Transcriptome Dynamics of Brassica juncea Leaves in Response to Omnivorous Beet Armyworm (Spodoptera exigua, Hübner).

Cruciferous plants manufacture glucosinolates (GSLs) as special and important defense compounds against insects. However, how insect feeding induces glucosinolates in Brassica to mediate insect resistance, and how plants regulate the strength of anti-insect defense response during insect feeding, remains unclear. Here, mustard (Brassica juncea), a widely cultivated Brassica plant, and beet armyworm (Spodoptera exigua), an economically important polyphagous pest of many crops, were used to analyze the changes in GSLs and transcriptome of Brassica during insect feeding, thereby revealing the plant-insect interaction in Brassica plants. The results showed that the content of GSLs began to significantly increase after 48 h of herbivory by S. exigua, with sinigrin as the main component. Transcriptome analysis showed that a total of 8940 DEGs were identified in mustard challenged with beet armyworm larvae. The functional enrichment results revealed that the pathways related to the biosynthesis of glucosinolate and jasmonic acid were significantly enriched by upregulated DEGs, suggesting that mustard might provide a defense against herbivory by inducing JA biosynthesis and then promoting GSL accumulation. Surprisingly, genes regulating JA catabolism and inactivation were also activated, and both JA signaling repressors (JAZs and JAMs) and activators (MYCs and NACs) were upregulated during herbivory. Taken together, our results indicate that the accumulation of GSLs regulated by JA signaling, and the regulation of active and inactive JA compound conversion, as well as the activation of JA signaling repressors and activators, collectively control the anti-insect defense response and avoid over-stunted growth in mustard during insect feeding.

A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness.

Maintaining fitness during pathogen infection is vital for host survival as an excessive response can be as detrimental as the infection itself. Fitness costs are frequently associated with insect hosts countering the toxic effect of the entomopathogenic bacterium Bacillus thuringiensis (Bt), which delay the evolution of resistance to this pathogen. The insect pest Plutella xylostella has evolved a mechanism to resist Bt toxins without incurring significant fitness costs. Here, we reveal that non-phosphorylated and phosphorylated forms of a MAPK-modulated transcription factor fushi tarazu factor 1 (FTZ-F1) can respectively orchestrate down-regulation of Bt Cry1Ac toxin receptors and up-regulation of non-receptor paralogs via two distinct binding sites, thereby presenting Bt toxin resistance without growth penalty. Our findings reveal how host organisms can co-opt a master molecular switch to overcome pathogen invasion with low cost, and contribute to understanding the underlying mechanism of growth-defense tradeoffs during host-pathogen interactions in P. xylostella.

RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

BACKGROUND: In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. RESULTS: As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. CONCLUSION: The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.

Insect cuticular compounds affect Conidiobolus coronatus (Entomopthorales) sporulation and the activity of enzymes involved in fungal infection.

Mycoses are a global problem that affects humans and animals. In the present study, the entomopathogenic soil fungus Conidiobolus coronatus (Entomophthorales), infecting in tropics also humans, sheep and horses, was cultivated with the addition of insect cuticular compounds (CCs) previously detected in the cuticle of C. coronatus-resistant fly species (C10-C30 fatty alcohols, butyl oleate, butyl stearate, glycerol oleate, squalene, tocopherol acetate). Our findings indicate that CCs have diversified and complex effects on the growth and sporulation of C. coronatus and its ability to infect the larvae of Galleria mellonella (Lepidoptera). The CCs affected protein content and cuticle-degrading enzymes (CDEs) activity in the conidia. Some CCs inhibited fungal growth (0.1% C10), decreased sporulation (C12, C16, C24, C28, C30, butyl stearate, squalene), virulence (C12, C14, butyl oleate, butyl stearate) and protein content (C18). They also reduced conidial CDE activity: elastase (C24, butyl oleate, butyl stearate, squalene, tocopherol acetate), chitobiosidase (C12, C14, C20) and lipase (C12, C18, C26, squalene, tocopherol acetate). Several CCs enhanced sporulation (C14, C18, C22, C26, C30), virulence (C18, C26, squalene), conidial protein content (C16, C24, C30, squalene) and CDE activity: elastase (C10, C16, C18), NAGase (C16, C20), chitobiosidase (C16) and lipase (C10, C14, C16, C20, butyl oleate). Our findings indicate that C. coronatus colonies grown on media supplemented with CCs employ various compensation strategies: colonies grown with C16 alcohol demonstrated reduced sporulation but greater conidial protein accumulation and increased elastase, NAGase, chitobiosidase and lipase activity, thus preserving high virulence. Also, colonies supplemented with C18 alcohol demonstrated high virulence and enhanced sporulation and elastase activity but slightly decreased conidial protein content. CCs that inhibit the activity of lipases and proteases show promise in the fight against conidiobolomycosis.

