Characterisation of lepidopteran geranylgeranyl diphosphate synthase as a putative pesticide target.

Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.

Cell penetrating peptides are versatile tools for enhancing multimodal uptake into cells from pest insects.

Cell penetrating peptides (CPPs) are small peptides defined by their ability to deliver molecular cargo into cells. While the subject of frequent investigation for pharmaceutical drug delivery, little consideration has been given to the possibility of CPPs for use as insecticides or insecticide enhancers. Here, we characterize the entry of four fluorescently tagged CPPs into two insect cell lines and dissected midgut tissues in terms of both total quantity and mode of penetration. Fluorescent microscopy showed that substantial amounts of CPPs penetrate the plasma membrane via endosomal uptake in ovarian (Sf9) and midgut derived (AW1) lepidopteran cells and that this process was sensitive to selected endocytosis inhibitors. Differences in the quantity of uptake was observed between CPPs, and further differences were found in the ability CPP-1838 to efficiently penetrate membranes through passive diffusion. These findings were extended to primary midgut derived cells and dissected tissues suggesting that CPPs show a preference for goblet cells and that CPP-1838 shows far higher rates of penetration. CPP-1838 thus shows extraordinary abilities to penetrate cells efficiency in both a diffusional and endocytotic manner. From these results more sophisticated delivery methods based on the utilization of CPPs can be developed.

A spatiotemporal atlas of the lepidopteran pest Helicoverpa armigera midgut provides insights into nutrient processing and pH regulation.

BACKGROUND: Caterpillars from the insect order Lepidoptera are some of the most widespread and destructive agricultural pests. Most of their impact is at the larval stage, where the midgut epithelium mediates the digestion and absorption of an astonishing amount of food. Although this tissue has been the subject of frequent investigation in Lepidoptera, a comprehensive expression atlas has yet to be generated. RESULTS: Here, we perform RNA-sequencing and proteomics on the gut of the polyphagous pest Helicoverpa armigera across, life stages, diet types, and compartments of the anterior-posterior axis. A striking relationship between the structural homology and expression pattern of a group of sugar transporters was observed in the early larval stages. Further comparisons were made among the spatial compartments of the midgut, which suggested a putative role for vATPases and SLC9 transporters in the generation of alkaline conditions in the H. armigera midgut. CONCLUSIONS: This comprehensive resource will aid the scientific community in understanding lepidopteran gut physiology in unprecedented resolution. It is hoped that this study advances the understanding of the lepidopteran midgut and also facilitates functional work in this field.

Characterization of a novel pesticide transporter and P-glycoprotein orthologues in Drosophila melanogaster.

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge about mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect Drosophila melanogaster. An RNAi screen was performed, which knocked down individual ABCs in specific epithelial tissues and examined the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This implicated a novel ABC drug transporter, CG4562, in spinosad transport, but also highlighted the P-glycoprotein orthologue Mdr65 as the most impactful ABC in terms of chemoprotection. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, transgenic Drosophila lines expressing P-glycoprotein from the major malaria vector Anopheles gambiae were used to establish a system for in vivo characterization of this transporter in non-model insects. This study provides the basis for establishing Drosophila as a model for toxicology research on drug transporters.

Using tissue specific P450 expression in Drosophila melanogaster larvae to understand the spatial distribution of pesticide metabolism in feeding assays.

