A Farnesyl Pyrophosphate Synthase Gene Is Expressed in Fat Body Regulates Cantharidin Synthesis in Male Epicauta impressicornis Blister Beetle.

Blister beetles of Epicauta impressicornis have attracted attention because they contain a large amount of cantharidin (CTD). To date, however, the synthesis and transfer of CTD in adults of E. impressicornis are largely unknown. Here, we showed that the larvae E. impressicornis are capable of synthesizing CTD and they consume CTD during pupation. Before sexual maturity, both male and female adults synthesized a small amount of CTD, while after sexual maturity, males produced larger amounts of CTD, but females did not. The newly synthesized CTD in males first appeared in the hemolymph and then accumulated in the reproductive system. During the mating, the males transferred CTD to the reproductive system of females. In addition, a farnesyl pyrophosphate synthase (FPPS) gene was identified in male E. impressicornis. RNA-seq analysis, quantitative RT-PCR, and RNA interference analyses were conducted to investigate expression patterns and the functional roles of E. impressicornis FPPS (EiFPPS). Our results indicate that EiFPPS is highly expressed in the fat body of males. Moreover, the knock-down of EiFPPS led to a significant decrease in CTD synthesis. The current study indicates that EiFPPS is expressed in the fat body to regulate CTD synthesis in male E. impressicornis blister beetles.

Investigating the thermal sensitivity of key enzymes involved in the energetic metabolism of three insect species.

The metabolic responses of insects to high temperatures have been linked to their mitochondrial substrate oxidation capacity. However, the mechanism behind this mitochondrial flexibility is not well understood. Here, we used three insect species with different thermal tolerances (the honey bee, Apis mellifera; the fruit fly, Drosophila melanogaster; and the potato beetle, Leptinotarsa decemlineata) to characterize the thermal sensitivity of different metabolic enzymes. Specifically, we measured activity of enzymes involved in glycolysis (hexokinase, HK; pyruvate kinase, PK; and lactate dehydrogenase, LDH), pyruvate oxidation and the tricarboxylic acid cycle (pyruvate dehydrogenase, PDH; citrate synthase, CS; malate dehydrogenase, MDH; and aspartate aminotransferase, AAT), and the electron transport system (Complex I, CI; Complex II, CII; mitochondrial glycerol-3-phosphate dehydrogenase, mG3PDH; proline dehydrogenase, ProDH; and Complex IV, CIV), as well as that of ATP synthase (CV) at 18, 24, 30, 36, 42 and 45°C. Our results show that at high temperature, all three species have significantly increased activity of enzymes linked to FADH2 oxidation, specifically CII and mG3PDH. In fruit flies and honey bees, this coincides with a significant decrease of PDH and CS activity, respectively, that would limit NADH production. This is in line with the switch from NADH-linked substrates to FADH2-linked substrates previously observed with mitochondrial oxygen consumption. Thus, we demonstrate that even though the three insect species have a different metabolic regulation, a similar response to high temperature involving CII and mG3PDH is observed, denoting the importance of these proteins for thermal tolerance in insects.

Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.

Horizontal gene transfer (HGT) provides an evolutionary shortcut for recipient organisms to gain novel functions. Although reports of HGT in higher eukaryotes are rapidly accumulating, in most cases the evolutionary trajectory, metabolic integration, and ecological relevance of acquired genes remain unclear. Plant cell wall degradation by HGT-derived enzymes is widespread in herbivorous insect lineages. Pectin is an abundant polysaccharide in the walls of growing parts of plants. We investigated the significance of horizontally acquired pectin-digesting polygalacturonases (PGs) of the leaf beetle Phaedon cochleariae. Using a CRISPR/Cas9-guided gene knockout approach, we generated a triple knockout and a quadruple PG-null mutant in order to investigate the enzymatic, biological, and ecological effects. We found that pectin-digestion 1) is exclusively linked to the horizontally acquired PGs from fungi, 2) became fixed in the host genome by gene duplication leading to functional redundancy, 3) compensates for nutrient-poor diet by making the nutritious cell contents more accessible, and 4) facilitates the beetles development and survival. Our analysis highlights the selective advantage PGs provide to herbivorous insects and demonstrate the impact of HGT on the evolutionary success of leaf-feeding beetles, major contributors to species diversity.

A beta-glucosidase of an insect herbivore determines both toxicity and deterrence of a dandelion defense metabolite.