Functional disruption of transferrin expression alters reproductive physiology in Anopheles culicifacies.

BACKGROUND: Iron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates and insects, but poorly studied in blood-feeding mosquitoes. RESULTS: We characterized a 2102 bp long transcript AcTrf1a with complete CDS of 1872bp, and 226bp UTR region, encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confirms the presence of an iron-binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a is abundantly expressed in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body but also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi-mediated knockdown of AcTrf1a causes a significant reduction in fecundity, confirming the important role of transferrin in oocyte maturation. CONCLUSION: All together our results advocate that detailed characterization of newly identified AcTrf1a transcript may help to select it as a unique target to impair the mosquito reproductive outcome.

Digestibility of insect meals for Pacific white shrimp (Litopenaeus vannamei) and their performance for growth, feed utilization and immune responses.

This study was conducted to examine digestibility of insect meals for Pacific white shrimp (Litopenaeus vannamei) and their utilization as fish meal substitutes. The tested insect meals were mealworm, silkworm, black soldier fly, rice grasshopper, two-spotted cricket, dynastid beetle and white-spotted flower chafer. Apparent digestibility coefficients of the tested insect meals were 83-89% for protein, 91-98% for lipid, 84-90% for energy, 77-81% for dry matter, 28-36% for chitin, 76-96% for amino acids and 89-93% for fatty acids. The amino acid availability of insect meals was high in taurine (93-96%), arginine (91-95%) and lysine (90-95%). Availability of fatty acids were 89-93% for saturated fatty acids, 90-93% for monounsaturated fatty acids and 88-93% for polyunsaturated fatty acids. For a feeding trial, a control diet was formulated using 27% tuna byproduct meal as a fish meal source and seven other diets were prepared replacing 10% tuna byproduct meal in the control diet with each insect meal. Triplicate groups of shrimp (initial body weight: 0.17 g) were fed the diets for 65 days. The growth performance was significantly improved when the shrimp were fed black soldier fly or dynastid beetle included diet. Dietary supplementation of insect meals significantly improved non-specific immune responses and antioxidant enzyme activity in the shrimp. These results indicate that the tested insect meals have high potentials to be used as a protein source that could replace fish meal in diets for the shrimp.

AOP Report: Inhibition of Chitin Synthase 1 Leading to Increased Mortality in Arthropods.