Drug metabolizing enzymes such as cytochrome P450s have often been implicated in influencing levels of pesticide toxicology and resistance. Consequently, a variety of different P450 genes and variants have been linked to pesticide metabolism. Substantially less is known in regards to which tissues these P450s contribute to pesticide metabolism. Here, we isolate the effect of different tissues in pesticide toxicology by driving the model P450 Cyp6g1 in specific tissues of Drosophila melanogaster. Fluorescent and luminescent assays were used to compare the strength of GAL4 lines specific to the midgut (Mex-GAL4), Malpighian tubules (UO-GAL4) and the fat body (LSP2-GAL4) with the widely used HR-GAL4 line which drives GAL4 expression in all three tissues simultaneously. These data suggested that GAL4 drivers specific for the midgut and fat body were of approximately equal strength to the HR-GAL4 line, while the Malpighian tubule specific line was significantly weaker. Multiple toxicology assays using the pesticides bendiocarb, imidacloprid and malathion were then performed to assess which tissues provide the most chemoprotection. In the long-term feeding assay, transgenic expression of Cyp6g1 specifically in the midgut accounted for the majority of the resistance caused by Cyp6g1 overexpression with the HR-GAL4 driver. Real-time toxicology assays on third instar larvae were also performed and showed variable contributions of tissues to acute toxicology response depending on which pesticide was used. These data suggest a strong influence of bioassay parameters such as life stage and dosing method on outcome but suggest a prominent role for the midgut in larval toxicology.

Comparative and functional genomics of the ABC transporter superfamily across arthropods.

BACKGROUND: The ATP-binding cassette (ABC) transporter superfamily is comprised predominantly of proteins which directly utilize energy from ATP to move molecules across the plasma membrane. Although they have been the subject of frequent investigation across many taxa, arthropod ABCs have been less well studied. While the manual annotation of ABC transporters has been performed in many arthropods, there has so far been no systematic comparison of the superfamily within this order using the increasing number of sequenced genomes. Furthermore, functional work on these genes is limited. RESULTS: Here, we developed a standardized pipeline to annotate ABCs from predicted proteomes and used it to perform comparative genomics on ABC families across arthropod lineages. Using Kruskal-Wallis tests and the Computational Analysis of gene Family Evolution (CAFE), we were able to observe significant expansions of the ABC-B full transporters (P-glycoproteins) in Lepidoptera and the ABC-H transporters in Hemiptera. RNA-sequencing of epithelia tissues in the Lepidoptera Helicoverpa armigera showed that the 7 P-glycoprotein paralogues differ substantially in their tissue distribution, suggesting a spatial division of labor. It also seems that functional redundancy is a feature of these transporters as RNAi knockdown showed that most transporters are dispensable with the exception of the highly conserved gene Snu, which is probably due to its role in cuticular formation. CONCLUSIONS: We have performed an annotation of the ABC superfamily across > 150 arthropod species for which good quality protein annotations exist. Our findings highlight specific expansions of ABC transporter families which suggest evolutionary adaptation. Future work will be able to use this analysis as a resource to provide a better understanding of the ABC superfamily in arthropods.

A transcriptomic and proteomic atlas of expression in the Nezara viridula (Heteroptera: Pentatomidae) midgut suggests the compartmentalization of xenobiotic metabolism and nutrient digestion.

BACKGROUND: Stink bugs are an emerging threat to crop security in many parts of the globe, but there are few genetic resources available to study their physiology at a molecular level. This is especially true for tissues such as the midgut, which forms the barrier between ingested material and the inside of the body. RESULTS: Here, we focus on the midgut of the southern green stink bug Nezara viridula and use both transcriptomic and proteomic approaches to create an atlas of expression along the four compartments of the anterior-posterior axis. Estimates of the transcriptome completeness were high, which led us to compare our predicted gene set to other related stink bugs and Hemiptera, finding a high number of species-specific genes in N. viridula. To understand midgut function, gene ontology and gene family enrichment analyses were performed for the most highly expressed and specific genes in each midgut compartment. These data suggested a role for the anterior midgut (regions M1-M3) in digestion and xenobiotic metabolism, while the most posterior compartment (M4) was enriched in transmembrane proteins. A more detailed characterization of these findings was undertaken by identifying individual members of the cytochrome P450 superfamily and nutrient transporters thought to absorb amino acids or sugars. CONCLUSIONS: These findings represent an initial step to understand the compartmentalization and physiology of the N. viridula midgut at a genetic level. Future studies will be able to build on this work and explore the molecular physiology of the stink bug midgut.