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid ß-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion (Taraxacum officinale) that deters its major root herbivore, the common cockchafer larva (Melolontha melolontha). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha ß-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions.

Plants produce certain substances to fend off attackers like plant-feeding insects. To stop these compounds from damaging their own cells, plants often attach sugar molecules to them. When an insect tries to eat the plant, the plant removes the stabilizing sugar, 'activating' the compounds and making them toxic or foul-tasting. Curiously, some insects remove the sugar themselves, but it is unclear what consequences this has, especially for insect behavior. Dandelions, Taraxacum officinale, make high concentrations of a sugar-containing defense compound in their roots called taraxinic acid ß-D-glucopyranosyl ester, or TA-G for short. TA-G deters the larvae of the Maybug ­ a pest also known as the common cockchafer or the doodlebug ­ from eating dandelion roots. When Maybug larvae do eat TA-G, it is found in their systems without its sugar. However, it is unclear whether it is the plant or the larva that removes the sugar. A second open question is how the sugar removal process affects the behavior of the Maybug larvae. Using chemical analysis and genetic manipulation, Huber et al. investigated what happens when Maybug larvae eat TA-G. This revealed that the acidity levels in the larvae's digestive system deactivate the proteins from the dandelion that would normally remove the sugar from TA-G. However, rather than leaving the compound intact, larvae remove the sugar from TA-G themselves. They do this using a digestive enzyme, known as a beta-glucosidase, that cuts through sugar. Removing the sugar from TA-G made the compound less toxic, allowing the larvae to grow bigger, but it also increased TA-G's deterrent effects, making the larvae less likely to eat the roots. Any organism that eats plants, including humans, must deal with chemicals like TA-G in their food. Once inside the body, enzymes can change these chemicals, altering their effects. This happens with many medicines, too. In the future, it might be possible to design compounds that activate only in certain species, or under certain conditions. Further studies in different systems may aid the development of new methods of pest control, or new drug treatments.

Near-infrared bioluminescence imaging of two cell populations in living mice.

Multicolor bioluminescence imaging using near-infrared emitting luciferases is an attractive application to detect two cell populations within one animal model. Herein, we describe how to distinguish dual-color bioluminescent signals co-localized in the same compartment. We tested CBG2 click beetle (λ = 660 nm) and CBR2 click beetle (λ = 730 nm) luciferases paired with NH2-NpLH2 luciferin. Following a spectral unmixing algorithm, single spectral contributions can be resolved and quantified, enabling the visualization of multiple cell types in deep tissue by injection of a single substrate. For complete details on the use and execution of this protocol, please refer to Zambito et al. (2020).

A novel thermostable beetle luciferase based cytotoxicity assay.

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed 'Matador-Glo assay' which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated -5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6-8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

Influence of the C-terminal domain on the bioluminescence activity and color determination in green and red emitting beetle luciferases and luciferase-like enzyme.

Beetle luciferases catalyze the bioluminescent oxidation of D-luciferin, producing bioluminescence colors ranging from green to red, using two catalytic steps: adenylation of D-luciferin to produce D-luciferyl-adenylate and PPi, and oxidation of D-luciferyl-adenylate, yielding AMP, CO2, and excited oxyluciferin, the emitter. Luciferases and CoA-ligases display a similar fold, with a large N-terminal domain, and a small C-terminal domain which undergoes rotation, closing the active site and promoting both adenylation and oxidative reactions. The effect of C-terminal domain deletion was already investigated for Photinus pyralis firefly luciferase, resulting in a red-emitting mutant with severely impacted luminescence activity. However, the contribution of C-terminal in the bioluminescence activities and colors of other beetle luciferases and related ancestral luciferases were not investigated yet. Here we compared the effects of the C-terminal domain deletion on green-emitting luciferases of Pyrearinus termitilluminans (Pte) click beetle and Phrixothrix vivianii railroadworm, and on the red-emitting luciferase of Phrixothrix hirtus railroadworm and luciferase-like enzyme of Zophobas morio. In all cases, the domain deletion severely impacted the overall bioluminescence activities and, slightly less, the oxidative activities, and usually red-shifted the bioluminescence colors. The results support the involvement of the C-terminal in shielding the active site from the solvent during the light emitting step. However, in Pte luciferase, the deletion caused only a 10 nm red-shift, indicating a distinctive active site which remains more shielded, independently of the C'-terminal. Altogether, the results confirm the main contribution of the C-terminal for the catalysis of the adenylation reaction and for active site shielding during the light emitting step.