Arthropods (including insects, crustaceans, and arachnids) rely on the synthesis of chitin to complete their life cycles (Merzendorfer 2011). The highly conserved chitin synthetic process and the absence of this process in vertebrates make it an exploitable target for pest management and veterinary medicines (Merzendorfer 2013; Junquera et al. 2019). Susceptible, nontarget organisms, such as insects and aquatic invertebrates, exposed to chitin synthesis inhibitors may suffer population declines, which may have a negative impact on ecosystems and associated services. Hence, it is important to properly identify, prioritize, and regulate relevant chemicals posing potential hazards to nontarget arthropods. The need for a more cost-efficient and mechanistic approach in risk assessment has been clearly evident and triggered the development of the adverse outcome pathway (AOP) framework (Ankley et al. 2010). An AOP links a molecular initiating event (MIE) through key events (KEs) to an adverse outcome. The mechanistic understanding of the underlying toxicological processes leading to a regulation-relevant adverse outcome is necessary for the utilization of new approach methodologies (NAMs) and efficient coverage of wider chemical and taxonomic domains. In the last decade, the AOP framework has gained traction and expanded within the (eco)toxicological research community. However, there exists a lack of mature invertebrate AOPs describing molting defect-associated mortality triggered by direct inhibition of relevant enzymes in the chitin biosynthetic pathway (chitin synthesis inhibitors) or interference with associated endocrine systems by environmental chemicals (endocrine disruptors). Arthropods undergo molting to grow and reproduce (Heming 2018). This process is comprised of the synthesis of a new exoskeleton, followed by the exuviation of the old exoskeleton (Reynolds 1987). The arthropod exoskeleton (cuticle) can be divided into 2 layers, the thin and nonchitinous epicuticle, which is the outermost layer of the cuticle, and the underlying chitinous procuticle. A single layer of epithelial cells is responsible for the synthesis and secretion of both cuticular layers (Neville 1975). The cuticle protects arthropods from predators and desiccation, acts as a physical barrier against pathogens, and allows for locomotion by providing support for muscular function (Vincent and Wegst 2004). Because the procuticle mainly consists of chitin microfibrils embedded in a matrix of cuticular proteins supplemented by lipids and minerals in insects (Muthukrishnan et al. 2012) and crustaceans (Cribb et al. 2009; Nagasawa 2012), chitin is a determinant factor for the appropriate composition of the cuticle and successful molting (Cohen 2001). A detailed overview of the endocrine mechanisms regulating chitin synthesis is given in Supplemental Data, Figure S1. The shedding of the old exoskeleton in insects is mediated by a sequence of distinct muscular contractions, the ecdysis motor program (EMP; Ayali 2009; Song et al. 2017a). Like the expression of chitin synthase isoform 1 (CHS-1), the expression of peptide hormones regulating the EMP is also controlled by ecdysteroids (Antoniewski et al. 1993; Gagou et al. 2002; Ayali 2009). Cuticular chitin is polymerized from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by the transmembrane enzyme CHS-1, which is localized in the epithelial plasma membrane in insects (Locke and Huie 1979; Binnington 1985; Merzendorfer and Zimoch 2003; Merzendorfer 2006). Because crustaceans are also dependent on the synthesis of chitin, the underlying mechanisms are believed to be similar, although less is known about different CHS isoforms and their localization (Rocha et al. 2012; Qian et al. 2014; Uddowla et al. 2014; Harðardóttir et al. 2019). Disruption of either chitin synthesis or the upstream endocrine pathways can lead to lethal molting disruption (Arakawa et al. 2008; Merzendorfer et al. 2012; Song et al. 2017a, 2017b). In the case of chitin synthesis inhibition, molting disruption can be referred to as "premature molting." If ecdysis cannot be completed because of decreased chitin synthesis, the organism may not successfully molt. Even if ecdysis can be completed on inhibition of chitin synthesis, the organism may not survive because of the poor integrity of the new cuticle. These effects are observed in arthropods following molting, which fail to survive subsequent molts (Arakawa et al. 2008; Chen et al. 2008) or animals being stuck in their exuviae (Wang et al. 2019) and ultimately dying as a result of insufficient food or oxygen intake (Camp et al. 2014; Song et al. 2017a). The term "premature molting" is used to differentiate from the term "incomplete ecdysis," which describes inhibition of ecdysis on a behavioral level, namely through reduction of the EMP (Song et al. 2017a). The present AOP describes molting-associated mortality through direct inhibition of the enzyme CHS-1. It expands the small but increasing number of invertebrate AOPs that have relevance to arthropods, the largest phylum within the animal kingdom (Bar-On et al. 2018). The development of this AOP will be useful in further research and regulatory initiatives related to assessment of CHS inhibitors and identification of critical knowledge gaps and may suggest new strategies for ecotoxicity testing efforts. Environ Toxicol Chem 2021;40:2112-2120. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Transgenic Cry1Ac/CpTI cotton assessment finds no detrimental effects on the insect predator Chrysoperla sinica.

The widespread commercialization of genetically modified (GM) cotton makes it important to assess the potential impact of this recombinant crop on non-target organisms. As important natural enemies of cotton field predators, green lacewing Chrysoperla sinica larvae are exposed to Bt insecticidal proteins expressed by GM cotton by feeding on herbivorous pests, and adults are directly exposed to Bt proteins by cotton pollen consumption. However, potential impacts of transgenic Bt cotton on C. sinica remain unclear. In this study, we evaluated the effects of two transgenic cotton varieties, CCRI41 and CCRI45, which express Cry1Ac (Bt toxin) and CpTI (Cowpea Trypsin Inhibitor), on C. sinica larvae and adults. After being fed with cotton aphids Aphis gossypii reared on transgenic cotton, the survival rate, developmental duration, pupation rate, and emergence rate of larvae were not adversely affected. After being fed two types of transgenic cotton pollen, the 7-day weight of adults and the preoviposition period and the cumulative oviposition of females were not significantly different from control specimen. Taken together, these results indicate that the potential risks of the two tested GM cotton varieties for the predator C. sinica are negligible. CAPSULE: Our study indicated that GM cotton varieties CCRI41 and CCRI45 have no adverse effects on insect predator C. sinica.