'What I cannot create, I do not understand': functionally validated synergism of metabolic and target site insecticide resistance.

The putative synergistic action of target-site mutations and enhanced detoxification in pyrethroid resistance in insects has been hypothesized as a major evolutionary mechanism responsible for dramatic consequences in malaria incidence and crop production. Combining genetic transformation and CRISPR/Cas9 genome modification, we generated transgenic Drosophila lines expressing pyrethroid metabolizing P450 enzymes in a genetic background along with engineered mutations in the voltage-gated sodium channel (para) known to confer target-site resistance. Genotypes expressing the yellow fever mosquito Aedes aegypti Cyp9J28 while also bearing the paraV1016G mutation displayed substantially greater resistance ratio (RR) against deltamethrin than the product of each individual mechanism (RRcombined: 19.85 > RRCyp9J28: 1.77 × RRV1016G: 3.00). Genotypes expressing Brassicogethes aeneus pollen beetle Cyp6BQ23 and also bearing the paraL1014F (kdr) mutation, displayed an almost multiplicative RR (RRcombined: 75.19 ≥ RRCyp6BQ23: 5.74 × RRL1014F: 12.74). Reduced pyrethroid affinity at the target site, delaying saturation while simultaneously extending the duration of P450-driven detoxification, is proposed as a possible underlying mechanism. Combinations of target site and P450 resistance loci might be unfavourable in field populations in the absence of insecticide selection, as they exert some fitness disadvantage in development time and fecundity. These are major considerations from the insecticide resistance management viewpoint in both public health and agriculture.

Sublethal larval exposure to imidacloprid impacts adult behaviour in Drosophila melanogaster.

Pesticides are now chronically found in numerous ecosystems incurring widespread toxic effects on multiple organisms. For insects, the larvae are very exposed to pesticide pollution and the acute effect of insecticides on larvae has been characterized in a range of species. However, the carry-on effects in adults of sublethal exposure occurring in larvae are not well characterized. Here, we use a collection of strains of Drosophila melanogaster differing in their larval resistance to a commonly used insecticide, imidacloprid, and we test the effect of larval exposure on behavioural traits at the adult stage. Focusing on locomotor activity and on courtship and mating behaviour, we observed a significant carry-on effect of imidacloprid exposure. The heritability of activity traits measured in flies exposed to imidacloprid was higher than measured in controls and in these, courtship traits were genetically less correlated from mating success. Altogether, we did not observe a significant effect of the larval insecticide resistance status on adult behavioural traits, suggesting that selection for resistance in larvae does not involve repeatable behavioural changes in adults. This lack of correlation between larval resistance and adult behaviour also suggests that resistance at the larval stage does not necessarily result in increased behavioural resilience at a later life stage. These findings imply that selection for resistance in larvae as well as for behavioural resilience to sublethal exposure in adult will combine and impose a greater evolutionary constraint. Our conclusions further substantiate the need to encompass multiple trait measures and life stages in toxicological assays to properly assess the environmental impact of pesticides.

Development of efficient RNAi in Nezara viridula for use in insecticide target discovery.

Stink bugs are an emerging pest in many regions of the world but their molecular biology is still poorly understood. While several transcriptomes are available, the lack of validated gene manipulation tools like RNA interference (RNAi) in species such as the southern green stinkbug Nezara viridula precludes the characterization of individual genes in vivo. Such tools are particularly useful in performing high-throughput screens to search for essential genes that can be prioritized as potential insecticide targets. Here, we developed and optimized an efficient RNAi in N. viridula for use in insecticide target discovery and beyond. The visible marker Sex combs reduced and the essential gene Actin were used to verify the usability and efficiency of RNAi by microinjection at both the adult and nymphal stages, respectively, with nymphal approach presenting significant advantages. Following validation, RNAi was then used to measure lethality following the knockdown (KD) of two genes that are known insecticide targets, Chitin synthase, and Acetyl-CoA carboxylase. The KD of each gene resulted in >75% corrected mortality. These results indicate that RNAi is an effective tool in N. viridula and set a benchmark to evaluate potential targets in future RNAi screens aimed at insecticide target discovery.

