A narrow host-range and lack of persistence in two non-target insect species of a bacterial symbiont exploited to deliver insecticidal RNAi in Western Flower Thrips.

Introduction: Insecticidal RNAi is a targeted pest insect population control measure. The specificity of insecticidal RNAi can theoretically be enhanced by using symbiotic bacteria with a narrow host range to deliver RNAi, an approach termed symbiont-mediated RNAi (SMR), a technology we have previously demonstrated in the globally-invasive pest species Western Flower Thrips (WFT). Methods: Here we examine distribution of the two predominant bacterial symbionts of WFT, BFo1 and BFo2, among genome-sequenced insects. Moreover, we have challenged two non-target insect species with both bacterial species, namely the pollinating European bumblebee, Bombus terrestris, and an insect predator of WFT, the pirate bug Orius laevigatus. Results: Our data indicate a very limited distribution of either symbiont among insects other than WFT. Moreover, whereas BFo1 could establish itself in both bees and pirate bugs, albeit with no significant effects on insect fitness, BFo2 was unable to persist in either species. Discussion: In terms of biosafety, these data, together with its more specific growth requirements, vindicate the choice of BFo2 for delivery of RNAi and precision pest management of WFT.

New Cell Lines Derived from Laboratory Colony Triatoma infestans and Rhodnius prolixus, Vectors of Trypanosoma cruzi, Do Not Harbour Triatoma Virus.

Triatomine bugs of the genera Triatoma and Rhodnius are vectors of Chagas disease, a neglected tropical disease of humans in South America caused by Trypanosoma cruzi. Triatoma virus (TrV), a natural pathogen of Triatoma infestans, has been proposed as a possible tool for the bio-control of triatomine bugs, but research into this virus has been hampered by a lack of suitable host cells for in vitro propagation. Here we report establishment and partial characterisation of continuous cell lines from embryos of T. infestans (TIE/LULS54) and Rhodnius prolixus (RPE/LULS53 and RPE/LULS57). RNAseq screening by a sequence-independent, single primer amplification approach confirmed the absence of TrV and other RNA viruses known to infect R. prolixus, indicating that these new cell lines could be used for propagation of TrV.

Bacterial Competition Influences the Ability of Symbiotic Bacteria to Colonize Western Flower Thrips.

Symbiont mediated RNAi (SMR) is a promising method for precision control of pest insect species such as Western Flower Thrips (WFT). Two species of bacteria are known to be dominant symbiotic bacteria in WFT, namely BFo1 and BFo2 (Bacteria from Frankliniella occidentalis 1 and 2), as we here confirm by analysis of next-generation sequence data derived to obtain a reference WFT genome sequence. Our first demonstration of SMR in WFT used BFo2, related to Pantoea, isolated from a domesticated Dutch thrips population. However, for successful use of SMR as a thrips control measure, these bacteria need to successfully colonize different environmental thrips populations. Here, we describe a United Kingdom thrips population that does not harbour BFo2, but does contain BFo1, a species related to Erwinia. Attempts to introduce BFo2 indicate that this bacterium is unable to establish itself in the United Kingdom thrips, in contrast to successful colonization by a strain of BFo1 expressing green fluorescent protein. Fluorescence microscopy indicates that BFo1 occupies similar regions of the thrips posterior midgut and hindgut as BFo2. Bacterial competition assays revealed that a barrier to BFo2 establishing itself in thrips is the identity of the resident BFo1; BFo1 isolated from the United Kingdom thrips suppresses growth of BFo2 to a greater extent than BFo1 from the Dutch thrips that is permissive for BFo2 colonization. The ability of the latter strain of BFo1 to colonize the United Kingdom thrips is also likely attributable to its ability to out-compete the resident BFo1. Lastly, we observed that United Kingdom thrips pre-exposed to the Dutch BFo1 could then be successfully colonized by BFo2. These results indicate, for the first time, that microbial competition and strain differences can have a large influence on how symbiotic bacteria can colonize different populations of an insect species.

