Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

8-HQA adjusts the number and diversity of bacteria in the gut microbiome of Spodoptera littoralis.

Quinolinic carboxylic acids are known for their metal ion chelating properties in insects, plants and bacteria. The larval stages of the lepidopteran pest, Spodoptera littoralis, produce 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in high concentrations from tryptophan in the diet. At the same time, the larval midgut is known to harbor a bacterial population. The motivation behind the work was to investigate whether 8-HQA is controlling the bacterial community in the gut by regulating the concentration of metal ions. Knocking out the gene for kynurenine 3-monooxygenase (KMO) in the insect using CRISPR/Cas9 eliminated production of 8-HQA and significantly increased bacterial numbers and diversity in the larval midgut. Adding 8-HQA to the diet of knockout larvae caused a dose-dependent reduction of bacterial numbers with minimal effects on diversity. Enterococcus mundtii dominates the community in all treatments, probably due to its highly efficient iron uptake system and production of the colicin, mundticin. Thus host factors and bacterial properties interact to determine patterns of diversity and abundance in the insect midgut.

Silencing of an ABC transporter, but not a cadherin, decreases the susceptibility of Colorado potato beetle larvae to Bacillus thuringiensis ssp. tenebrionis Cry3Aa toxin.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.

Sugar transporters enable a leaf beetle to accumulate plant defense compounds.

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Malpighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.

Characterization of calcium signaling proteins from the fat body of the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae): Implications for diapause and lipid metabolism.

Calcium (Ca2+) regulates many cellular and physiological processes from development to reproduction. Ca2+ is also an important factor in the metabolism of lipids, the primary energy source used during insect starvation and diapause. Ca2+ signaling proteins bind to Ca2+ and maintain intracellular Ca2+ levels. However, knowledge about Ca2+ signaling proteins is mostly restricted to the model Drosophila melanogaster and the response of Ca2+ signaling genes to starvation or diapause is not known. In this study, we identified three Ca2+ signaling proteins; the primary Ca2+ binding protein Calmodulin (LdCaM), phosphatase Calcineurin B (LdCaNB), and the senescence marker protein Regucalcin (LdRgN), from the fat body of the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). This insect is a major pest of potato worldwide and overwinters under hibernation diapause as adults while utilizing lipids as the primary energy source. Putative EF-hand domains involved in Ca2+ binding were present in LdCaM, LdCaNB, but absent in LdRgN. LdCaM and LdCaNB were expressed in multiple tissues, while LdRgN was primarily expressed in the fat body. LdCaM was constitutively-expressed throughout larval development and at the adult stage. LdCaNB was primarily expressed in feeding larvae, and LdRgN in both feeding larvae and adults at comparable levels; however, both genes were down-regulated by molting. A response to starvation was observed only for LdRgN. Transcript abundance analysis in the entire body in relation to diapause revealed differential regulation with a general suppression during diapause, and higher mRNA levels in favor of females at post-diapause for LdCaM, and in favor of males at non-diapause for LdCaNB. Fat body-specific transcript abundance was not different between non-diapause and post-diapause for LdCaNB, but both LdCaM and LdRgN were down-regulated in males and both sexes, respectively by post-diapause. Silencing LdCaNB or LdRgN in larvae led to decreased fat content, indicating their involvement in lipid accumulation, while RNAi of LdCaM led to lethality.

Two calcium-binding chaperones from the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause.

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin-type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N-globular domain, a P-arm domain, and a highly charged C-terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause-related expression analysis in the whole body revealed an upregulation of both genes by post-diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause-related expression pattern in the entire body for both genes. Fat body-specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post-diapause. This study suggests that calcium-binding chaperones play similar and possibly gender-specific roles during diapause.

A look into Colorado potato beetle lipid metabolism through the lens of lipid storage droplet proteins.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) inflicts serious damage to potato plants by feeding ravenously on their leaves. Adult L.decemlineata have a photoperiod-induced dormancy response, also known as diapause, which allows them to survive severe winter conditions by digging into soil. Most insects that undergo diapause accumulate abundant lipid reserves prior to diapause and utilize most of them during the diapause. This process is likely to be governed by the interplay of lipid storage droplet proteins (LSDs), also known as perilipins, with the help of other proteins. Here, genes encoding L. decemlineata LSD1 and LSD2 were identified. Both were expressed primarily in the fat body with LdLSD1 and LdLSD2 being primarily expressed in adult and larval stages, respectively. LdLSD1 was up-regulated in starving larvae, while LdLSD2 was primarily expressed in feeding larvae. The expression pattern of LdLSD1 in adults during feeding, diapause and post-diapause contrasted to the total body fat levels, while the expression pattern of LdLSD2 was positively correlated with total body fat levels. RNA interference (RNAi) of LdLSD2 in larvae suggested a core role for LSD2 in the protection/assembly of storage lipids as this treatment reduced overall lipid droplet volume. These data shed light on the functions of these proteins in L. decemlineata and their roles in both diapause and during starvation.

Adaptation by copy number variation increases insecticide resistance in the fall armyworm.

Understanding the genetic basis of insecticide resistance is a key topic in agricultural ecology. The adaptive evolution of multi-copy detoxification genes has been interpreted as a cause of insecticide resistance, yet the same pattern can also be generated by the adaptation to host-plant defense toxins. In this study, we tested in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), if adaptation by copy number variation caused insecticide resistance in two geographically distinct populations with different levels of resistance and the two host-plant strains. We observed a significant allelic differentiation of genomic copy number variations between the two geographic populations, but not between host-plant strains. A locus with positively selected copy number variation included a CYP gene cluster. Toxicological tests supported a central role for CYP enzymes in deltamethrin resistance. Our results indicate that copy number variation of detoxification genes might be responsible for insecticide resistance in fall armyworm and that evolutionary forces causing insecticide resistance could be independent of host-plant adaptation.

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Genetic basis of allochronic differentiation in the fall armyworm.

BACKGROUND: Very little is known on how changes in circadian rhythms evolve. The noctuid moth Spodoptera frugiperda (Lepidoptera: Noctuidae) consists of two strains that exhibit allochronic differentiation in their mating time, which acts as a premating isolation barrier between the strains. We investigated the genetic basis of the strain-specific timing differences to identify the molecular mechanisms of differentiation in circadian rhythms. RESULTS: Through QTL analyses we identified one major Quantitative trait chromosome (QTC) underlying differentiation in circadian timing of mating activity. Using RADtags, we identified this QTC to be homologous to Bombyx mori C27, on which the clock gene vrille is located, which thus became the major candidate gene. In S. frugiperda, vrille showed strain-specific polymorphisms. Also, vrille expression differed significantly between the strains, with the rice-strain showing higher expression levels than the corn-strain. In addition, RT-qPCR experiments with the other main clock genes showed that pdp1, antagonist of vrille in the modulatory feedback loop of the circadian clock, showed higher expression levels in the rice-strain than in the corn-strain. CONCLUSIONS: Together, our results indicate that the allochronic differentiation in the two strains of S. frugiperda is associated with differential transcription of vrille or a cis-acting gene close to vrille, which contributes to the evolution of prezygotic isolation in S. frugiperda.

Geographic variation in sexual attraction of Spodoptera frugiperda corn- and rice-strain males to pheromone lures.

The corn- and rice-strains of Spodoptera frugiperda exhibit several genetic and behavioral differences and appear to be undergoing ecological speciation in sympatry. Previous studies reported conflicting results when investigating male attraction to pheromone lures in different regions, but this could have been due to inter-strain and/or geographic differences. Therefore, we investigated whether corn- and rice-strain males differed in their response to different synthetic pheromone blends in different regions in North America, the Caribbean and South America. All trapped males were strain-typed by two strain-specific mitochondrial DNA markers. In the first experiment, we found a nearly similar response of corn- and rice-strain males to two different 4-component blends, resembling the corn- and rice-strain female blend we previously described from females in Florida. This response showed some geographic variation in fields in Canada, North Carolina, Florida, Puerto Rico, and South America (Peru, Argentina). In dose-response experiments with the critical secondary sex pheromone component (Z)-7-dodecenyl acetate (Z7-12:OAc), we found some strain-specific differences in male attraction. While the response to Z7-12:OAc varied geographically in the corn-strain, rice-strain males showed almost no variation. We also found that the minor compound (Z)-11-hexadecenyl acetate (Z11-16:OAc) did not increase attraction of both strains in Florida and of corn-strain males in Peru. In a fourth experiment, where we added the stereo-isomer of the critical sex pheromone component, (E)-7-dodecenyl acetate, to the major pheromone component (Z)-9-tetradecenyl acetate (Z9-14:OAc), we found that this compound was attractive to males in North Carolina, but not to males in Peru. Overall, our results suggest that both strains show rather geographic than strain-specific differences in their response to pheromone lures, and that regional sexual communication differences might cause geographic differentiation between populations.

Pheromonal divergence between two strains of Spodoptera frugiperda.

Spodoptera frugiperda consists of two genetically and behaviorally different strains, the corn- and the rice-strain, which seem to be in the process of sympatric speciation. We investigated the role of strain-specific sexual communication as a prezygotic mating barrier between both strains by analyzing strain-specific variation in female pheromone composition of laboratory and field strains, and also male attraction in wind tunnel and field experiments. Laboratory-reared and field-collected females from Florida exhibited strain-specific differences in their relative amount of (Z)-7-dodecenyl acetate (Z7-12:OAc) and (Z)-9-dodecenyl acetate (Z9-12:OAc). In wind tunnel assays, we did not find strain-specific attraction of males to females. However, in field experiments in Florida, we observed some differential attraction to synthetic pheromone blends. In a corn field, the corn-strain blend attracted more males of both strains than the rice-strain blend, but both blends were equally attractive in a grass field. Thus, habitat-specific volatiles seemed to influence male attraction to pheromones. In dose-response experiments, corn-strain males were more attracted to 2 % Z7-12:OAc than other doses tested, while rice-strain males were attracted to a broader range of Z7-12:OAc (2-10 %). The attraction of corn-strain males to the lowest dose of Z7-12:OAc corresponds to the production of this compound by females; corn-strain females produced significantly smaller amounts of Z7-12:OAc than rice-strain females. Although corn-strain individuals are more restricted in their production of and response to pheromones than rice-strain individuals, it seems that differences in sexual communication between corn- and rice-strain individuals are not strong enough to cause assortative mating.