Using CRISPR/Cas9 genome modification to understand the genetic basis of insecticide resistance: Drosophila and beyond.

Chemical insecticides are a major tool for the control of many of the world's most damaging arthropod pests. However, their intensive application is often associated with the emergence of resistance, sometimes with serious implications for sustainable pest control. To mitigate failure of insecticide-based control tools, the mechanisms by which insects have evolved resistance must be elucidated. This includes both identification and functional characterization of putative resistance genes and/or mutations. Research on this topic has been greatly facilitated by using powerful genetic model insects like Drosophila melanogaster, and more recently by advances in genome modification technology, notably CRISPR/Cas9. Here, we present the advances that have been made through the application of genome modification technology in insecticide resistance research. The majority of the work conducted in the field to date has made use of genetic tools and resources available in D. melanogaster. This has greatly enhanced our understanding of resistance mechanisms, especially those mediated by insensitivity of the pesticide target-site. We discuss this progress for a series of different insecticide targets, but also report a number of unsuccessful or inconclusive attempts that highlight some inherent limitations of using Drosophila to characterize resistance mechanisms identified in arthropod pests. We also discuss an experimental framework that may circumvent current limitations while retaining the genetic versatility and robustness that Drosophila has to offer. Finally, we describe examples of direct CRISPR/Cas9 use in non-model pest species, an approach that will likely find much wider application in the near future.

Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling.

Trypanosomatids are single-celled parasites responsible for human and animal disease. Typically, colonization of an insect host is required for transmission. Stable attachment of parasites to insect tissues via their single flagellum coincides with differentiation and morphological changes. Although attachment is a conserved stage in trypanosomatid life cycles, the molecular mechanisms are not well understood. To study this process, we elaborate upon an in vitro model in which the swimming form of the trypanosomatid Crithidia fasciculata rapidly differentiates following adhesion to artificial substrates. Live imaging of cells transitioning from swimming to attached shows parasites undergoing a defined sequence of events, including an initial adhesion near the base of the flagellum immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like attachment plaque between the tip of the shortened flagellum and the substrate. Quantitative proteomics of swimming versus attached parasites suggests differential regulation of cyclic adenosine monophosphate (cAMP)-based signaling proteins. We have localized two of these proteins to the flagellum of swimming C. fasciculata; however, both are absent from the shortened flagellum of attached cells. Pharmacological inhibition of cAMP phosphodiesterases increased cAMP levels in the cell and prevented attachment. Further, treatment with inhibitor did not affect the growth rate of either swimming or established attached cells, indicating that its effect is limited to a critical window during the early stages of adhesion. These data suggest that cAMP signaling is required for attachment of C. fasciculata and that flagellar signaling domains may be reorganized during differentiation and attachment.IMPORTANCETrypanosomatid parasites cause significant disease burden worldwide and require insect vectors for transmission. In the insect, parasites attach to tissues, sometimes dividing as attached cells or producing motile, infectious forms. The significance and cellular mechanisms of attachment are relatively unexplored. Here, we exploit a model trypanosomatid that attaches robustly to artificial surfaces to better understand this process. This attachment recapitulates that observed in vivo and can be used to define the stages and morphological features of attachment as well as conditions that impact attachment efficiency. We have identified proteins that are enriched in either swimming or attached parasites, supporting a role for the cyclic AMP signaling pathway in the transition from swimming to attached. As this pathway has already been implicated in environmental sensing and developmental transitions in trypanosomatids, our data provide new insights into activities required for parasite survival in their insect hosts.

