resistance to insecticides (over 50 different compounds in all major classes, in some cases within the first year of use). To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species, using whole-genome sequencing, transcriptome sequencing, and a large community-driven annotation effort. Results We present a 140x coverage whole genome sequence from a single female L. decemlineata, with a reference gene set of 24,740 genes. Transposable elements comprise at least 17% of the genome, and are heavily represented in an analysis of rapidly evolving gene families compared to other Coleoptera. Population genetic analyses provide evidence of high levels of nucleotide diversity, local geographic structure, and recent population growth in pest populations, pointing to the availability of considerable standing genetic variation. These factors may play an important role in rapid evolutionary change. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes (e.g. cysteine peptidase genes) in gut tissues, as well as expansions of the gustatory receptors for bitter tasting plants in the nightshade family, Solanaceae. Despite its notoriety for adapting to insecticides, L. decemlineata has a similar suite of genes involved in resistance (metabolic detoxification and cuticle penetration) compared to other beetles, although expansions in specific cytochrome P450 subfamilies are known to be associated with insecticide resistance. Finally, this beetle has interesting duplications in RNAi genes that might be linked to its high sensitivity to RNAi and could be important in the future development of gene targeted pesticides. Conclusions As a representative of one of the most evolutionarily diverse lineages, the L. decemlineata genome will undoubtedly provide new opportunities for deeper understanding on the ecology, evolution, and management of this species, as well as new opportunities to leverage genomic technologies to understand the basis of a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Distribution of glycan motifs at the surface of midgut cells in the cotton leafworm (Spodoptera littoralis) demonstrated by lectin binding

Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA), GalNAc/Gal (RSA and SSA), GlcNAc (WGA and Nictaba) and Neu5Ac(α-2,6)Gal/GalNAc (SNA-I)], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and Nictaba bound more strongly to the membrane of these undifferentiated cells compared to the microvillar pole of the columnar cells, while SSA, HHA, WGA, and SNA-I showed stronger binding to the microvilli. Our results indicated that polarization of the midgut cells is also reflected by a specific distribution of glycans, especially between the basal and microvillar pole. The data are discussed in relation to the functioning and development of the insect midgut.