Decrypting the molecular basis of cellular drug phenotypes by dose-resolved expression proteomics.

Proteomics is making important contributions to drug discovery, from target deconvolution to mechanism of action (MoA) elucidation and the identification of biomarkers of drug response. Here we introduce decryptE, a proteome-wide approach that measures the full dose-response characteristics of drug-induced protein expression changes that informs cellular drug MoA. Assaying 144 clinical drugs and research compounds against 8,000 proteins resulted in more than 1 million dose-response curves that can be interactively explored online in ProteomicsDB and a custom-built Shiny App. Analysis of the collective data provided molecular explanations for known phenotypic drug effects and uncovered new aspects of the MoA of human medicines. We found that histone deacetylase inhibitors potently and strongly down-regulated the T cell receptor complex resulting in impaired human T cell activation in vitro and ex vivo. This offers a rational explanation for the efficacy of histone deacetylase inhibitors in certain lymphomas and autoimmune diseases and explains their poor performance in treating solid tumors.

Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum.

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

Scintillation and Optical Characterization of CsCu2I3 Single Crystals from 10 to 400 K.

Currently only Eu2+-based scintillators have approached the light yield needed to improve the 2% energy resolution at 662 keV of LaBr3:Ce3+,Sr2+. Their major limitation, however, is the significant self-absorption due to Eu2+. CsCu2I3 is an interesting new small band gap scintillator. It is nonhygroscopic and nontoxic, melts congruently, and has an extremely low afterglow, a density of 5.01 g/cm3, and an effective atomic number of 50.6. It shows self-trapped exciton emission at room temperature. The large Stokes shift of this emission ensures that this material is not sensitive to self-absorption, tackling one of the major problems of Eu2+-based scintillators. An avalanche photo diode, whose optimal detection efficiency matches the 570 nm mean emission wavelength of CsCu2I3, was used to measure pulse height spectra. From the latter, a light yield of 36 000 photons/MeV and energy resolution of 4.82% were obtained. The scintillation proportionality of CsCu2I3 was found to be on par with that of SrI2:Eu2+. Based on temperature-dependent emission and decay measurements, it was demonstrated that CsCu2I3 emission is already about 50% quenched at room temperature. Using temperature-dependent pulse height measurements, it is shown that the light yield can be increased up to 60 000 photons/MeV by cooling to 200 K, experimentally demonstrating the scintillation potential of CsCu2I3. Below this temperature, the light yield starts to decrease, which can be linked to the unusually large increase in the band gap energy of CsCu2I3.

Micro-Twinning in IN738LC Manufactured with Laser Powder Bed Fusion.

Components manufactured with Metal Laser Powder Bed Fusion (PBF-LB/M) are built in a layerwise fashion. The PBF-LB/M build orientation affects grain morphology and orientation. Depending on the build orientation, microstructures from equiaxed to textured grains can develop. In the case of a textured microstructure, a clear anisotropy of the mechanical properties affecting short- and long-term mechanical properties can be observed, which must be considered in the component design. Within the scope of this study, the IN738LC tensile and creep properties of PBF-LB/M samples manufactured in 0° (perpendicular to build direction), 45° and 90° (parallel to build direction) build orientations were investigated. While the hot tensile results (at 850 °C) are as expected, where the tensile properties of the 45° build orientation lay between those of 0° and 90°, the creep results (performed at 850 °C and 200 MPa) of the 45° build orientation show the least time to rupture. This study discusses the microstructural reasoning behind the peculiar creep behavior of 45° oriented IN738LC samples and correlates the results to heat-treated microstructures and the solidification conditions of the PBF-LB/M process itself.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus.

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.

Ovariole-specific Yellow-g and Yellow-g2 proteins are required for fecundity and egg chorion rigidity in the red flour beetle, Tribolium castaneum.