Concentration ratios of 238U and 226Ra for insects and amphibians living in the vicinity of the closed uranium mine at Ningyo-toge, Japan.

There is still a scarcity of data on the transfer of naturally occurring radionuclides to wildlife in various ecosystems. In the present study, concentration ratios (CRwo-media) of 238U and 226Ra were obtained for grasshoppers, frogs and newts in terrestrial and freshwater ecosystems. Soil, water and animal samples were collected for 2 years in the vicinity of the closed uranium mine at Ningyo-toge, Japan. Three sites with different 238U and 226Ra levels were of interest: (i) pond and its shore (PO); (ii) low-level stream and its shore near overburden dump (OD); and (iii) uranium mill tailings pond and its shore (MP). The activity concentrations in both soil and water were PO ≈ OD < MP for 238U, and PO < OD < MP for 226Ra. Regarding the wildlife, 238U was able to be determined for all samples, but the detection of 226Ra was observed only for part of the samples. The means and standard deviations of CRwo-soil or CRwo-water were then calculated and may indicate the insignificant dependence of CRwo-media on environmental conditions characterized by the tested sites. The present data on CRwo-media were compared to the corresponding data or surrogate data from the IAEA's database, showing both agreement and discrepancy. Our data contribute to enhancing the available data for those radionuclides and animals. In particular, the transfer to amphibians, one of the main links in common food webs, is reported here for the first time.

Bioaccumulation and Dispersion of Uranium by Freshwater Organisms.

Uranium is the heaviest naturally occurring element on Earth. Uranium mining may result in ground and surface water contamination with potential bioaccumulation and dispersion by aquatic invertebrates with aerial stages. We investigated the effects of uranium contamination at community level in terms of abundance, richness, the composition of invertebrate communities, and functional traits. We also investigated uranium mobility across aquatic food webs and its transfer to land via the emergence of aquatic insects. We sampled water, sediment, biofilm, macrophytes, aquatic invertebrates, adult insects, and spiders in the riparian zone across sites with a gradient of uranium concentrations in stream water (from 2.1 to 4.7 µg L-1) and sediments (from 10.4 to 41.8 µg g-1). Macroinvertebrate assemblages differed between sites with a higher diversity and predominance of Nemouridae and Baetidae at the reference site and low diversity and predominance of Chironomidae in sites with the highest uranium concentration. Uranium concentrations in producers and consumers increased linearly with uranium concentration in stream water and sediment (p < 0.05). The highest accumulation was found in litter (83.76 ± 5.42 µg g-1) and macrophytes (47.58 ± 6.93 µg g-1) in the most contaminated site. Uranium was highest in scrapers (14.30 ± 0.98 µg g-1), followed by shredders (12.96 ± 0.81 µg g-1) and engulfer predators (7.01 ± 1.3 µg g-1). Uranium in adults of aquatic insects in the riparian zone in all sites ranged from 0.25 to 2.90 µg g-1, whereas in spiders it ranged from 0.96 to 1.73 µg g-1, with no differences between sites (p > 0.05). There was a negative relationship between δ15N and uranium, suggesting there is no biomagnification along food webs. We concluded that uranium is accumulated by producers and consumers but not biomagnified nor dispersed to land with the emergence of aquatic insects.

Effects of Selenium on Mercury Bioaccumulation in a Terrestrial food Chain from an Abandoned Mercury Mining Region.

Few reports of the relationship exist between mercury (Hg) and selenium (Se) from locations of severe Hg contamination in terrestrial environments. Here, we report the concentrations of Hg and Se as well as Se:Hg molar ratios in biotic samples collected from a region with a long history of Hg mining. Nitrogen isotopes (δ15N) were analyzed to confirm the trophic levels. Results showed that bird feathers at the top trophic level exhibited the highest Hg concentrations, while the lowest concentrations were found in herbivorous insects, demonstrating a significant biomagnification across the food chain. In contrast, herbivorous insects of different types (generalists vs. specialized rice pests) exhibited both the highest and the lowest concentrations of Se, indicating a lack of biomagnification. Indeed, Se was correlated positively with Hg when Se:Hg ratios were greater than one, suggesting Se:Hg molar ratios can be a controlling influence on Hg in terrestrial organisms.