Symbiont-Mediated RNA Interference (SMR): Using Symbiotic Bacteria as Vectors for Delivering RNAi to Insects.

RNA interference (RNAi) has emerged as a widely used approach for reverse genetic analysis in eukaryotes. In insects, RNAi also has an application in the control of insect pests. Several methods have been developed for delivery of interfering RNA in insects, with varying outcomes for different species. Here we describe how a bacterial symbiont can be exploited for continuous synthesis of interfering double-stranded RNA (dsRNA) in its insect host. This approach, termed symbiont-mediated RNAi (SMR), can overcome problems associated with instability of dietary dsRNA due to action of salivary or foregut nucleases. As insects do not possess RNA-dependent RNA polymerase activity that can amplify and extend RNAi in other organisms, SMR also offers the possibility of long-term systemic RNAi not afforded by single applications of dsRNA to insects by other delivery methods. Here, we describe how SMR can be applied in a globally distributed agricultural pest species, western flower thrips (Frankliniella occidentalis).

Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci.

In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well-formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double-strand RNA (dsRNA)-expressing recombinant bacteria. In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA-expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting "naked" dsRNA or dsRNA-expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding "naked" dsRNA to an artificial diet was an unreliable form of RNAi delivery in our hands due to dsRNA degradation. Delivery via host plants was more successful, and the new diet formulation was suitable for symbiont-mediated dsRNA delivery, which we believe is the most convenient approach for large-scale knockdown experiments. This study, therefore, provides alternative methodologies for thrips rearing, dietary RNAi delivery, and insights into the challenges of performing dietary RNAi in nonmodel insects.

Novel RNAi delivery systems in the control of medical and veterinary pests.

RNA interference (RNAi) is a transformative technology with great potential to control, study or even protect insects and acarines through the knockdown of target gene expression. RNAi offers unprecedented levels of control, but fundamental to its successful deployment is the need to deliver 'trigger' RNA in an appropriate fashion giving due consideration to potential barriers of RNAi efficiency, safety, and the intended purpose of the knockdown. This short review focusses on recent innovations in RNAi delivery that are designed for, or could be adapted for use with, insect and acarine pests of medical or veterinary importance.

Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella.

This study examines how the dynamics of fungus-insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well-studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme-based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4-7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents.

Re-evaluation of insect melanogenesis research: Views from the dark side.

Melanins (eumelanin and pheomelanin) are synthesized in insects for several purposes including cuticle sclerotization and color patterning, clot formation, organogenesis, and innate immunity. Traditional views of insect immunity detail the storage of pro-phenoloxidases inside specialized blood cells (hemocytes) and their release upon recognition of foreign bodies. Activated phenoloxidases convert monophenols into reactive quinones in a two-step enzymatic reaction, and until recently, the mechanism of tyrosine hydroxylation remained a mystery. Herein, we present our interpretations of these enzyme-substrate complexes. The resultant melanins are deposited onto the surface of microbes to immobilize, agglutinate, and suffocate them. Phenoloxidase activity and melanin production are not limited to the blood (hemolymph) or cuticle, as recent evidence points to more diverse, sophisticated interactions in the gut and with the resident symbionts. This review offers insight into the somewhat neglected areas of insect melanogenesis research, particularly in innate immunity, its role in beneficial insects such as pollinators, the functional versatility of phenoloxidases, and the limitations of common experimental approaches that may impede progress inadvertently.

Gene silencing in non-model insects: Overcoming hurdles using symbiotic bacteria for trauma-free sustainable delivery of RNA interference: Sustained RNA interference in insects mediated by symbiotic bacteria: Applications as a genetic tool and as a biocide.

Insight into animal biology and development provided by classical genetic analysis of the model organism Drosophila melanogaster was an incentive to develop advanced genetic tools for this insect. But genetic systems for the over one million other known insect species are largely undeveloped. With increasing information about insect genomes resulting from next generation sequencing, RNA interference is now the method of choice for reverse genetics, although it is constrained by the means of delivery of interfering RNA. A recent advance to ensure sustained delivery with minimal experimental intervention or trauma to the insect is to exploit commensal bacteria for symbiont-mediated RNA interference. This technology not only offers an efficient means for RNA interference in insects in laboratory conditions, but also has potential for use in the control of human disease vectors, agricultural pests and pathogens of beneficial insects.

Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis.

Microevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susceptible insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management and immune defense in the fat body. Following oral infection with Bt, the expression of these genes was further elevated in the fat body and midgut of both lines and to a greater extent some of them in resistant line than the susceptible line. This gene expression analysis reveals a pattern of resistance mechanisms targeted to sites damaged by Bt with the insect placing greater emphasis on tissue repair as revealed by elevated expression of these genes in both the fat body and midgut epithelium. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affects the viability of other gut bacteria. Remarkably the resistant line employs multifactorial adaptations for resistance to Bt without any detected negative trade off since the insects exhibited higher fecundity.

Symbiont-mediated RNA interference in insects.

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects.

A novel bacterial infection of the edible crab, Cancer pagurus.

There are few reports of bacterial diseases in crabs. A juvenile edible crab (Cancer pagurus) with a rickettsial-like infection was found in the intertidal zone at Freshwater East in South West Wales in July, 2012. Large numbers of bacteria-like particles were found in the haemolymph and within fixed phagocytes of the hepatopancreas. Molecular sequencing and subsequent phylogenetic analysis showed that the infectious agent was a member of the order Rhizobiales and therefore distinct to bacteria classified as rickettsia.

Draft Genomes, Phylogenetic Reconstruction, and Comparative Genomics of Two Novel Cohabiting Bacterial Symbionts Isolated from Frankliniella occidentalis.

Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola. BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae. The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea.

Disease profiles of juvenile edible crabs (Cancer pagurus L.) differ at two geographically-close intertidal sites.

The prevalence of disease in edible crabs (Cancer pagurus) was assessed at two sites in South West Wales; one estuarine (Pembroke Ferry) and another facing open water (Freshwater East). Diseases included pink crab disease caused by Hematodinium sp., an infection of the antennal gland caused by Paramikrocytos canceri and an idiopathic inflammatory condition of the connective tissue surrounding the anterior ganglionic masses. This latter condition was only found in crabs from Pembroke Ferry. There was a significantly higher prevalence of pink crab disease at Freshwater East than Pembroke Ferry, although both sites had similar levels of infection by P. canceri.

Cuticles of European and American lobsters harbor diverse bacterial species and differ in disease susceptibility.

Diseases of lobster shells have a significant impact on fishing industries but the risk of disease transmission between different lobster species has yet to be properly investigated. This study compared bacterial biofilm communities from American (Homarus americanus) and European lobsters (H. gammarus), to assess both healthy cuticle and diseased cuticle during lesion formation. Culture-independent molecular techniques revealed diversity in the bacterial communities of cuticle biofilms both within and between the two lobster species, and identified three bacterial genera associated with shell lesions plus two putative beneficial bacterial species (detected exclusively in healthy cuticle or healing damaged cuticle). In an experimental aquarium shared between American and European lobsters, heterospecific transmission of potentially pathogenic bacteria appeared to be very limited; however, the claws of European lobsters were more likely to develop lesions when reared in the presence of American lobsters. Aquarium biofilms were also examined but revealed no candidate pathogens for environmental transmission. Aquimarina sp. 'homaria' (a potential pathogen associated with a severe epizootic form of shell disease) was detected at a much higher prevalence among American than European lobsters, but its presence correlated more with exacerbation of existing lesions rather than with lesion initiation.

A comparison of the structure of American (Homarus americanus) and European (Homarus gammarus) lobster cuticle with particular reference to shell disease susceptibility.