Transgenic Drosophila to Functionally Validate Fall Armyworm ABCC2 Mutations Conferring Bt Resistance.

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae) is an invasive agricultural pest with a global distribution, causing major crop losses annually. Its control strategies largely rely on chemical insecticides and transgenic crops expressing Bacillus thuringiensis insecticidal proteins (Cry and Vip toxins); however, the development of high resistance poses a significant issue. The ATP-binding cassette transporter C2 (ABCC2) has been linked to Cry toxin pore formation, acting as a receptor of some Cry toxins. Recently detected mutations in the SfABCC2 gene in extracellular loop 4 (ECL4) have been associated with Bt toxin resistance in FAW. In the present study, we expressed the SfABCC2 gene in Drosophila melanogaster, a species normally unaffected by the Bt toxins. We demonstrate that susceptibility can be introduced by the ectopic and tissue-specific expression of wildtype SfABCC2. Next, we introduced mutations into ECL4-both individually and in combination-that have been recently described in Brazilian FAW and functionally validated by toxicity bioassays against the foliar Bt product Xentari. Our results provide an efficient demonstration of the suitability of transgenic Drosophila for validating FAW ABCC2 resistance mutations in ECL4 against Bt toxins, and potential cross-resistance issues between closely related proteins that use ABCC2.

Identification of Helicoverpa armigera promoters for biotechnological applications.

Helicoverpa armigera and Helicoverpa zea are highly polyphagous major agricultural pests with a global distribution. Their control is based on insecticides, however, new, effective, and environmentally friendly control tools are required to be developed and validated. In an effort to facilitate the development of advanced biotechnological tools in these species that will take advantage of new powerful molecular biology techniques like CRISPR/Cas9, we used available transcriptomic data and literature resources, in order to identify RNA polymerase II and III promoters active in RP-HzGUT-AW1(MG), a midgut derived cell line from Helicoverpa zea. Following functional analysis in insect cell lines, four RNA polymerase II promoters from the genes HaLabial, HaTsp-2A, HaPtx-I and HaCaudal were found to exhibit high transcriptional activity in vitro. The HaTsp-2A promoter did not exhibit any activity in the non-midgut derived cell lines Sf-9 and Hi-5 despite high sequence conservation among Lepidoptera, suggesting that it may function in a gut specific manner. Furthermore, considering the utility of RNA polymerase III U6 promoters in methodologies such as RNAi and CRISPR/Cas9, we identified and evaluated four different U6 promoters of H. armigera. In vitro experiments based on luciferase and GFP reporter assays, as well as in vivo experiments targeting an essential gene of Helicoverpa, indicate that these U6 promoters are functional and can be used to experimentally silence or knockout target genes through the expression of shRNAs and sgRNAs respectively. Taking our findings together, we provide a set of promoters useful for the genetic manipulation of Helicoverpa species, that can be used in various applications in the context of agricultural biotechnology.

Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation.

BACKGROUND: Canine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. METHODS: This study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA. RESULTS: Malpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed. CONCLUSIONS: Our data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity.

Functional characterization of putative ecdysone transporters in lepidopteran pests.

The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone enters cells through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone importer (EcI) from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that EcI likely appeared only at the root of the arthropod lineage. Partial disruption of EcI in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of EcI in the lepidoptera cell line RP-HzGUT-AW1(MG) demonstrated this protein's ability to control ecdysone mediated signaling in gene regulation, its role in ecdysone mediated cell death, and its sensitivity to rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates.

Can the mammalian organoid technology be applied to the insect gut?

Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self-organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance. © 2020 Society of Chemical Industry.

Functional characterization and transcriptomic profiling of a spheroid-forming midgut cell line from Helicoverpa zea (Lepidoptera: Noctuidae).