Assessment of the potential of non-thermal atmospheric pressure plasma discharge and microwave energy against Tribolium castaneum and Trogoderma granarium.

This study was carried out to investigate the efficacy of the non-thermal atmospheric pressure plasma produced with dielectric barrier discharge (APPD) using air as a processing gas and microwave energy to control Tribolium castaneum and Trogoderma granarium adults and larvae in wheat grains. Insects' mortality was found to be power and time-dependent. The results indicated that non-thermal APPD and the microwave have enough insecticidal effect on the target pests. From the bioassay, LT50's and LT90's levels were estimated, T. granarium larvae appeared more tolerant to non-thermal APPD and the microwave energy than adults 7 days post-exposure. The germination percentage of wheat grains increased as the time of exposure to the non-thermal APPD increased. On the contrary, the germination percentage of wheat grains decreased as the time of exposure to the microwave increased. In addition, changes in antioxidant enzyme activities, catalase (CAT), glutathione S-transferase (GST) and peroxidase, in adults and larvae were examined after 24 h post-treatment to non-thermal APPD at 15.9 W power level, which caused 50% mortality. The activity of CAT, GST and lipid peroxide in the treated larvae showed a significant increase post-exposure to the non-thermal APPD at 15.9 W power level. On the other hand, no significant change in GSH-Px activity was observed. Reductions in the level of glutathione (GSH) and protein content occurred in treated larvae in comparison with the control.

Erucin modulates digestive enzyme release via crustacean cardioactive peptide in the elm leaf beetle Xanthogaleruca luteola (Coleoptera: Chrysomelidae).

Plant secondary metabolites influence the feeding in insects through several modes of action. In this study, the physiological effects of erucin isothiocyanate were investigated on the elm leaf beetleXanthogaleruca luteola(Müller) (Coleoptera: Chrysomelidae) via impact on crustacean cardioactive peptide (CCAP) and midgut digestive enzymes. Third instar larvae of elm leaf beetle were fed on leaves impregnated with erucin for three days. The results showed that erucin decreasedα-amylase, lipase, and protease release. Western blot analysis and competitive ELISA showed that erucin decreased CCAP content of the midgut, brain, and hemolymph. Moreover, incubation of dissected midgut with CCAP and also its injection into the hemocoel increased digestive enzyme release. It could be concluded that erucin isothiocyanate decreases CCAP content that itself led to a decrease in digestive enzyme release. Also, it suggests that CCAP could be one of the factors, regulating feeding activities in the elm leaf beetle. This report shows that CCAP is both a midgut factor and a neuropeptide that regulates digestive enzyme release in the elm leaf beetle and could be used to study erucin effects in insects.

Horizontally acquired antibacterial genes associated with adaptive radiation of ladybird beetles.

BACKGROUND: Horizontal gene transfer (HGT) has been documented in many herbivorous insects, conferring the ability to digest plant material and promoting their remarkable ecological diversification. Previous reports suggest HGT of antibacterial enzymes may have contributed to the insect immune response and limit bacterial growth. Carnivorous insects also display many evolutionary successful lineages, but in contrast to the plant feeders, the potential role of HGTs has been less well-studied. RESULTS: Using genomic and transcriptomic data from 38 species of ladybird beetles, we identified a set of bacterial cell wall hydrolase (cwh) genes acquired by this group of beetles. Infection with Bacillus subtilis led to upregulated expression of these ladybird cwh genes, and their recombinantly produced proteins limited bacterial proliferation. Moreover, RNAi-mediated cwh knockdown led to downregulation of other antibacterial genes, indicating a role in antibacterial immune defense. cwh genes are rare in eukaryotes, but have been maintained in all tested Coccinellinae species, suggesting that this putative immune-related HGT event played a role in the evolution of this speciose subfamily of predominant predatory ladybirds. CONCLUSION: Our work demonstrates that, in a manner analogous to HGT-facilitated plant feeding, enhanced immunity through HGT might have played a key role in the prey adaptation and niche expansion that promoted the diversification of carnivorous beetle lineages. We believe that this represents the first example of immune-related HGT in carnivorous insects with an association with a subsequent successful species radiation.

An insect acetylcholinesterase biosensor utilizing WO3/g-C3N4 nanocomposite modified pencil graphite electrode for phosmet detection in stored grains.