Species-Specific Interactions between Plant Metabolites and Insect Juvenile Hormone Receptors.

Because juvenile hormone (JH) controls insect development and its analogs are used as insecticides, juvenile hormone disruptors (JHDs) represent potential sources from which novel pesticides can be developed. Many plant species harbor JHD activity, which has previously been attributed plant secondary metabolites (i.e., diterpenes) that disrupt insect development by interfering with the JH-mediated heterodimer formation of insect juvenile receptor complexes. The results of the present study indicate that plant JHD activity is also concentrated in certain plant groups and families and that plant metabolites have insect group-specific activity. These findings suggest that reciprocal diversification has occurred between plants and insects through the evolution of the plant metabolites and JH receptors, respectively, and that plant metabolites could be developed into insect group-specific pesticides with limited effects on non-target species.

The B vitamin nutrition of insects: the contributions of diet, microbiome and horizontally acquired genes.

Insects generally cannot synthesize eight B vitamins that function as co-enzymes in various required enzymatic reactions. Most insects derive their B vitamin requirements from the diet, microbial symbionts, or a combination of these complementary sources. Exceptionally, the genomes of a few insects bear genes in vitamin B5 (pantothenate) and B7 (biotin) synthesis, horizontally acquired from bacteria. Biomarkers of B vitamin deficiency (e.g. vitamin titers, activity of vitamin-dependent enzymes) offer routes to investigate the incidence and the physiological and fitness consequences of B vitamin deficiency in laboratory and field populations of insects.

Nutritional and reproductive signaling revealed by comparative gene expression analysis in Chrysopa pallens (Rambur) at different nutritional statuses.

BACKGROUND: The green lacewing, Chrysopa pallens Rambur, is one of the most important natural predators because of its extensive spectrum of prey and wide distribution. However, what we know about the nutritional and reproductive physiology of this species is very scarce. RESULTS: By cDNA amplification and Illumina short-read sequencing, we analyzed transcriptomes of C. pallens female adult under starved and fed conditions. In total, 71236 unigenes were obtained with an average length of 833 bp. Four vitellogenins, three insulin-like peptides and two insulin receptors were annotated. Comparison of gene expression profiles suggested that totally 1501 genes were differentially expressed between the two nutritional statuses. KEGG orthology classification showed that these differentially expression genes (DEGs) were mapped to 241 pathways. In turn, the top 4 are ribosome, protein processing in endoplasmic reticulum, biosynthesis of amino acids and carbon metabolism, indicating a distinct difference in nutritional and reproductive signaling between the two feeding conditions. CONCLUSIONS: Our study yielded large-scale molecular information relevant to C. pallens nutritional and reproductive signaling, which will contribute to mass rearing and commercial use of this predaceous insect species.

Dietary and taxonomic controls on incorporation of microbial carbon and phosphorus by detritivorous caddisflies.

Heterotrophic microbes on detritus play critical roles in the nutrition of detritivorous animals, yet few studies have examined factors controlling the acquisition of microbial nutrients toward detritivore growth, which is termed "incorporation". Here, we assessed effects of detrital substrate identity (leaf type), background nutrients, and detritivore species identity on detritivore incorporation of microbial carbon (C) and phosphorus (P) in leaf litter diets. We fed oak and maple litter conditioned under two nutrient concentrations (50 or 500 µg P L(-1)) to the detritivorous caddisfly larvae Ironoquia spp., Lepidostoma spp., and Pycnopsyche lepida and used the radioisotopes 14C as glucose and 33P as phosphate to dually trace incorporation of microbial C and P by caddisflies. Incorporation efficiencies of microbial C (mean ± SE = 12.3 ± 1.3%) were one order of magnitude higher than gross growth efficiencies for bulk detrital C from recent studies (1.05 ± 0.08%). Litter type did not affect incorporation of microbial nutrients; however, caddisflies incorporated microbial P 11 % less efficiently when fed litter from the higher P concentration. Two lower body C:P species (Pycnopsyche and Ironoquia) exhibited 9.9 and 7.1% greater microbial C and 19.0 and 17.7% greater microbial P incorporation efficiencies, respectively, than the higher body C:P species (Lepidostoma). Our findings support ecological stoichiometry theory on post-ingestive regulation that animals fed lower C:P diets should reduce P incorporation efficiency due to excess diet P or alleviation of P-limited growth, and that lower C:P species must incorporate dietary C and P more efficiently to support fast growth of P-rich tissues.

