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.

Characterization of the swede midge, Contarinia nasturtii, first instar larval salivary gland transcriptome.

Proteins in saliva of gall-forming insect larvae govern insect-host plant interactions. Contarinia nasturtii, the swede midge, is a pest of brassicaceous vegetables (cabbage, cauliflower, broccoli) and canola. We examined the salivary gland (SG) transcriptome of first instar larvae reared on Brassica napus and catalogued genes encoding secreted proteins that may contribute to the initial stages of larval establishment, the synthesis of plant growth hormones, extra-oral digestion and evasion of host defenses. A significant portion of the secreted proteins with unknown functions were unique to C. nasturtii and were often members of larger gene families organized in genomic clusters with conservation patterns suggesting that they are undergoing selection.

Genetic variation and structural diversity in major seed proteins among and within Camelina species.

MAIN CONCLUSION: Genetic variation in seed protein composition, seed protein gene expression and predictions of seed protein physiochemical properties were documented in C. sativa and other Camelina species. Seed protein diversity was examined in six Camelina species (C. hispida, C. laxa, C. microcarpa, C. neglecta, C. rumelica and C. sativa). Differences were observed in seed protein electrophoretic profiles, total seed protein content and amino acid composition between the species. Genes encoding major seed proteins (cruciferins, napins, oleosins and vicilins) were catalogued for C. sativa and RNA-Seq analysis established the expression patterns of these and other genes in developing seed from anthesis through to maturation. Examination of 187 C. sativa accessions revealed limited variation in seed protein electrophoretic profiles, though sufficient to group the majority into classes based on high MW protein profiles corresponding to the cruciferin region. C. sativa possessed four distinct types of cruciferins, named CsCRA, CsCRB, CsCRC and CsCRD, which corresponded to orthologues in Arabidopsis thaliana with members of each type encoded by homeologous genes on the three C. sativa sub-genomes. Total protein content and amino acid composition varied only slightly; however, RNA-Seq analysis revealed that CsCRA and CsCRB genes contributed > 95% of the cruciferin transcripts in most lines, whereas CsCRC genes were the most highly expressed cruciferin genes in others, including the type cultivar DH55. This was confirmed by proteomics analyses. Cruciferin is the most abundant seed protein and contributes the most to functionality. Modelling of the C. sativa cruciferins indicated that each type possesses different physiochemical attributes that were predicted to impart unique functional properties. As such, opportunities exist to create C. sativa cultivars with seed protein profiles tailored to specific technical applications.

Sources of genomic diversity in the self-fertile plant pathogen, Sclerotinia sclerotiorum, and consequences for resistance breeding.

The ascomycete, Sclerotinia sclerotiorum, has a broad host range and causes yield loss in dicotyledonous crops world wide. Genomic diversity was determined in a population of 127 isolates obtained from individual canola (Brassica napus) fields in western Canada. Genotyping with 39 simple sequence repeat (SSR) markers revealed each isolate was a unique haplotype. Analysis of molecular variance showed 97% was due to isolate and 3% due to geographical location. Testing of mycelium compatibility among 133 isolates identified clones of mutually compatible isolates with 86-95% similar SSR haplotype, whereas incompatible isolates were highly diverse. In the Province of Manitoba, 61% of isolates were compatible forming clones and stings of pairwise compatible isolates not described before. In contrast, only 35% of isolates were compatible in Alberta without forming clones and strings, while 39% were compatible in Saskatchewan with a single clone, but no strings. These difference can be explained by wetter growing seasons and more susceptible crop species in Manitoba favouring frequent mycelium interaction and more life cycles over time, which might also explain similar differences observed in other geographical areas and host crops. Analysis of linkage disequilibrium rejected random recombination, consistent with a self-fertile fungus, restricted outcrossing due to mycelium incompatibility, and only a single annual opportunity for genomic recombination during meiosis in the ascospore stage between non-sister chromatids in the rare event nuclei from different isolates come together. More probable sources of genomic diversity is slippage during DNA replication and point mutation affecting single nucleotides that accumulate and likely increase mycelium incompatibility in a population over time. A phylogenetic tree based on SSR haplotype grouped isolates into 17 sub-populations. Aggressiveness was tested by inoculating one isolate from each sub-population onto B. napus lines with quantitative resistance. Analysis of variance was significant for isolate, line, and isolate by line interaction. These isolates represent the genomic and pathogenic diversity in western Canada, and are suitable for resistance screening in canola breeding programs.

Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds.

Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6-13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

What You Eat Matters: Nutrient Inputs Alter the Metabolism and Neuropeptide Expression in Egyptian Cotton Leaf Worm, Spodoptera littoralis (Lepidoptera: Noctuidae).

Lipids and carbohydrates are the two primary energy sources for both animals and insects. Energy homeostasis is under strict control by the neuroendocrine system, and disruption of energy homeostasis leads to the development of various disorders, such as obesity, diabetes, fatty liver syndrome, and cardiac dysfunction. One critical factor in this respect is feeding habits and diet composition. Insects are good models to study the physiological and biochemical background of the effect of diet on energy homeostasis and related disorders; however, most studies are based on a single model species, Drosophila melanogaster. In the current study, we examined the effects of four different diets, high fat (HFD), high sugar (HSD), calcium-rich (CRD), and a plant-based (PBD) on energy homeostasis in younger (third instar) and older (fifth instar) larvae of the Egyptian cotton leafworm, Spodoptera littoralis (Lepidoptera: Noctuidae) in comparison to a regular artificial bean diet. Both HSD and HFD led to weight gain, while CRD had the opposite effect and PBD had no effect in fifth instar larvae and pupae. The pattern was the same for HSD and CRD in third instar larvae while a reduction in weight was detected with HFD and PBD. Larval development was shortest with the HSD, while HFD, CRD, and PBD led to retardation compared to the control. Triglyceride (TG) levels were higher with HFD, HSD, and PBD, with larger lipid droplet sizes, while CRD led to a reduction of TG levels and lipid droplet size. Trehalose levels were highest with HSD, while CRD led to a reduction at third instar larvae, and HFD and PBD had no effect. Fifth instar larvae had similar levels of trehalose with all diets. There was no difference in the expression of the genes encoding neuropeptides SpoliAKH and SpoliILP1-2 with different diets in third instar larvae, while all three genes were expressed primarily with HSD, and SpolisNPF was primarily expressed with HFD in fifth instar larvae. In summary, different diet treatments alter the development of insects, and energy and metabolic pathways through the regulation of peptide hormones.

A Comparative Perspective on Functionally-Related, Intracellular Calcium Channels: The Insect Ryanodine and Inositol 1,4,5-Trisphosphate Receptors.

Calcium (Ca2+) homeostasis is vital for insect development and metabolism, and the endoplasmic reticulum (ER) is a major intracellular reservoir for Ca2+. The inositol 1,4,5- triphosphate receptor (IP3R) and ryanodine receptor (RyR) are large homotetrameric channels associated with the ER and serve as two major actors in ER-derived Ca2+ supply. Most of the knowledge on these receptors derives from mammalian systems that possess three genes for each receptor. These studies have inspired work on synonymous receptors in insects, which encode a single IP3R and RyR. In the current review, we focus on a fundamental, common question: "why do insect cells possess two Ca2+ channel receptors in the ER?". Through a comparative approach, this review covers the discovery of RyRs and IP3Rs, examines their structures/functions, the pathways that they interact with, and their potential as target sites in pest control. Although insects RyRs and IP3Rs share structural similarities, they are phylogenetically distinct, have their own structural organization, regulatory mechanisms, and expression patterns, which explains their functional distinction. Nevertheless, both have great potential as target sites in pest control, with RyRs currently being targeted by commercial insecticide, the diamides.

Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

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.

De Novo Whole-Genome Assembly of the Swede Midge (Contarinia nasturtii), a Specialist of Brassicaceae, Using Linked-Read Sequencing.

The swede midge, Contarinia nasturtii, is a cecidomyiid fly that feeds specifically on plants within the Brassicaceae. Plants in this family employ a glucosinolate-myrosinase defense system, which can be highly toxic to nonspecialist feeders. Feeding by C. nasturtii larvae induces gall formation, which can cause substantial yield losses thus making it a significant agricultural pest. A lack of genomic resources, in particular a reference genome, has limited deciphering the mechanisms underlying glucosinolate tolerance in C. nasturtii, which is of particular importance for managing this species. Here, we present an annotated, scaffolded reference genome of C. nasturtii using linked-read sequencing from a single individual and explore systems involved in glucosinolate detoxification. The C. nasturtii genome is similar in size and annotation completeness to that of the Hessian fly, Mayetiola destructor, but has greater contiguity. Several genes encoding enzymes involved in glucosinolate detoxification in other insect pests, including myrosinases, sulfatases, and glutathione S-transferases, were found, suggesting that C. nasturtii has developed similar strategies for feeding on Brassicaceae. The C. nasturtii genome will, therefore, be integral to continued research on plant-insect interactions in this system and contribute to effective pest management strategies.

Gene expression patterns in shoots of Camelina sativa with enhanced salinity tolerance provided by plant growth promoting bacteria producing 1-aminocyclopropane-1-carboxylate deaminase or expression of the corresponding acdS gene.

Growth of plants in soil inoculated with plant growth promoting bacteria (PGPB) producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase or expression of the corresponding acdS gene in transgenic lines reduces the decline in shoot length, shoot weight and photosynthetic capacity triggered by salt stress in Camelina sativa. Reducing the levels of ethylene attenuated the salt stress response as inferred from decreases in the expression of genes involved in development, senescence, chlorosis and leaf abscission that are highly induced by salt to levels that may otherwise have a negative effect on plant growth and productivity. Growing plants in soil treated with Pseudomonas migulae 8R6 negatively affected ethylene signaling, auxin and JA biosynthesis and signalling, but had a positive effect on the regulation of genes involved in GA signaling. In plants expressing acdS, the expression of the genes involved in auxin signalling was positively affected, while the expression of genes involved in cytokinin degradation and ethylene biosynthesis were negatively affected. Moreover, fine-tuning of ABA signaling appears to result from the application of ACC deaminase in response to salt treatment. Moderate expression of acdS under the control of the root specific rolD promoter or growing plants in soil treated with P. migulae 8R6 were more effective in reducing the expression of the genes involved in ethylene production and/or signaling than expression of acdS under the more active Cauliflower Mosaic Virus 35S promoter.

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.

Subunit composition affects formation and stabilization of o/w emulsions by 11S seed storage protein cruciferin.

The 11S globulin cruciferin is the major storage protein in Brassicaceae/Cruciferae seeds and exists as a hexamer in its natural configuration. Arabidopsis thaliana cruciferin is composed of CRUA, CRUB and CRUC subunits. Wild type (WT) cruciferin and cruciferins composed only of identical CRUA, CRUB and CRUC subunits were examined for their ability to form and stabilize oil-in-water (o/w) emulsions. All proteins (0.9% at pH 7.4 and 2.0), except CRUC, formed stable canola oil or triolein emulsions with a dispersed phase volume fraction of 22-23%. A fine emulsion was formed by CRUB at pH 7.4 with droplet sizes of 6.8 and 8.6 µm for canola oil and triolein, respectively. The presence of 0.5 M NaCl reduced the level of adsorbed protein and protein load at the interface at pH 7.4, and resulted in emulsions that were less stable. Emulsions of CRUA and CRUB (pH 7.4, zero ionic strength, canola oil or triolein) had higher stability than emulsions with WT cruciferin up to 15 days after formation. CRUC formed a stable emulsion only at pH 2.0. The low solubility, low surface hydrophobicity and compact structure of the CRUC protein may contribute to its inferior emulsifying properties at neutral pH; however, acidic pH-induced dissociation of the hexameric assembly improved these properties. The abundance and exposure of hydrophobic residues in the hypervariable regions, extended loop regions, and solvent exposed surfaces of cruciferin are critical factors affecting o/w interface stabilization.

Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors.