Most insects reproduce by laying eggs that have an eggshell/chorion secreted by follicle cells, which serves as a protective barrier for developing embryos. Thus, eggshell formation is vital for reproduction. Insect yellow family genes encode for secreted extracellular proteins that perform different, context-dependent functions in different tissues at various stages of development involving, for example, cuticle/eggshell coloration and morphology, molting, courtship behavior and embryo hatching. In this study we investigated the function of two of this family's genes, yellow-g (TcY-g) and yellow-g2 (TcY-g2), on the formation and morphology of the eggshell of the red flour beetle, Tribolium castaneum. Real-time PCR analysis revealed that both TcY-g and TcY-g2 were specifically expressed in the ovarioles of adult females. Loss of function produced by injection of double-stranded RNA (dsRNA) for either TcY-g or TcY-g2 gene resulted in failure of oviposition. There was no effect on maternal survival. Ovaries dissected from those dsRNA-treated females exhibited ovarioles containing not only developing oocytes but also mature eggs in their egg chambers. However, the ovulated eggs were collapsed and ruptured, resulting in swollen lateral oviducts and calyxes. TEM analysis showed that lateral oviducts were filled with electron-dense material, presumably from some cellular content leakage out of the collapsed eggs. In addition, morphological abnormalities in lateral oviduct epithelial cells and the tubular muscle sheath were evident. These results support the hypothesis that both TcY-g and TcY-g2 proteins are required for maintaining the rigidity and integrity of the chorion, which is critical for resistance to mechanical stress and/or rehydration during ovulation and egg activation in the oviducts of T. castaneum. Because Yellow-g and Yellow-g2 are highly conserved among insect species, both genes are potential targets for development of gene-based insect pest population control methods.

Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Avoiding concentration quenching and self-absorption in Cs4EuX6 (X = Br, I) by Sm2+ doping.

The benefits of doping Cs4EuBr6 and Cs4EuI6 with Sm2+ are studied for near-infrared scintillator applications. It is shown that undoped Cs4EuI6 suffers from a high probability of self-absorption, which is almost completely absent in Cs4EuI6:2% Sm. Sm2+ doping is also used to gain insight in the migration rate of Eu2+ excitations in Cs4EuBr6 and Cs4EuI6, which shows that concentration quenching is weak, but still significant in the undoped compounds. Both self-absorption and concentration quenching are linked to the spectral overlap of the Eu2+ excitation and emission spectra which were studied between 10 K and 300 K. The scintillation characteristics of Cs4EuI6:2% Sm is compared to that of the undoped samples. An improvement of energy resolution from 11% to 7.5% is found upon doping Cs4EuI6 with 2% Sm and the scintillation decay time shortens from 4.8 s to 3.5 s in samples of around 3 mm in size.

Temporal changes in the physical and mechanical properties of beetle elytra during maturation.

Changes in physical properties of Tenebrio molitor and Tribolium castaneum elytra (hardened forewings) were studied to understand how the development of microstructure and chemical interactions determine cuticle mechanical properties. Analysis of these properties supports a model in which cuticular material is continuously secreted from epidermal cells to produce an extracellular matrix so that the outermost layers mature first. It is hypothesized that enzymatic crosslinking and pigmentation reactions along with dehydration help to stabilize the protein-chitin network within the initial layers of cuticle shortly after eclosion. Mature layers are proposed to bear most of the mechanical loads. The frequency dependence of the storage modulus and the tan δ values decreased during the beginning of maturation, reaching constant values after 48 h post-eclosion. A decrease of tan δ indicates an increase in crosslinking of the material. The water content declined from 75% to 31%, with a significant portion lost from within the open spaces between the dorsal and ventral cuticular layers. Dehydration had a less significant influence than protein crosslinking on the mechanical properties of the elytron during maturation. When Tribolium cuticular protein TcCP30 expression was decreased by RNAi, the tan δ and frequency dependence of E' of the elytron did not change during maturation. This indicates that TcCP30 plays a role in the crosslinking process of the beetle's exoskeleton. This study was inspired by previous work on biomimetic multicomponent materials and helps inform future work on creating robust lightweight materials derived from natural sources. STATEMENT OF SIGNIFICANCE: Examination of changes in the physical properties of the elytra (hardened forewings) of two beetle species advanced understanding of how the molecular interactions influence the mechanical properties of the elytra. Physical characterization, including dynamic mechanical analysis, determined that the outer portion of the elytra matured first, while epidermal cells continued to secrete reactive components until the entire structure reached maturation. RNA interference was used to identify the role of a key protein in the elytra. Suppression of its expression reduced the formation of crosslinked polymeric components in the elytra. Identifying the molecular interactions in the matrix of proteins and polysaccharides in the elytra together with their hierarchical architecture provides important design concepts in the development of biomimetic materials.

Chitin deacetylases are necessary for insect femur muscle attachment and mobility.