Complete nutrient content of four species of commercially available feeder insects fed enhanced diets during growth.

Commercially raised feeder insects used to feed captive insectivores are a good source of many nutrients but are deficient in several key nutrients. Current methods used to supplement insects include dusting and gut-loading. Here, we report on the nutrient composition of four species of commercially raised feeder insects fed a special diet to enhance their nutrient content. Crickets, mealworms, superworms, and waxworms were analyzed for moisture, crude protein, fat, ash, acid detergent fiber, total dietary fiber, minerals, amino acids, fatty acids, vitamins, taurine, carotenoids, inositol, and cholesterol. All four species contained enhanced levels of vitamin E and omega 3 fatty acids when compared to previously published data for these species. Crickets, superworms, and mealworms contained ß-carotene although using standard conversion factors only crickets and superworms would likely contain sufficient vitamin A activity for most species of insectivores. Waxworms did not contain any detectable ß-carotene but did contain zeaxanthin which they likely converted from dietary ß-carotene. All four species contained significant amounts of both inositol and cholesterol. Like previous reports all insects were a poor source of calcium and only superworms contained vitamin D above the limit of detection. When compared to the nutrient requirements as established by the NRC for growing rats or poultry, these species were good sources of most other nutrients although the high fat and low moisture content of both waxworms and superworms means when corrected for energy density these two species were deficient in more nutrients than crickets or mealworms. These data show the value of modifying the diet of commercially available insects as they are growing to enhance their nutrient content. They also suggest that for most insectivores properly supplemented lower fat insects such as crickets, or smaller mealworms should form the bulk of the diet.

Phytochemical diversity drives plant-insect community diversity.

What are the ecological causes and consequences of variation in phytochemical diversity within and between plant taxa? Despite decades of natural products discovery by organic chemists and research by chemical ecologists, our understanding of phytochemically mediated ecological processes in natural communities has been restricted to studies of either broad classes of compounds or a small number of well-characterized molecules. Until now, no studies have assessed the ecological causes or consequences of rigorously quantified phytochemical diversity across taxa in natural systems. Consequently, hypotheses that attempt to explain variation in phytochemical diversity among plants remain largely untested. We use spectral data from crude plant extracts to characterize phytochemical diversity in a suite of co-occurring plants in the tropical genus Piper (Piperaceae). In combination with 20 years of data focused on Piper-associated insects, we find that phytochemical diversity has a direct and positive effect on the diversity of herbivores but also reduces overall herbivore damage. Elevated chemical diversity is associated with more specialized assemblages of herbivores, and the cascading positive effect of phytochemistry on herbivore enemies is stronger as herbivore diet breadth narrows. These results are consistent with traditional hypotheses that predict positive associations between plant chemical diversity, insect herbivore diversity, and trophic specialization. It is clear from these results that high phytochemical diversity not only enhances the diversity of plant-associated insects but also contributes to the ecological predominance of specialized insect herbivores.

The 2014 Bernard B. Brodie award lecture-epoxide hydrolases: drug metabolism to therapeutics for chronic pain.

Dr. Bernard Brodie's legacy is built on fundamental discoveries in pharmacology and drug metabolism that were then translated to the clinic to improve patient care. Similarly, the development of a novel class of therapeutics termed the soluble epoxide hydrolase (sEH) inhibitors was originally spurred by fundamental research exploring the biochemistry and physiology of the sEH. Here, we present an overview of the history and current state of research on epoxide hydrolases, specifically focusing on sEHs. In doing so, we start with the translational project studying the metabolism of the insect juvenile hormone mimic R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene], which led to the identification of the mammalian sEH. Further investigation of this enzyme and its substrates, including the epoxyeicosatrienoic acids, led to insight into mechanisms of inflammation, chronic and neuropathic pain, angiogenesis, and other physiologic processes. This basic knowledge in turn led to the development of potent inhibitors of the sEH that are promising therapeutics for pain, hypertension, chronic obstructive pulmonary disorder, arthritis, and other disorders.

Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.

Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity.