Identification, Expression Patterns and RNA Interference of Aquaporins in Dendroctonus armandi (Coleoptera: Scolytinae) Larvae During Overwintering.
ABSTRACT: The ability to survive annual temperature minima could be a key determinant of distribution limits for insects under global climate change. Recent studies have suggested that insect aquaporins are indispensable for cellular water management under conditions that lead to dehydration and cold stress. Aquaporins are integral membrane water channel proteins in the major intrinsic protein superfamily and promote selected solutes and the movement of water across biological membranes. We cloned and characterized nine full-length aquaporins from Dendroctonus armandi (DaAqps), the most destructive forest pest in the Qinling Mountains of Shaanxi Province, China. Eight of the DaAqps belong to three classical aquaporin grades, including the Drosophila integral protein, the Pyrocoelia rufa integral protein, the entomoglyceroporins and one that belongs to the unorthodox grade of aquaporin 12-like channels. The DaAqps were increasingly expressed during different developmental stages and in different larval tissues, and expression peaked in mid-winter. They were tested under cold conditions for different lengths of time, and the expression of almost all DaAqps was down regulated with decreasing temperatures and long-term exposure to cold conditions. However, when the lowest temperatures were reached, the levels were immediately upregulated. These genes indicate that cold tolerance can improve through mortality responses at low temperatures after RNA interference of DaAqps. In our study, we analyzed the molecular response, expression patterns, and RNA interference of DaAqps and clarified the crucial role of protective compounds (aquaporins) underlying D. armandi cold tolerance and provide a new pest control method.
Project description:The Chinese white pine beetle, Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae), is a serious pest of coniferous forests in China. Thus, there is considerable interest in developing eco-friendly pest-control methods, with the use of semiochemicals as a distinct possibility. Olfaction is extremely important for fitness of D. armandi because it is the primary mechanism through which the insect locates hosts and mates. Thus, here we characterized nine full-length genes encoding chemosensory proteins (CSPs) from D. armandi. The genes were ubiquitously and multiply expressed across different developmental stages and adult tissues, indicating various roles in developmental metamorphosis, olfaction, and gustation. Ligand-binding assays implied that DarmCSP2 may be the carrier of D. armandi pheromones and various plant host volatiles. These volatiles were identified through RNA interference of DarmCSP2 as: (+)-?-pinene, (+)-?-pinene, (-)-?-pinene, (+)-camphene, (+)-3-carene, and myrcene. The systematic chemosensory functional analysis of DarmCSP2 in this study clarified the molecular mechanisms underlying D. armandi olfaction and provided a theoretical foundation for eco-friendly pest control.
Project description:The Chinese white pine beetle is arguably the most destructive forest insect in the Qinling Mountains in Northern China. Little is known about the structure of the fungal communities associated with Dendroctonus armandi, even though this wood-boring insect plays important roles in ecosystem and biological invasion processes that result in huge economic losses in pine forests. The aim of this study was to investigate the fungal community structure present in the galleries and guts of D. armandi at different developmental stages using PCR-denaturing gradient gel electrophoresis (DGGE). Analysis of PCR-amplified 18S rRNA gene fragments of fungi from the guts of D. armandi revealed fungal communities of low complexity that differed according to the developmental stage. Yeast of the genus Candida and the filamentous fungi Ophiostoma predominated in D. armandi and its galleries. In particular, Candida accounted for 56% of the fungal community in the pupal stage. Characterizing the evolution and content of the intestinal microbial community structure in D. armandi may facilitate the development of new pest control strategies.
Project description:The Chinese white pine beetle Dendroctonus armandi Tsai and Li, is arguably the most destructive forest insect in the Qinling Mountains in Northern China. Little is known about the structure of the bacterial communities associated with D. armandi even though this wood-boring insect plays important roles in ecosystem and biological invasion processes that result in huge economic losses in pine forests. The aim of this study was to investigate the composition of the bacterial communities present in the guts of D. armandi at different developmental stages using a culture-independent method involving PCR-denaturing gradient gel electrophoresis (DGGE). Analysis of PCR-amplified 16S rRNA gene fragments of bacteria from the guts of larvae, pupae, and male and female adults revealed bacterial communities of low complexity that differed according to the developmental stage. Citrobacter spp. and Pantoea spp. predominated in larvae and adults, whereas Methylobacterium was the dominant genus at the pupal stage. The main difference between the guts of male and female adults was the greater dominance of Citrobacter in females. Previous studies suggest that the bacterial community associated with D. armandi guts may influence insect development. The data obtained in this study regarding the phylogenetic relationships and the community structure of intestinal bacteria at different developmental stages of the D. armandi life cycle contribute to our understanding of D. armandi and could aid the development of new pest control strategies.
Project description:BACKGROUND: The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. RESULTS: We report on the characterization of Aquaporin-11 (AQP11). AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. CONCLUSION: AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain.
Project description:Water homeostasis is fundamental for cell survival. Transport of water across cellular membranes is governed by aquaporins-tetrameric integral membrane channels that are highly conserved throughout the prokaryotic and eukaryotic kingdoms. In eukaryotes, specific regulation of these channels is required and is most commonly carried out by shuttling the protein between cellular compartments (trafficking) or by opening and closing the channel (gating). Structural and functional studies have revealed phosphorylation as a ubiquitous mechanism in aquaporin regulation by both regulatory processes. In this review we summarize what is currently known about the phosphorylation-dependent regulation of mammalian aquaporins. Focusing on the water-specific aquaporins (AQP0?AQP5), we discuss how gating and trafficking are controlled by phosphorylation and how phosphorylation affects the binding of aquaporins to regulatory proteins, thereby highlighting structural details and dissecting the contribution of individual phosphorylated residues when possible. Our aim is to provide an overview of the mechanisms behind how aquaporin phosphorylation controls cellular water balance and to identify key areas where further studies are needed.
Project description:The aquaporin superfamily of hydrophobic integral membrane proteins constitutes water channels essential to the movement of water across the cell membrane, maintaining homeostatic equilibrium. During the passage of water between the extracellular and intracellular sides of the cell, aquaporins act as ultra-sensitive filters. Owing to their hydrophobic nature, aquaporins self-assemble in phospholipids. If a proper choice of lipids is made then the aquaporin biomimetic membrane can be used in the design of an artificial kidney. In combination with graphene, the aquaporin biomimetic membrane finds practical application in desalination and water recycling using mostly Escherichia coli AqpZ. Recently, human aquaporin 1 has emerged as an important biomarker in renal cell carcinoma. At present, the ultra-sensitive sensing of renal cell carcinoma is cumbersome. Hence, we discuss the use of epitopes from monoclonal antibodies as a probe for a point-of-care device for sensing renal cell carcinoma. This device works by immobilizing the antibody on the surface of a single-layer graphene, that is, as a microfluidic device for sensing renal cell carcinoma.
Project description:Aquaporins are integral membrane proteins that facilitate the diffusion of water and other small, uncharged solutes across the cellular membrane and are widely distributed in organisms from humans to bacteria. However, the characteristics of prokaryotic aquaporins remain largely unknown. We investigated the distribution and sequence characterization of aquaporins in prokaryotic organisms and summarized the transport characteristics, physiological functions, and regulatory mechanisms of prokaryotic aquaporins. Aquaporin homologues were identified in 3315 prokaryotic genomes retrieved from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, but the protein clustering pattern is not completely congruent with the phylogeny of the species that carry them. Moreover, prokaryotic aquaporins display diversified aromatic/arginine constriction region (ar/R) amino acid compositions, implying multiple functions. The typical water and glycerol transport characterization, physiological functions, and regulations have been extensively studied in Escherichia coli AqpZ and GlpF. A Streptococcus aquaporin has recently been verified to facilitate the efflux of endogenous H2O2, which not only contributes to detoxification but also to species competitiveness, improving our understanding of prokaryotic aquaporins. Furthermore, recent studies revealed novel regulatory mechanisms of prokaryotic aquaporins at post-translational level. Thus, we propose that intensive investigation on prokaryotic aquaporins would extend the functional categories and working mechanisms of these ubiquitous, intrinsic membrane proteins.
Project description:BACKGROUND: Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown. RESULTS: The screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur. CONCLUSION: The zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.
Project description:In this study, a polymerase chain reaction (PCR) based on homology genes of Orco was utilized to identify DarmOrco, which is essential for olfaction in D. armandi. The results showed that DarmOrco shares significant sequence homology with Orco proteins had known in other insects. Quantitative real-time PCR (qRT-PCR) analysis suggested that DarmOrco was abundantly expressed in adult D. armandi; by contrast, DarmOrco showed trace amounts of expression level in other stages. Of different tissues, DarmOrco expression level was the highest in the antennae. In order to understand the functional significance of Orco, we injected siRNA of DarmOrco into the conjunctivum between the second and third abdominal segments, and evaluated its expression after siRNA injected for 24 h, 48 h and 72 h. The results of qRT-PCR demonstrated that the reduction of mRNA expression level was significant (~80%) in DarmOrco siRNA-treated D. armandi than in water-injected and non-injected controls. The electroantennogram responses of females and males to 11 major volatiles of its host, were also reduced (30~68% for females; 16~70% for males) in siRNA-treated D. armandi compared with the controls. These results suggest that DarmOrco is crucial in mediating odorant perception.
Project description:Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.