The integument of arthropods is an important first-line defence against the invasion of parasites and pathogens. Once damaged, this can be subject to colonisation by microbial agents from the surrounding environment, which in crustaceans can lead to a condition termed shell disease syndrome. This condition has been reported in several crustacean species, including crabs and lobsters. The syndrome is a progressive condition where the outer cuticle becomes pitted and eroded, and in extreme cases is compromised, leaving animals susceptible to septicaemia. This study examined the susceptibility of juvenile American (Homarus americanus) and European (Homarus gammarus) lobsters to shell disease, as a result of mechanical damage. Scanning electron microscopy was used as a method to identify differences in the cuticle structure and consequences of mechanical damage. Claw regions were aseptically punctured, whilst carapaces were abraded using sterile sandpaper, to mimic natural damage. After a period of between 10 and 12 weeks, lobsters were sacrificed, fixed and stored for later examination. The carapace and claws of juvenile American lobsters were shown to be thinner and more vulnerable to abrasion damage than their European counterparts. In addition, the number and distribution of setal pits and pore canal openings also differed between the two species of lobster. Mechanical damage resulted in the formation of shell disease lesions on the claw and carapace of both lobster species. However, American lobsters, unlike their European counterparts, had extensive bacterial colonisation on the margins of these lesions. Overall, it is concluded that the cuticle of the American lobster is more susceptible to damage and resulting microbial colonisation. This may have implications for susceptibility of both species of lobster to shell disease syndrome.

Comparative susceptibility of different biological forms of Anopheles stephensi to Plasmodium berghei ANKA strain.

BACKGROUND: There are varying degrees of compatibility between malaria parasite-mosquito species, and understanding this compatibility may be crucial for developing effective transmission-blocking vaccines. This study investigates the compatibility of different biological forms of a malaria vector, Anopheles stephensi, to Plasmodium berghei ANKA strain. METHODS: Several biologically different and allopatric forms of A. stephensi were studied. Three forms were isolated from different regions of southern Iran: the variety mysorensis, the intermediate form and the native type form, and an additional type form originated from India (Beech strain).The mosquitoes were experimentally infected with P. berghei to compare their susceptibility to parasitism. Anti-mosquito midgut antiserum was then raised in BALB/cs mice immunized against gut antigens from the most susceptible form of A. stephensi (Beech strain), and the efficacy of the antiserum was assessed in transmission-blocking assays conducted on the least susceptible mosquito biological form. RESULTS: The susceptibility of different biological forms of A. stephensi mosquito to P. berghei was specifically inter-type varied. The Beech strain and the intermediate form were both highly susceptible to infection, with higher oocyst and sporozoite infection rates than intermediate and mysorensis forms. The oocyst infection, and particularly sporozite infection, was lowest in the mysorensis strain. Antiserum raised against midgut proteins of the Indian Beech type form blocked infection in this mosquito population, but it was ineffective at blocking both oocyst and sporozoite development in the permissive but geographically distant intermediate form mosquitoes. This suggests that a strong degree of incompatibility exists between the mosquito strains in terms of midgut protein(s) acting as putative ookinete receptors. CONCLUSIONS: The incompatibility in the midgut protein profiles between two biological forms of A. stephensi demonstrates a well-differentiated population structure according to geographical origin. Therefore, the design of potential transmission-blocking strategies should incorporate a more thorough understanding of intra-species variations in host-parasite interactions.

Draft Genome Sequence of Rhodococcus rhodnii Strain LMG5362, a Symbiont of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the Principle Vector of Trypanosoma cruzi.

We report the 4,385,577-bp high-quality draft assembly of the bacterial symbiont Rhodococcus rhodnii strain LMG5362, isolated from the gut of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the principle vector of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This sequence might provide useful information for subsequent studies of the symbiotic relationship between Rd. prolixus and Rc. rhodnii, while also providing a starting point for the development of biotechnological applications for the control of Rd. prolixus.

Can insects develop resistance to insect pathogenic fungi?

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25(th) generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses.