Electroporation-mediated RNA interference reveals a role of the multicopper oxidase 2 gene in dragonfly cuticular pigmentation

Dragonflies are colorful insects, and recent RNA sequencing studies have identified a number of candidate genes potentially involved in their color pattern formation and color vision. However, functional aspects of such genes have not been assessed due to the lack of molecular genetic tools applicable to dragonflies. We established an electroporation-mediated RNA interference (RNAi) procedure using the tiny dragonfly Nannophya pygmaea Rambur, 1842 (Odonata: Libellulidae) that targets the multicopper oxidase 2 gene (MCO2; also known as laccase2 gene) responsible for cuticular pigmentation in many insects. RNA sequencing of N. pygmaea and genomic survey of the dragonfly Ladona fulva identified four multicopper oxidase family genes: MCO1, MCO2, MCO3 and multicopper oxidase-related protein gene (MCORP). In N. pygmaea, MCO2 was specifically expressed around the cuticular pigmentation period, whereas MCO1 was constantly expressed. MCORP was expressed at adult stages, and MCO3 was scarcely expressed. When we applied in vivo electroporation, final instar larvae injected with MCO2 small interfering RNA became adults with patchy unpigmented regions. RNAi without in vivo electroporation did not affect cuticular pigmentation, suggesting that dragonflies do not show a systemic RNAi response. These results indicate that MCO2 is required for cuticular pigmentation across diverse insects, and highlight the usefulness of the electroporation-mediated RNAi method in dragonflies.     

Effects of time and environmental conditions on the quality of DNA extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest

Extraction of DNA from non-invasive samples (feces) has been used increasingly in genetic research on wildlife. For effective and reliable genetic analyses, knowledge about which samples should be selected in the field is essential. For this reason, we examined the process of DNA degradation in feces of deer. We collected fresh fecal pellets from three wild deer living in a warm temperate forest. We then assessed the effects of time (3, 5, and 10 days) under three environmental conditions (on the forest floor, on exposed ground, and inside the laboratory) on the rates of correct genotyping (CG), amplification failure (NA), genotyping error among positive amplification (ER), false alleles (FA), and allelic dropout (AD) of 15 microsatellite loci. The rate of CG significantly decreased, and those of NA and FA increased with increasing lapse of time. Rates of CG tended to be highest and those of NA, ER, FA, and AD to be lowest in feces kept inside, followed by those on the forest floor. Suitability of samples for DNA extraction was lowest in fecal pellets left on exposed ground, and we suspect that rain may hasten DNA degradation. NA rate could serve as a reliable indicator of the quality of fecal pellets because it was significantly positively correlated with ER rate. For efficient genetic analyses using deer feces in warm temperate zones, we recommend collecting fecal pellets within 3 days of defecation, during periods without rainfall and from under the cover of trees.     

Biosynthesis and therapeutic implications of iridoid glycosides from <Emphasis Type="Italic">Picrorhiza</Emphasis> genus: the road ahead

Picrorhiza genus is emerging as an important paradigm for herbal drug formulations due to its versatile iridoid glycosides exhibition and robustness in the treatment of diverse infections including hepatic amoebiasis, cancer, malaria, ulcerative colitis and cerebral ischemia reperfusion injury. Owing to the superiority of these bioactivities, iridoid glycosides from Picrorhiza have become a hot research area over the years. A metabolic pathway for the formation of iridoid glycosides has been proposed. However, some enzymes and genes of this route are still unidentified and demand the enumeration of facilitating pathways contributing to the biosynthesis of iridoid glycosides. This review summarizes the current knowledge of all naturally occurring iridoid glycosides from Picrorhiza, their biosynthesis and pharmacological capabilities which could provide the insight into metabolic regulation and the basis for the development of new drugs.     

Damage by <Emphasis Type="Italic">Tibraca limbativentris</Emphasis> Stål (Pentatomidae) to Upland Rice Cultivated in Amazon Rainforest Region (Brazil) at Different Growth Stages

In this study, we evaluated the damage caused in the field by Tibraca limbativentris Stål adults at different levels of infestation (0, 1, 2, and 4 stink bugs) per three rice plants during three growth stages (V8, V13, and R4 stages) of upland rice cultivated in southwestern of Pará State, Amazon Rainforest region, Brazil. Heading time (panicle exertion) was affected by T. limbativentris infestations mainly in the vegetative stage and the whiteheads percentage in treatments ranged from 18.2 to 38%. The dead hearts percentages varied between 0 and 21.5%, and the mean number of primary branches (ramifications) ranged from 5.9?±?0.4 to 12.3?±?0.2. The number of empty spikelets was only affected in infestations with four insects/three plants, while the quantity of filled grains per panicle was affected only when infestations occurred during the vegetative stage. The total number of spikelets (filled?+?empty) per panicle decreased significantly in all phenological stages, and the percentage of damage ranged from 17 to 44% among treatments. Based on the proportion of damage observed, we suggest doubling the number of insects presently used as action threshold to 2 and 4 stink bugs per 15 stalks sampled for the vegetative stage, and of 1 or 2 stink bugs per 15 stalks sampled at the beginning of reproductive stage (R3/R4). Also, the field should be monitored during the entire vegetative stage, since most damage was observed in this phenological stage.     