Muscle attachment sites (MASs, apodemes) in insects and other arthropods involve specialized epithelial cells, called tendon cells or tenocytes, that adhere to apical extracellular matrices containing chitin. Here, we have uncovered a function for chitin deacetylases (CDAs) in arthropod locomotion and muscle attachment using a double-stranded RNA-mediated gene-silencing approach targeted toward specific CDA isoforms in the red flour beetle, Tribolium castaneum (Tc). Depletion of TcCDA1 or the alternatively spliced TcCDA2 isoform, TcCDA2a, resulted in internal tendon cuticle breakage at the femur-tibia joint, muscle detachment from both internal and external tendon cells, and defective locomotion. TcCDA deficiency did not affect early muscle development and myofiber growth toward the cuticular MASs but instead resulted in aborted microtubule development, loss of hemiadherens junctions, and abnormal morphology of tendon cells, all features consistent with a loss of tension within and between cells. Moreover, simultaneous depletion of TcCDA1 or TcCDA2a and the zona pellucida domain protein, TcDumpy, prevented the internal tendon cuticle break, further supporting a role for force-dependent interactions between muscle and tendon cells. We propose that in T. castaneum, the absence of N-acetylglucosamine deacetylation within chitin leads to a loss of microtubule organization and reduced membrane contacts at MASs in the femur, which adversely affect musculoskeletal connectivity, force transmission, and physical mobility.

AA15 lytic polysaccharide monooxygenase is required for efficient chitinous cuticle turnover during insect molting.

Microbial lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of crystalline polysaccharides including chitin and cellulose. The discovery of a large assortment of LPMO-like proteins widely distributed in insect genomes suggests that they could be involved in assisting chitin degradation in the exoskeleton, tracheae and peritrophic matrix during development. However, the physiological functions of insect LPMO-like proteins are still undetermined. To investigate the functions of insect LPMO15 subgroup I-like proteins (LPMO15-1s), two evolutionarily distant species, Tribolium castaneum and Locusta migratoria, were chosen. Depletion by RNAi of T. castaneum TcLPMO15-1 caused molting arrest at all developmental stages, whereas depletion of the L. migratoria LmLPMO15-1, prevented only adult eclosion. In both species, LPMO15-1-deficient animals were unable to shed their exuviae and died. TEM analysis revealed failure of turnover of the chitinous cuticle, which is critical for completion of molting. Purified recombinant LPMO15-1-like protein from Ostrinia furnacalis (rOfLPMO15-1) exhibited oxidative cleavage activity and substrate preference for chitin. These results reveal the physiological importance of catalytically active LPMO15-1-like proteins from distant insect species and provide new insight into the enzymatic mechanism of cuticular chitin turnover during molting.

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-ß-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Heat management of a compact x-ray source for microbeam radiotherapy and FLASH treatments.

BACKGROUND: Microbeam and x-ray FLASH radiation therapy are innovative concepts that promise reduced normal tissue toxicity in radiation oncology without compromising tumor control. However, currently only large third-generation synchrotrons deliver acceptable x-ray beam qualities and there is a need for compact, hospital-based radiation sources to facilitate clinical translation of these novel treatment strategies. PURPOSE: We are currently setting up the first prototype of a line-focus x-ray tube (LFxT), a promising technology that may deliver ultra-high dose rates (UHDRs) of more than 100 Gy/s from a table-top source. The operation of the source in the heat capacity limit allows very high dose rates with micrometer-sized focal spot widths. Here, we investigate concepts of effective heat management for the LFxT, a prerequisite for the performance of the source. METHODS: For different focal spot widths, we investigated the temperature increase numerically with Monte Carlo simulations and finite element analysis (FEA). We benchmarked the temperature and thermal stresses at the focal spot against a commercial x-ray tube with similar power characteristics. We assessed thermal loads at the vacuum chamber housing caused by scattering electrons in Monte Carlo simulations and FEA. Further, we discuss active cooling strategies and present a design of the rotating target. RESULTS: Conventional focal spot widths led to a temperature increase dominated by heat conduction, while very narrow focal spots led to a temperature increase dominated by the heat capacity of the target material. Due to operation in the heat capacity limit, the temperature increase at the focal spot was lower than for the investigated commercial x-ray tube. Hence, the thermal stress at the focal spot of the LFxT was considered uncritical. The target shaft and the vacuum chamber housing require active cooling to withstand the high heat loads. CONCLUSIONS: The heat capacity limit allows very high power densities at the focal spot of the LFxT and thus facilitates very high dose rates. Numerical simulations demonstrated that the heat load imparted by scattering electrons requires active cooling.

Photocatalytic Activity of Fibrous Ti/Ce Oxides Obtained by Hydrothermal Impregnation of Short Flax Fibers.

Fibrous Ti/Ce oxide photocatalysts were prepared for the first time by a biomimetic solution process using short flax fibers (flax straw processing waste) as a biotemplate. Titanium polyhydroxy complex solutions with 3% and 5% cerium were used as precursors. Flax fibers were impregnated in an autoclave under hydrothermal conditions. Ti/Ce oxides were obtained from the biotemplate by annealing at 600 °C. The photocatalytic activity of the Ti/Ce oxides was studied by the adsorption and decomposition of the dye rhodamine B under UV irradiation. The photocatalytic decomposition of the dye was 50% and 75% faster for Ti/Ce oxides with 3% and 5% Ce, respectively, than for the analogous undoped fibrous TiO2. The morphologies, textures, and structures of the photocatalysts were studied by scanning electron microscopy, low temperature N2 adsorption/desorption, UV-Vis spectroscopy, and X-ray and XPS analytical methods. It was shown that the introduction of Ce into the precursor solution increased the surface irregularity of the Ti/Ce oxide crystallites compared to pure TiO2. This effect scaled with the Ce concentration. Ce improved the UV light absorption of the material. The Ti/Ce oxides contained Ce4+/Ce3+ pairs that played an important role in redox processes and intensified the photocatalytic activity.

Optimized photoluminescence quantum yield in upconversion composites considering the scattering, inner-filter effects, thickness, self-absorption, and temperature.

Optimizing upconversion (UC) composites is challenging as numerous effects influence their unique emission mechanism. Low scattering mediums increase the number of dopants excited, however, high scattering mediums increase the UC efficiency due to its non-linear power dependency. Scattering also leads to greater thermal effects and emission saturation at lower excitation power density (PD). In this work, a photoluminescence quantum yield (PLQY) increase of 270% was observed when hexagonal NaYF4:(18%)Yb3+,(2%)Er3+ phosphor is in air compared to a refractive index-matched medium. Furthermore, the primary inner-filter effect causes a 94% PLQY decrease when the excitation focal point is moved from the front of the phosphor to 8.4 mm deep. Increasing this effect limits the maximum excitation PD, reduces thermal effects, and leads to emission saturation at higher excitation PDs. Additionally, self-absorption decreases the PLQY as the phosphor's thickness increases from 1 to 9 mm. Finally, in comparison to a cuboid cuvette, a 27% PLQY increase occurs when characterizing the phosphor in a cylindrical cuvette due to a lensing effect of the curved glass, as supported by simulations. Overall, addressing the effects presented in this work is necessary to both maximize UC composite performance as well as report their PLQY more reliably.

Formation of Defect-Dicubane-Type NiII 2LnIII 2 (Ln = Tb, Er) Clusters: Crystal Structures and Modeling of the Magnetic Properties.

In the field of molecular nanoclusters, cubane and defect-dicubane, or butterfly structures, are typical examples of tetranuclear metal core architectures. In this work, a halogenated and anionic Schiff-base ligand (L2-) is utilized as it is predisposed to chelate within a cluster core to both 3d and 4f metal ions, in different binding configurations (H2L = 4-chloro-2-(2-hydroxy-3-methoxybenzyliden amino)phenol). The phenolate oxygen atoms of the deprotonated ligand can act in µ-O and µ3-O bridging binding modes for the intramolecular assembly of metal ions. Based on that, two tetranuclear and isostructural compounds [Ni2Tb2(L)4(NO3)2(DMF)2]·2CH3CN (1) and [Ni2Er2(L)4(NO3)2(DMF)2]·0.5CH3CN (2) were synthesized and structurally characterized. Magnetic susceptibility and magnetization data indicate the occurrence of dominant intramolecular ferromagnetic interactions between the spin centers. Particular emphasis is given to the theoretical description of the magnetic behavior, taking into account the Ln-Ni and Ni-Ni coupling paths and the magnetic anisotropy of the LnIII and NiII ions. The study is distinguished for its discussion of two distinct models, whereby model A relies on the uniaxial B 20 Stevens term describing the lanthanide anisotropy and model B is based on point-charge model calculations. Importantly, the physical meaning of the obtained parameters for both models was critically scrutinized.

Yellow-y Functions in Egg Melanization and Chorion Morphology of the Asian Tiger Mosquito, Aedes albopictus.