Insect cell lines have been frequently used in insect science research in recent years. Establishment of cell lines from specialized tissues like the lepidopteran midgut is expected to facilitate research efforts towards the understanding of uptake and metabolic properties, as well as the design of assays for use in pesticide discovery. However, the number of available lines from specialized tissues of insects and the level of understanding of the biological processes taking place in insect cells is far behind mammalian systems. In this study we examine two established cell lines of insect midgut origin, investigate their growth parameters and amenability to transfection and genetic manipulation, and test their potential to form spheroid-like 3D structures. Our results indicate that a midgut-derived cell line from Helicoverpa zea, RP-HzGUT-AW1, is amenable to genetic manipulation by transfection with a standard insect expression vector and has excellent ability to form spheroids. To further investigate the differentiation status of this line, we examined for expression of several candidate marker genes from different midgut cell types, enterocytes (ECs), Goblet cells (GCs), enteroendocrine cells (EEs) and intestinal stem cells (ISCs), indicating that both certain ISC and certain differentiated cell markers were present. To acquire a more detailed perspective of the differentiation landscape of the specific cells, we performed an RNAseq analysis of RP-HzGUT-AW1 grown either in 2D or 3D cultures. We hypothesize that RP-HzGUT-AW1 are in an "arrested" developmental stage between ISC and terminal differentiation. Furthermore, an enrichment of stress response and oxidoreductase genes was observed in the spheroid samples while no significant difference was evident in differentiation markers between cells grown in 2D and 3D. These results render RP-HzGUT-AW1 as the most well-characterized insect gut derived cell line so far, and lay the groundwork for future work investigating midgut cell lines application potential.

The Identification and Evolutionary Trends of the Solute Carrier Superfamily in Arthropods.

The solute carrier (SLC) transporter superfamily comprises an ancient and ubiquitous group of proteins capable of translocating a range of nutrients, endogenous molecules, and xenobiotics. Although the group has been the subject of intense investigation in both bacteria and mammals, its systematic identification in arthropods has not yet been undertaken. Here, we present a genome-wide identification of all 66 human SLC families in 174 arthropod species. A pipeline (SLC_id) was constructed to identify and group SLCs using a combination of hidden Markov model and BLAST searches followed by filtering based on polypeptide length and the number of transmembrane domains. Comparative analysis of the number of transporters in each family across diverse arthropod lineages was accomplished using one-way analysis of variance (ANOVA) and the Computational Analysis of gene Family Evolution (CAFE). These results suggested that many SLC families have undergone expansions or contractions in particular evolutionary lineages. Notably, the sugar transporting SLC2 family was significantly larger in insects compared with arachnids. This difference may have been complemented by a rapid expansion of the SLC60 family in arachnids which also acts on dietary sugars. Furthermore, the SLC33 family underwent a recent and drastic expansion in aphids, although the biological relevance of this expansion was not possible to infer. Information on specific SLC transporter families across arthropod species can be accessed through an R shiny web application at http://chrysalida.imbb.forth.gr : 3838/Arthropod_SLC_Database/. The present study greatly facilitates further investigation of the diverse group of SLC transporters in arthropods.

How do oral insecticidal compounds cross the insect midgut epithelium?

The use of oral insecticidal molecules (small molecules, peptides, dsRNA) via spray or plant mediated applications represents an efficient way to manage damaging insect species. With the exception of Bt toxins that target the midgut epithelium itself, most of these compounds have targets that lie within the hemocoel (body) of the insect. Because of this, one of the greatest factors in determining the effectiveness of an oral insecticidal compound is its ability to traverse the gut epithelium and enter the hemolymph. However, for many types of insecticidal compounds, neither the pathway taken across the gut nor the specific genes which influence uptake are fully characterized. Here, we review how different types of insecticidal compounds enter or cross the midgut epithelium through passive (diffusion) or active (transporter based, endocytosis) routes. A deeper understanding of how insecticidal molecules cross the gut will help to best utilize current insecticides and also provide for more rational design of future ones.