Taxonomy and First Records of Two <Emphasis Type="Italic">Megaselia</Emphasis> Rondani Species (Diptera: Phoridae) Preying upon Eggs of <Emphasis Type="Italic">Phyllomedusa iheringii</Emphasis> Boulenger (Anura: Phyllomedusidae)

Non-aquatic reproductive modes have evolved among frogs possibly favored by some advantages such as the avoidance of aquatic predators. These reproductive modes, however, make the egg clutches susceptible to terrestrial predators, among which Diptera larvae are some of the most harmful. The present work reports the predation by phorid flies of 22 egg clutches of Phyllomedusa iheringii Boulenger in the South of Brazil. Phorid specimens were identified as Megaselia bruchiana (Borgmeier & Schmitz) and Megaselia necrophaga (Enderlein), species that were reported previously to be associated with ants and dead beetles, respectively. Frog-feeding in these species is hypothesized to be use of an alternative seasonal food source. We amend the diagnoses of both Megaselia species and provide new illustrations to facilitate their identification. We also describe the male of M. bruchiana for the first time and associate males with females of both species.     

The Role of DNA Methylation in Lens Development and Cataract Formation

Epigenetics pertains to heritable alterations in genomic structural modifications without altering genomic DNA sequence. The studies of epigenetic mechanisms include DNA methylation, histone modifications, and microRNAs. DNA methylation may contribute to silencing gene expression which is a major mechanism of epigenetic gene regulation. DNA methylation regulatory mechanisms in lens development and pathogenesis of cataract represent exciting areas of research that have opened new avenues for association with aging and environment. This review addresses our current understanding of the major mechanisms and function of DNA methylation in lens development, age-related cataract, secondary cataract, and complicated cataract. By understanding the role of DNA methylation in the lens disease and development, it is expected to open up a new therapeutic approach to clinical treatment of cataract.     

Vimentin is important in the neural differentiation of PC12 cells promoted by sialylation

Sialic acid modification is a kind of post-translational modification. To investigate the regulation effect of sialic acid on neural differentiation, we used CycloManN propanyl perac (CycloManN pro), a metabolic precursor of sialic acid, to treat PC12 cells. We noted that CycloManN pro indeed robustly promoted global sialylation detected by MAL II lectin blot in PC12 cells. Simultaneously, we interestingly found that the neurite outgrowth of PC12 cells was significantly promoted by the CycloManN pro treatment. The profile analysis of sialylated proteins showed that a protein band at 55KD was greatly enhanced especially in PC12L cells after CycloManN pro treatment. After enrichment with lectin MAL II, the proteins in this band were analyzed by mass spectrometry. The results showed that 23 proteins were in the band, but the score of vimentin was the highest among them. To investigate further the role of vimentin in the process of neurite differentiation, vimentin construct was transfected into PC12 cells. We interestingly observed that ectopic expression of vimentin significantly enhanced the neurite outgrowth induced by CycloManN pro. However, after three potential glycosylation sites (Ser-7, Thr-33, Ser-34:) of vimentin were mutated to alanine, overexpression of the mutated vimentin completely lost the enhancement activity for the neural differentiation even in the presence of CycloManN pro. Taken together, our study demonstrated that vimentin was important in the induction of neural differentiation by CycloManN pro.     

Gene duplications and the evolution of c-type lysozyme during adaptive radiation of East African cichlid fish

Given the reported degraded nature of DOC in the Rio Negro, and low oxygen, pH, and bacterial riverine levels, we hypothesized: (1) DOC would have strong humic and fulvic acid fluorescence signals with high aromaticity and large mean molecular weight; and (2) photo-oxidation rates would be slow, and reactive oxygen species (ROS) concentrations low, producing no oxidative stress in biota. We surveyed the environment and properties of DOC and explored DOC photo-oxidation and fish sensitivity to DOC products. DOC properties were investigated using absorption and fluorescence indices and parallel factor analysis (PARAFAC) of excitation–emission matrices. ROS concentrations were measured spectrophotometrically. A native fish, Hemigrammus levis, was exposed to photo-oxidizing DOC and its tissues (brain, gill, liver) assayed for changes in antioxidant and biotransformation enzymes. With respect to our hypotheses, (1) DOC was highly terrigenous, with high SAC₃₄₀ values (aromaticity), high capacity to produce ROS, and high tryptophan-like fluorescence (bacterial, autochthonous signal); (2) photo-oxidation rates were appreciable, while products were related to mean UV-radiation levels (total radiation was constant). ROS levels were often higher than freshwater averages, yet fish experienced no oxidative stress. Results suggest photo-oxidation influences patterns in C-cycling, bacterial production and community dynamics between wet and dry seasons.     

Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5?±?0.1 (n?=?2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.     

Changes in cardiac Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase expression and activity in female rats fed a high-fat diet

The aim of this study was to investigate whether the presence of endogenous estradiol alters the effects of a high-fat (HF) diet on activity/expression of the cardiac Na⁺/K⁺-ATPase, via PI3K/IRS and RhoA/ROCK signalling cascades in female rats. For this study, female Wistar rats (8 weeks old, 150–200 g) were fed a standard diet or a HF diet (balanced diet for laboratory rats enriched with 42% fat) for 10 weeks. The results show that rats fed a HF diet exhibited a decrease in phosphorylation of the α₁ subunit of Na⁺/K⁺-ATPase by 30% (p < 0.05), expression of total α₁ subunit of Na⁺/K⁺-ATPase by 31% (p < 0.05), and association of IRS1 with p85 subunit of PI3K by 42% (p < 0.05), while the levels of cardiac RhoA and ROCK2 were significantly increased by 84% (p < 0.01) and 62% (p < 0.05), respectively. Our results suggest that a HF diet alters cardiac Na⁺/K⁺-ATPase expression via molecular mechanisms involving RhoA/ROCK and IRS-1/PI3K signalling in female rats.