Alimentary microbes of winter‐form Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a damaging pest of fruit. Reproductively diapausing adults overwinter in woodlands and remain active on warmer winter days. It is unknown if this adult phase of the lifecycle feeds during the winter period, and what the food source may be. This study characterized the flora in the digestive tract of D. suzukii using a metagenomics approach. Live D. suzukii were trapped in four woodlands in the south of England and their guts dissected for DNA extraction and amplicon‐based metagenomics sequencing (internal transcribed spacer and 16S rRNA). Analysis at genus and family taxonomic levels showed high levels of diversity with no differences in digestive tract bacterial or fungal biota between woodland sites of winter‐form D. suzukii. Female D. suzukii at one site appeared to have higher bacterial diversity in the alimentary canal than males, but there was a site, sex interaction. Many of the biota were associated with cold, wet climatic conditions and decomposition. This study provides the first evidence that winter‐form D. suzukii may be opportunistic feeders during the winter period and are probably exploiting food sources associated with moisture on decomposing vegetation during this time. A core gut microbiome has been identified for winter‐form D. suzukii.     

Genomic targeting by recombinase‐mediated cassette exchange in the spotted wing drosophila,Drosophila suzukii

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon‐mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase‐mediated cassette exchange (RMCE) system for D. suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon‐mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D. suzukii, and quantitative PCR determined that polyubiquitin‐regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology‐directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.     

Wolbachia‐mediated protection against viruses in the invasive pest Drosophila suzukii

The maternally inherited bacterium Wolbachia is well known for spreading in natural populations by manipulating the reproduction of its arthropod hosts, but can also have mutualist effects that increase host fitness. In mosquitoes and Drosophila some Wolbachia strains can lead to an increase in survival of virus‐infected insects, and in most cases this is associated with reduced accumulation of the virus in host tissues. We investigated if the Wolbachia strain wSuz, which naturally infects Drosophila suzukii, is able to confer protection against Drosophila C virus and Flock House virus in different host genetic backgrounds. We found that this strain can increase host survival upon infection with these two viruses. In some cases this effect was associated with lower viral titres, suggesting that it confers resistance to the viruses rather than allowing the flies to tolerate infection. Our results indicate that, in D. suzukii, the antiviral protection provided by Wolbachia is not correlated to its density as found in other Drosophila species. This study demonstrates a phenotypic effect induced by wSuz on its native host which could explain its maintenance in natural populations of D. suzukii.     

mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity

The epithelial Na⁺ channel (ENaC) is essential for Na⁺ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na⁺ and K⁺ transport in WT and Sgk1^–/– mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na⁺ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K⁺ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na⁺ homeostasis through SGK1-dependent modulation of ENaC activity.     

Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper

In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference‐based insect pest management.     

Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity

Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo-sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo-sensitive TRP channels in cultured odontoblast-like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast-like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca²⁺]_i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca²⁺]_i which could be blocked by TRP-channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.     

Phenotypic variation of autosomal recessive pseudohypoaldosteronism type I: a case in point

We present a 27‐month‐old male infant with pseudohypoaldosteronism, with two novel α‐subunits, epithelial sodium channel (ENaC) mutations. Despite the presence of the ENaC in the lungs, kidneys, and exocrine glands, he continues to only have renal and exocrine involvement, stressing differential effects of the mutation in each organ.     

In vitro vascularization of a combined system based on a 3D printing technique

A vital challenge in complex organ manufacturing is to vascularize large combined tissues. The aim of this study is to vascularize in vitro an adipose‐derived stem cell (ADSC)/fibrin/collagen incorporated three‐dimensional (3D) poly(d,l ‐lactic‐co‐glycolic acid) (PLGA) scaffold (10 × 10 × 10 mm³) with interconnected channels. A low‐temperature 3D printing technique was employed to build the PLGA scaffold. A step‐by‐step cocktail procedure was designed to engage or steer the ADSCs in the PLGA channels towards both endothelial and smooth muscle cell lineages. The combined system had sufficient mechanical properties to support the cell/fibrin/collagen hydrogel inside the predefined PLGA channels. The ADSCs encapsulated in the fibrin/collagen hydrogel differentiated to endothelial and smooth muscle cell lineage, respectively, corresponding to their respective locations in the construct and formed vascular‐like structures. This technique allows in vitro vascularization of the predefined PLGA channels and provides a choice for complex organ manufacture. Copyright © 2014 John Wiley & Sons, Ltd.     

Silencing of the DNA methyltransferase 1 associated protein 1 (DMAP1) gene in the invasive ladybird Harmonia axyridis implies a role of the DNA methyltransferase 1‐DMAP1 complex in female fecundity

The invasive harlequin ladybird Harmonia axyridis is a textbook example of polymorphism and polyphenism as the temperature during egg development determines the frequency of melanic morphs and the number and size of black spots in nonmelanic morphs. Recent concepts in evolutionary biology suggest that epigenetic mechanisms can translate environmental stimuli into heritable phenotypic changes. To investigate whether epigenetic mechanisms influence the penetrance and expressivity of colour morphs in H. axyridis, we used RNA interference to silence key enzymes required for DNA methylation and histone modification. We found that neither of these epigenetic mechanisms affected the frequency of different morphs, but there was a significant impact on life‐history traits such as longevity and fecundity. Strikingly, we found that silencing the gene encoding for DNA methyltransferase 1 associated protein 1 (DMAP1) severely reduced female fecundity, which correlated with an abundance of degenerated ovaries in DMAP1‐knockdown female beetles. Finally, we observed significant differences in DMAP1 expression when we compared native and invasive H. axyridis populations with a biocontrol strain differing in egg‐laying capacity, suggesting that the DNA methyltransferase 1‐DMAP1 complex may influence the invasive performance of this ladybird.     

Olfactory co‐receptor is involved in host recognition and oviposition in Ophraella communa (Coleoptera: Chrysomelidae)

Common ragweed (Ambrosia artemisiifolia) is a notorious invasive weed that has spread across most temperate regions of the world. The beetle (Ophraella communa) is considered to be an effective control agent against A. artemisiifolia. As an oligophagous insect, its olfactory system is extremely important for host seeking in the wild. To the best of our knowledge, there is no report on the molecular mechanisms underlying olfaction recognition in this beetle. Hence, in this study, we characterized the odorant receptor co‐receptor of O. communa and named it as ‘OcomORco’. Real‐time quantitative PCR (qRT‐PCR) showed that, compared to the control treatment, RNA interference (RNAi) strongly reduced the expression of OcomORco by 89% in male and 90% in female beetles. Electroantennogram assay showed that the antennal response of both male and female beetles to four volatiles of A. artemisiifolia was significantly reduced. The injected male or female beetles lost their preference for plant leaves as observed in the behavioural tests. In addition, disruption of the expression of OcomORco resulted in a reduction of oviposition, while there was no difference in larval hatching rate between control and knockdown females. We demonstrated that OcomORco plays a vital role in olfactory perception and host search in O. communa, and it is involved in oviposition in an indirect way.     

Dexmedetomidine enhances human lung fluid clearance through improving alveolar sodium transport

Accumulating evidence shows that dexmedetomidine can attenuate lung edema with acute lung injury in experimental mouse and rat models, but the mechanisms of dexmedetomidine on human alveolar fluid transport are still unknown. We measured the effects of dexmedetomidine on alveolar fluid clearance in human lung lobes ex vivo. Moreover, we measured the regulation of transepithelial Na⁺ transport by dexmedetomidine in H441 cells by electrophysiological technique and Western blot method. Our results showed that intratracheal instillation of dexmedetomidine markedly increased the reabsorption of 5% bovine serum albumin instillate (19.8 ± 1.4%, P < 0.01 vs. Control, n = 5). Further studies suggested that dexmedetomidine increased amiloride‐sensitive short‐circuit currents in permeabilized H441 monolayers and whole cell amiloride‐sensitive Na⁺ currents in a dose‐dependent fashion. Real‐time PCR and Western blot results showed that dexmedetomidine could enhance the mRNA and protein expression of α‐ENaC subunit, while inhibiting the phosphorylation of ERK_1/2. These data demonstrate that dexmedetomidine could improve human lung fluid clearance and lung epithelial Na⁺ channel activity, and these effects may be mediated through the enhancement of α‐ENaC expression and inhibition of ERK_1/2 pathway.