This study was undertaken to assess the potential of Tribolium castaneum (Red flour beetle) acetylcholinesterase (Tc-AChE) based electrochemical biosensor integrating WO3/g-C3N4 nanocomposite modified Pencil graphite electrode to detect an organophosphate insecticide, Phosmet. The WO3/g-C3N4 nanocomposite provides a non-toxic, biocompatible surface for binding the enzyme on the electrode surface, attributed to its large surface area, high conductivity, and low ohmic resistance. The proposed biosensor shows a very good analytical performance with LOD 3.6 nM for Phosmet and effectively determined Phosmet in wheat with a 99% recovery rate. Furthermore, molecular docking deciphers the binding interactions of Phosmet with Tc-AChE using a modified AutoDock LGA algorithm and an AMBER03 force field in YASARA. The kinetic parameters strongly suggest the high potency of inhibitor with the enzyme. This study presents an adaptable, rapid, and straightforward approach that opens ways towards real progress in developing commercial biosensors for pesticide detection.

RNA interference-mediated silencing of vATPase subunits A and E affect survival and development of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.

RNA interference (RNAi) has emerged as a powerful tool for developing novel management strategies for controlling insect pests. The 28-spotted ladybeetle, Henosepilachna vigintioctopunctata is one of the most important pests attacking solanaceous plants in Asia. In this study, the potential of dietary RNAi to manage H. vigintioctopunctata was investigated using both in vitro synthesized and bacterially expressed double-stranded RNAs (dsRNAs) of HvvATPase A and HvvATPase E. The expression levels of HvvATPase A and HvvATPase E were higher in Malpighian tubules than in other tissue types. The silencing of HvvATPase A and HvvATPase E led to significant mortality in H. vigintioctopunctata larvae. In addition, the ingestion of HvvATPase A and HvvATPase E significantly deterred feeding behavior and subsequently arrested the development of H. vigintioctopunctata. Notably, the bacterially expressed dsRNAs consistently caused higher mortality in larvae and adults. Finally, the nontarget effects of the dsRNAs of H. vigintioctopunctata on the predatory ladybeetle Propylaea japonica were evaluated. P. japonica 1st instar larvae were administered vATPase A and vATPase E dsRNAs from H. vigintioctopunctata and P. japonica under the worst-case scenario, in which dsGFP served as negative control. There were significant effects of dsHvvATPase A on P. japonica at the transcriptional level but not at the organismal level, whereas dsHvvATPase E did not effect P. japonica at either the transcriptional or the organismal level. Collectively, the results of the study suggest that HvvATPase A and HvvATPase E can act as novel molecular targets for the control of H. vigintioctopunctata.

RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production.

The pinyon ips beetle, Ips confusus (LeConte) is a highly destructive pest in pine forests in western North America. When colonizing a new host tree, I. confusus beetles coordinate a mass attack to overcome the tree's defenses using aggregation pheromones. Ips confusus, as with other Ips spp. beetles, biosynthesize ipsdienol and ipsenol in a specific enantiomeric blend and ratio as aggregation pheromones. While several of the initial steps in the pheromone biosynthetic pathway have been well defined, the final steps were unknown. We used comparative RNA-Seq analysis between fed and unfed male I. confusus midgut tissue to identify candidate genes involved in pheromone biosynthesis. The 12,995 potentially unique transcripts showed a clear separation based on feeding state. Differential expression analysis identified gene groups that were tightly connected. This analysis identified all known pheromone biosynthetic genes and suggested a novel monoterpene double bond reductase, ipsdienone reductase (IDONER), with pheromone biosynthetic gene expression patterns. IDONER cDNA was cloned, expressed, and functionally characterized. The coding DNA sequence has an ORF of 1101 nt with a predicted translation product of 336 amino acids. The enzyme has a molecular weight of 36.7 kDa with conserved motifs of the medium chain dehydrogenases/reductase (MDR) superfamily in the leukotriene B4 dehydrogenases/reductases (LTB4R) family. Tagged recombinant protein was expressed and purified. Enzyme assays and GC/MS analysis showed IDONER catalyzed the reduction of ipsdienone to form ipsenone. This study shows that IDONER is a monoterpene double bond reductase involved in I. confusus pheromone biosynthesis.

Treating green pea aphids, Myzus persicae, with azadirachtin affects the predatory ability and protective enzyme activity of harlequin ladybirds, Harmonia axyridis.

As a natural enemy of green peach aphids, harlequin ladybirds, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), are also indirectly affected by azadirachtin. In this study, we evaluated the effects of ladybird exposure to azadirachtin through azadirachtin-treated aphids. About 2 mg/L azadirachtin treated aphid can deliver the azadirachtin to ladybird larvae in 12 and 24 h. And azadirachtin treatment affected the rate at which fourth instar larvae and adult ladybirds preyed on aphids. Furthermore, the antifeedant effect increased with increasing azadirachtin concentrations. Twelve hours after exposing fourth instar ladybird larvae to aphids treated with 10 mg/L azadirachtin, the antifeedant effect was 47.70%. Twelve hours after exposing adult ladybirds to aphids treated with 2 mg/L azadirachtin, the antifeedant effect was 67.49%. Forty-eight hours after exposing ladybird larvae to azadirachtin-treated aphids, their bodyweights were 8.37 ± 0.044 mg (2 mg/L azadirachtin), 3.70 ± 0.491 mg (10 mg/L azadirachtin), and 2.39 ± 0.129 mg (50 mg/L azadirachtin). Treatment with azadirachtin affected the ability of ladybirds to prey on aphids. The results indicated that the instant attack rate of ladybird larvae and adults and the daily maximum predation rate were reduced by azadirachtin treatment. Superoxide dismutase (SOD), peroxidase (POD), and peroxide (CAT) enzyme activities of ladybirds were affected after feeding on aphids treated with azadirachtin. Azadirachtin has certain antifeedant effects on ladybirds and affects the ability of ladybirds to prey on aphids and the activities of SOD, POD, and CAT enzymes, which results in inhibition of normal body development.

Colorado potato beetle alpha-amylase: Purification, action pattern and subsite mapping for exploration of active centre.

Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the active centre of Colorado potato beetle (Leptinotarsa decemlineata) α-amylase (LdAmy). Bond cleavage frequency values for LdAmy were determined by HPLC product analysis on a chromophore labelled maltooligomer substrate series. Binding energies between amino acid moieties of subsites and glucose residues of substrate were calculated. Active site contains six subsites in the binding region of LdAmy; four glycone- (-4, -3, -2, -1) and two aglycone-binding sites (+1, +2). Subsite map calculation resulted in apparent binding energies -11.8 and - 11.0 kJ/mol for subsites (+2) and (-3), respectively, which revealed very favorable interactions at these positions. Structures of binding sites of LdAmy and mammalian α-amylases show similarity, but there are variations in the binding energies at subsite (-2) and (-4). Differences were interpreted by comparison of amino acid sequences of human salivary α-amylase (HSA) and porcine pancreatic α-amylase (PPA) and two insect (Leptinotarsa decemlineata and Tenebrio molitor) enzymes. The observed substitution of positively charged His305 in HSA at subsite (-2) with an acidic Asp in LdAmy in the same position may explain the obtained energy reduction.

Molecular characterization and expression of two enzymes from Dendroctonus armandi, with phloem feeding and juvenile hormone.

The Chinese white pine beetle, Dendroctonus armandi Tsai and Li, is a serious native pest in the Qinling Mountains of China. exo-Bevicomin, as the main component of bark beetle pheromone, is released by the female D. armandi. In this paper, we identified two genes encoding, (Z)-6-nonen-2-ol dehydrogenase and CYP6CR, that are known to be involved in xo-brevicomin synthesis to improve the understanding of exo-brevicomin biosynthesis in the Chinese white pine beetle. The two protiens had high homology with their orthologs in the exo-brevicomin biosynthesis pathway from D. ponderosae. The expression profiles of CYP6CR12 and DaZnoDH in D. armandi females are closely correlated with exo-brevicomin biosynthesis. The expression levels of CYP6CR12 and DaZnoDH are also regulated by feeding behavior and juvenile hormone levels. Since they are also expressed in males, CYP6CR12 and DaZnoDH are not only important for exo-brevicomin biosynthesis that this might be potential role for the semichemical biosysthesis pathways.

The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase.

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonusrhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.

Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging.

PURPOSE: Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different D-luciferin (D-LH2) analogues in vivo. PROCEDURES: Transduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software. RESULTS: Our in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was D-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with D-LH2. CONCLUSION: We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne.

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval-pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval-pupal and pupal-adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.