The Asian tiger mosquito, Aedes albopictus, is one of the most serious public health pests, which can transmit various vector-borne diseases. Eggs from this mosquito species become dark black shortly after oviposition and exhibit high desiccation resistance. Some of the Yellow proteins that act as dopachrome conversion enzymes (DCEs) are involved in the tyrosine-mediated tanning (pigmentation and sclerotization) metabolic pathway that significantly accelerates melanization reactions in insects. In this research, we analyzed the function of one of the yellow genes, yellow-y (AalY-y), in eggshell/chorion melanization of Ae. albopictus eggs. Developmental and tissue-specific expression measured by real-time PCR showed that AalY-y transcripts were detected at all stages of development analyzed, with significantly higher levels in the ovaries from blood-fed adult females. Injection of double-stranded RNA for AalY-y (dsAalY-y) had no significant effect on fecundity. However, unlike dsEGFP-treated control eggs that become black by 2-3 h after oviposition (HAO), dsAalY-y eggs were yellow-brown at 2 HAO, and reddish-brown even at 48 HAO. dsEGFP eggs exhibited resistance to desiccation at 48 HAO, whereas approximately 50% of the dsAalY-y eggs collapsed when they were moved to a low humidity condition. In addition, TEM analysis revealed an abnormal morphology and ultrastructure of the outer-endochorion in the dsAalY-y eggs. These results support the hypothesis that AalY-y is involved in the tyrosine-induced melanin biosynthetic pathway, plays an important role in black melanization of the chorion and functions in conferring proper morphology of the outer-endochorion, a structure that is presumably required for egg desiccation resistance in Ae. albopictus.

Yellow-g and Yellow-g2 proteins are required for egg desiccation resistance and temporal pigmentation in the Asian tiger mosquito, Aedes albopictus.

Eggs from Aedes mosquitoes exhibit desiccation resistance that helps them to survive and spread as human disease vectors throughout the world. Previous studies have suggested that eggshell/chorion melanization and/or serosal cuticle formation are important for desiccation resistance. In this study, using dsRNAs for target genes, we analyzed the functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the resistance to egg desiccation of the Asian tiger mosquito, Aedes albopictus, a species in which neither the timing of the melanization nor temporal development of the serosal cuticle is correlated with desiccation resistance. Injections of dsAalY-g, dsAalY-g2 or dsAalY-g/g2 (co-injection) into adult females have no effect on their fecundity. However, initial melanization is delayed by 1-2 h with the eggshells eventually becoming black similar to that observed in eggs from dsEGFP-injected control females. In addition, the shape of the eggs from dsAalY-g, -g2 and -g/g2-treated females is abnormally crescent-shaped and the outermost exochorion is more fragile and partially peeled off. dsEGFP control eggs, like those from the wild-type strain, acquire resistance to desiccation between 18 and 24 h after oviposition (HAO). In contrast, ~80% of the 24 HAO dsAalY-g and dsAalY-g2 eggs collapse when they are transferred to a low humidity environment. In addition, there is no electron-dense outer endochorion evident in either dsAalY-g or dsAalY-g2 eggs. These results support the hypothesis that AalY-g and AalY-g2 regulate the timing of eggshell darkening and are required for integrity of the exochorion as well as for rigidity, normal morphology and formation of the outer endochorion, a structure that apparently is critical for desiccation resistance of the Ae. albopictus egg.

Gene functions in adult cuticle pigmentation of the yellow mealworm, Tenebrio molitor.

In many arthropod species including insects, the cuticle tanning pathway for both pigmentation and sclerotization begins with tyrosine and is responsible for production of both melanin- and quinoid-type pigments, some of which are major pigments for body coloration. In this study we identified and cloned cDNAs of the yellow mealworm, Tenebrio molitor, encoding seven key enzymes involved in this pathway including tyrosine hydroxylase (TmTH), DOPA decarboxylase (TmDDC), laccase 2 (TmLac2), Yellow-y (TmY-y), arylalkylamine N-acetyltransferase (TmAANAT1), aspartate 1-decarboxylase (TmADC) and N-ß-alanyldopamine synthase (Tmebony). Expression profiles of these genes during development were analyzed by real-time PCR, revealing development-specific patterns of expression. Loss of function mediated by RNAi of either 1) TmTH or TmLac2, 2) TmDDC or TmY-y, and 3) TmAANAT1, TmADC or Tmebony resulted in pale/white, light yellow/brown and dark/black adult body coloration, respectively. In addition, there are three distinct layer/regional pigmentation differences in rigid types of adult cuticle, a brownish outer exocuticle (EX), a dark pigmented middle mesocuticle (ME) and a transparent inner endocuticle (EN). Decreases in pigmentation of the EX and/or ME layers were observed after RNAi of TmDDC or TmY-y. In TmADC- or Tmebony-deficient adults, a darker pigmented EX layer was observed. In TmAANAT1-deficient adults, trabeculae formed between the dorsal and ventral elytral cuticles as well as the transparent EN layer became highly pigmented. These results demonstrate that knocking down the level of gene expression of specific enzymes of this tyrosine metabolic pathway leads to abnormal pigmentation in individual layers and substructure of the rigid adult exoskeleton of T. molitor.