Sclerotinia sclerotiorum is a characteristic necrotrophic plant pathogen and is dependent on the induction of host cell death for nutrient acquisition. To identify necrosis-inducing effectors, the genome of S. sclerotiorum was scanned for genes encoding small, secreted, cysteine-rich proteins. These potential effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium-mediated expression and for cellular localization in host cells. Six novel proteins were discovered, of which all but one required a signal peptide for export to the apoplast for necrotizing activity. Virus-induced gene silencing revealed that the five necrosis-inducing effectors with a requirement for secretion also required the plant co-receptor-like kinases Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) and Suppressor of BAK1-Interacting Receptor-like Kinase 1 (SOBIR1) for the induction of necrosis. S. sclerotiorum necrosis-inducing effector 2 (SsNE2) represented a new class of necrosis-inducing proteins as orthologs were identified in several other phytopathogenic fungi that were also capable of inducing necrosis. Substitution of conserved cysteine residues with alanine reduced, but did not abolish, the necrotizing activity of SsNE2 and full-length protein was required for function as peptides spanning the entire protein were unable to induce necrosis. These results illustrate the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the role of host extracellular receptor(s) in effector-triggered susceptibility to this pathogen.

A journey into the world of insect lipid metabolism.

Lipid metabolism is fundamental to life. In insects, it is critical, during reproduction, flight, starvation, and diapause. The coordination center for insect lipid metabolism is the fat body, which is analogous to the vertebrate adipose tissue and liver. Fat body contains various different cell types; however, adipocytes and oenocytes are the primary cells related to lipid metabolism. Lipid metabolism starts with the hydrolysis of dietary lipids, absorption of lipid monomers, followed by lipid transport from midgut to the fat body, lipogenesis or lipolysis in the fat body, and lipid transport from fat body to other sites demanding energy. Lipid metabolism is under the control of hormones, transcription factors, secondary messengers and posttranscriptional modifications. Primarily, lipogenesis is under the control of insulin-like peptides that activate lipogenic transcription factors, such as sterol regulatory element-binding proteins, whereas lipolysis is coordinated by the adipokinetic hormone that activates lipolytic transcription factors, such as forkhead box class O and cAMP-response element-binding protein. Calcium is the primary-secondary messenger affecting lipid metabolism and has different outcomes depending on the site of lipogenesis or lipolysis. Phosphorylation is central to lipid metabolism and multiple phosphorylases are involved in lipid accumulation or hydrolysis. Although most of the knowledge of insect lipid metabolism comes from the studies on the model Drosophila; other insects, in particular those with obligatory or facultative diapause, also have great potential to study lipid metabolism. The use of these models would significantly improve our knowledge of insect lipid metabolism.

CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.

BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.

Examining population structure of a bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), outbreak in western North America: Implications for gene flow and dispersal.

The bertha armyworm (BAW), Mamestra configurata, is a significant pest of canola (Brassica napus L. and B. rapa L.) in western North America that undergoes cyclical outbreaks every 6-8 years. During peak outbreaks millions of dollars are spent on insecticidal control and, even with control efforts, subsequent damage can result in losses worth millions of dollars. Despite the importance of this pest insect, information is lacking on the dispersal ability of BAW and the genetic variation of populations from across its geographic range which may underlie potential differences in their susceptibility to insecticides or pathogens. Here, we examined the genetic diversity of BAW populations during an outbreak across its geographic range in western North America. First, mitochondrial cytochrome oxidase 1 (CO1) barcode sequences were used to confirm species identification of insects captured in a network of pheromone traps across the range, followed by haplotype analyses. We then sequenced the BAW genome and used double-digest restriction site associated DNA sequencing, mapped to the genome, to identify 1000s of single nucleotide polymorphisms (SNP) markers. CO1 haplotype analysis identified 9 haplotypes distributed across 28 sample locations and three laboratory-reared colonies. Analysis of genotypic data from both the CO1 and SNP markers revealed little population structure across BAW's vast range. The CO1 haplotype pattern showed a star-like phylogeny which is often associated with species whose population abundance and range has recently expanded and combined with pheromone trap data, indicates the outbreak may have originated from a single focal point in central Saskatchewan. The relatively recent introduction of canola and rapid expansion of the canola growing region across western North America, combined with the cyclical outbreaks of BAW caused by precipitous population crashes, has likely selected for a genetically homogenous BAW population adapted to this crop.