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.

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.

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.

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.

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.

The major yolk protein vitellogenin interferes with the anti-plasmodium response in the malaria mosquito Anopheles gambiae.

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.

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.

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).

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.

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.

Molecular and cellular components of the mating machinery in Anopheles gambiae females.

Anopheles gambiae mosquitoes are the principal vectors of malaria. A major determinant of the capacity of these mosquitoes as disease vectors is their high reproductive rate. Reproduction depends on a single insemination, which profoundly changes the behavior and physiology of females. To identify factors and mechanisms relevant to the fertility of A. gambiae, we performed a comprehensive analysis of the molecular and cellular machinery associated with copulation in females. Initial whole-body microarray experiments comparing virgins with females at 2 h, 6 h, and 24 h after mating detected large transcriptional changes. Analysis of tissue localization identified a subset of genes whose expression was strikingly regulated by mating in the lower reproductive tract and, surprisingly, the gut. In the atrium of virgin females, where the male seminal fluid is received, our studies revealed a "mating machinery" consisting of molecular and structural components that are turned off or collapse after copulation, suggesting that this tissue loses its competence for further insemination. In the sperm storage organ, we detected a number of mating-responsive genes likely to have a role in the maintenance and function of stored sperm. These results identify genes and mechanisms regulating the reproductive biology of A. gambiae females, highlighting considerable differences with Drosophila melanogaster. Our data inform vector control strategies and reveal promising targets for the manipulation of fertility in field populations of these important disease vectors.

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.

Fz2 and cdc42 mediate melanization and actin polymerization but are dispensable for Plasmodium killing in the mosquito midgut.

The midgut epithelium of the mosquito malaria vector Anopheles is a hostile environment for Plasmodium, with most parasites succumbing to host defenses. This study addresses morphological and ultrastructural features associated with Plasmodium berghei ookinete invasion in Anopheles gambiae midguts to define the sites and possible mechanisms of parasite killing. We show by transmission electron microscopy and immunofluorescence that the majority of ookinetes are killed in the extracellular space. Dead or dying ookinetes are surrounded by a polymerized actin zone formed within the basal cytoplasm of adjacent host epithelial cells. In refractory strain mosquitoes, we found that formation of this zone is strongly linked to prophenoloxidase activation leading to melanization. Furthermore, we identify two factors controlling both phenomena: the transmembrane receptor frizzled-2 and the guanosine triphosphate-binding protein cell division cycle 42. However, the disruption of actin polymerization and melanization by double-stranded RNA inhibition did not affect ookinete survival. Our results separate the mechanisms of parasite killing from subsequent reactions manifested by actin polymerization and prophenoloxidase activation in the A. gambiae-P. berghei model. These latter processes are reminiscent of wound healing in other organisms, and we propose that they represent a form of wound-healing response directed towards a moribund ookinete, which is perceived as damaged tissue.

Competency of Anopheles stephensi mysorensis strain for Plasmodium vivax and the role of inhibitory carbohydrates to block its sporogonic cycle.

BACKGROUND: Despite the abundance of studies conducted on the role of mosquitoes in malaria transmission, the biology and interaction of Plasmodium with its insect host still holds many mysteries. This paper provides the first study to follow the sporogonic cycle of Plasmodium vivax in a wild insecticide-resistant mysorensis strain of Anopheles stephensi, a major vector of vivax malaria in south-eastern Iran. The study subsequently demonstrates that host-parasite sugar binding interactions are critical to the development of this parasite in the salivary glands of its mosquito host. The identity of the receptors or sugars involved was revealed by a receptor "pre-saturation" strategy in which sugars fed to the mosquitoes inhibited normal host-parasite interactions. METHODS: Anopheles stephensi mysorensis mosquitoes were artificially infected with P. vivax by feeding on the blood of gametocytaemic volunteers reporting to local malaria clinics in the Sistan-Baluchistan province of south-eastern Iran. In order to determine the inhibitory effect of carbohydrates on sporogonic development, vector mosquitoes were allowed to ingest blood meals containing both gametocytes and added carbohydrates. The carbohydrates tested were GlcNAc, GalNAc, arabinose, fucose, mannose, lactose, glucose and galactose. Sporogonic development was assessed by survival of the parasite at both the oocyst and sporozoite stages. RESULTS: Oocyst development was observed among nearly 6% of the fed control mosquitoes but the overall number of mosquitoes exhibiting sporozoite invasion of the salivary glands was 47.5% lower than the number supporting oocysts in their midgut. Of the tested carbohydrates, only arabinose and fucose slightly perturbed the development of P. vivax oocysts at the basal side of the mosquito midgut, and the remaining sugars caused no reductions in oocyst development. Strikingly however, sporozoites were completely absent from the salivary glands of mosquitoes treated with mannose, GalNAc, and lactose. CONCLUSION: The study indicates that An. stephensi in southern Iran has the potential to survive long enough to be re-infected and transmit vivax malaria several times, based on the average adult female longevity (about 30 days) and its gonotrophic cycle (2-3 days) during the malaria transmission season. Certain sugar binding interactions are important for the development of P. vivax sporozoites, and this information may be instrumental for the development of transmission blocking strategies.

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.

Differential modulation of Rhodnius prolixus nitric oxide activities following challenge with Trypanosoma rangeli, T. cruzi and bacterial cell wall components.

Nitric oxide (NO) is a key immune effector and signaling molecule in many organisms. However, the contribution NO makes towards insect immunity has received little attention, particularly in non-dipteran species. In this study, tissue- and time-specific alterations in NO synthase (NOS) gene expression and NO production were documented in the hemipteran vector of Chagas' disease, Rhodnius prolixus, following in vivo immune challenge by Trypanosoma cruzi, T. rangeli and crude bacterial lipopolysaccharide (LPS). The most pronounced reactions to crude LPS occurred in major immune tissues (fat body and blood cells), while tissues of the digestive tract were most responsive to infections by T. cruzi and T. rangeli. The data suggest the NO-mediated immune responses of R. prolixus are pathogen-specific and independently modified both at the transcriptional and enzyme levels.

Exploring the role of insect host factors in the dynamics of Trypanosoma cruzi-Rhodnius prolixus interactions.

Members of the subfamily Triatominae, family Reduviidae, comprise a large number of insect species of which some are vectors of Trypanosoma cruzi, the causative agent of Chagas' disease. This article outlines research on the process of transformation and the dynamics of developmental stages of Trypanosoma cruzi in the triatomine insect hosts. Special attention is given to the interactions of parasites with gut molecules, and the gut environment, and with host developmental physiology and intestinal organization. The vector insect's permissiveness to Trypanosoma cruzi, which develops in the vector gut, largely depends on the host nutritional state, the parasite strain, trypanolytic compounds, digestive enzymes, lectins, resident bacteria in the gut and the endocrine system of the insect vector. Finally, the mechanisms of these interactions and their significance for Trypanosoma cruzi transmission are discussed.

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.