A Short Peptide Designed from Late Embryogenesis Abundant Protein Enhances Acid Tolerance in Escherichia coli.

Unsuitable pH is a major limiting factor for all organisms, and a low pH can lead to organism death. Late embryogenesis abundant (LEA) peptides confer tolerance to abiotic stresses including salinity, drought, high and low temperature, and ultraviolet radiation same as the LEA proteins from which they originate. In this study, LEA peptides derived from group 3 LEA proteins of Polypedilum vanderplanki were used to enhance low pH tolerance. Recombinant Escherichia coli BL21 (DE3) cells expressing the five designed LEA peptides were grown at pH 4, 3, and 2. The transformants showed higher growth capacity at low pH as compared to control cells. These results indicate that LEA peptide could prevent E. coli cell death under low pH conditions.

The Antioxidant System in the Anhydrobiotic Midge as an Essential, Adaptive Mechanism for Desiccation Survival.

One of the major damaging factors for living organisms experiencing water insufficiency is oxidative stress. Loss of water causes a dramatic increase in the production of reactive oxygen species (ROS). Thus, the ability for some organisms to survive almost complete desiccation (called anhydrobiosis) is tightly related to the ability to overcome extraordinary oxidative stress. The most complex anhydrobiotic organism known is the larva of the chironomid Polypedilum vanderplanki. Its antioxidant system shows remarkable features, such as an expansion of antioxidant genes, their overexpression, as well as the absence or low expression of enzymes required for the synthesis of ascorbate and glutathione and their antioxidant function. In this chapter, we summarize existing data about the antioxidant system of this insect, which is able to cope with substantial oxidative damage, even in an intracellular environment that is severely disturbed due to water loss.

Physicochemical Aspects of the Biological Functions of Trehalose and Group 3 LEA Proteins as Desiccation Protectants.

In this review, we first focus on the mechanism by which the larva of the sleeping chironomid, Polypedilum vanderplanki, survives an extremely dehydrated state and describe how trehalose and probably late embryogenesis abundant (LEA) proteins work as desiccation protectants. Second, we summarize the solid-state and solution properties of trehalose and discuss why trehalose works better than other disaccharides as a desiccation protectant. Third, we describe the structure and function of two model peptides based on group 3 LEA proteins after a short introduction of native LEA proteins themselves. Finally, we present our conclusions and a perspective on the application of trehalose and LEA model peptides to the long-term storage of biological materials.

Embryo development. A cysteine-clamp gene drives embryo polarity in the midge Chironomus.

In the fruit fly Drosophila, head formation is driven by a single gene, bicoid, which generates head-to-tail polarity of the main embryonic axis. Bicoid deficiency results in embryos with tail-to-tail polarity and no head. However, most insects lack bicoid, and the molecular mechanism for establishing head-to-tail polarity is poorly understood. We have identified a gene that establishes head-to-tail polarity of the mosquito-like midge, Chironomus riparius. This gene, named panish, encodes a cysteine-clamp DNA binding domain and operates through a different mechanism than bicoid. This finding, combined with the observation that the phylogenetic distributions of panish and bicoid are limited to specific families of flies, reveals frequent evolutionary changes of body axis determinants and a remarkable opportunity to study gene regulatory network evolution.

Hsp70 expression in Chironomus ramosus exposed to gamma radiation.

PURPOSE: A tropical species of midge, Chironomus ramosus has been recently reported to be one of the radio-tolerant groups of organisms. The present study was undertaken to examine the protein profile and expression of Heat shock protein-70 (Hsp70) in gamma radiation stress, which has also been reported as a common biomarker for different type of stressors. MATERIALS AND METHODS: Metabolic labelling of salivary gland (SG) proteins with [(35)S]-methionine showed over-expression of a 70 kDa protein band up to 4 hours (h) of observation in the post exposure recovery period. For confirmation of the expression of Hsp70 in SG cells after gamma radiation exposure, semi-quantitative real-time-polymerase chain reaction (RT-PCR), Western blotting and immuno-fluorescence detection of Hsp70 were carried out. RESULTS: Results showed elevated levels of Hsp70 mRNA and protein in SG cells of larvae immediately after gamma radiation exposure. The levels dropped to basal values by 48 h in the recovery period. CONCLUSIONS: The present study confirmed that radio-tolerant midge, C. ramosus expressed Hsp70 upon gamma radiation exposure and Hsp70 might be one of the gamma radiation-induced stress proteins required during the early stages of radiation stress management in aquatic midge larvae. This is the first report of its kind from the juvenile stage of any aquatic insect group.

Cells from an anhydrobiotic chironomid survive almost complete desiccation.

Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science. As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210. Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply. We also attempted dry-preservation of organs isolated from P. vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation.

Fish assemblages in stream stretches occupied by cattail (Typhaceae, Angiospermae) stands in Southeast Brazil

Macrophytes are a major component of lentic and lotic aquatic ecosystems. As consequences of environmental degradation, species of cattail (genus Typha) may become dominant along streams. The purpose of this study was to investigate the structure and feeding of fish assemblages in stream stretches under influence of Typha sp., also addressing the influences of temporal variation on composition, abundance, biomass, diversity, species richness, and feeding of fish. Six streams (labelled S1-S6) in the upper rio Turvo basin, southeast Brazil, with dense stands of Typha sp. in the instream and riparian habitat, were studied in six campaigns during three central months in both of the dry and wet periods, by using a standardized collection effort. Thirty-seven fish species were registered, totaling 4,228 individuals and 3.9 kg of biomass. Abundance, biomass, diversity, and species richness was higher in the wet period, but only the temporal variation in the species richness revealed to be statistically significant. Cluster analyses with composition and abundance showed little temporal similarity, but indicated two groups of streams (S1-S2-S5 and S3-S4-S6), that were corroborated along the axis 1 of the ordination analysis. Resident species was represented by six species, most of them considered tolerant and generalists. Chironomidae aquatic larvae and detritus were the most important items in the fish diet. These results suggest that the fish populations are opportunistic in exploring stream stretches occupied by cattail.

Macrófitas são um importante componente de ecossistemas aquáticos lóticos e lênticos. Como consequências da degradação ambiental, espécies de taboas (gênero Typha) podem se tornar dominantes em riachos. O objetivo do presente estudo foi investigar a estrutura e alimentação de uma comunidade de peixes em trechos de riachos com influência de Typha sp., bem como a variação temporal na composição, abundância, biomassa, diversidade, riqueza de espécies e alimentação da ictiofauna. Seis riachos (nomeados S1-S6) na bacia do alto rio Turvo, sudeste do Brasil, com densos bancos de Typha sp. no habitat interno e ripário, foram estudados em seis campanhas durante os três meses centrais dos períodos seco e chuvoso, por meio de um esforço padronizado de coleta. Trinta e sete espécies foram registradas, totalizando 4.228 indivíduos e 3,9 kg de biomassa. Abundância, biomassa, diversidade e riqueza de espécies foram maiores no período chuvoso, mas somente as diferenças temporais na riqueza de espécies se mostraram estatisticamente significativas. As análises de agrupamentos com a composição e abundância mostraram baixa similaridade temporal, mas indicaram dois grupos de riachos (S1-S2-S5 e S3-S4-S6), o que foi corroborado ao longo do eixo 1 da análise de ordenação. A fauna de peixes residente foi representada por seis espécies, a maioria tolerante e com hábitos generalistas. Larvas aquáticas de Chironomidae e detritos foram os itens mais importantes na dieta dos peixes. Os resultados obtidos sugerem que as populações de peixes exploram os trechos de riachos com Typha de forma oportunística.

Gamma radiation tolerance of a tropical species of midge, Chironomus ramosus Chaudhuri (Diptera: Chironomidae).

PURPOSE: The Chironomid midges are known to thrive well under adverse environmental conditions and are even found inhabiting in areas contaminated by radioactive wastes. Studies were therefore undertaken to find out the radiosensitivity of different developmental stages of the Indian tropical midge, Chironomus ramosus. MATERIALS AND METHODS: In order to determine the threshold levels of lethality, eggs, larvae, pupae and adults of C. ramosus were exposed to varying dosages of gamma radiation (60Co radiation source) ranging from 0-3500 Gray (Gy) at dose-rate of 5.5 Gy/minute. The post-irradiation studies were conducted at three different time points: (a) Immediately after the end of irradiation, (b) 24 hours (h), and (c) 48 h after the end of radiation treatments. Determination of the lethal dose required to kill 50% (LD50), 90% (LD90) and 100% population was carried out using the log-probit analysis. RESULTS: Different developmental stages showed variable threshold levels of radiosensitivity. The radiation doses required to cause 100% mortality immediately after radiation exposure of egg, larva, pupa and adult stages were 1000 Gy, 3000 Gy, 3200 Gy and 3500 Gy, respectively, indicating eggs as the most sensitive stage. Detailed analysis of the LD50 values of different post-irradiation time points indicated that pupal stages were also sensitive at 48 h post-irradiation amongst all the post-embryonic stages as described in many other insects. Interestingly detailed analysis of data indicated that amongst the adult population, females were the most radioresistant, compared to the males as reported in many other insect groups in the literature. CONCLUSIONS: The Indian tropical midge C. ramosus was found to tolerate higher dose of gamma radiation as compared to other known dipteran insects. It is evident from the present findings that C. ramosus falls in the category of radiation-tolerant group of insects.

Purification and properties of ferrochelatase from Chironomidae larvae.

Ferrochelatase with an Mr of 42,700 Da and a pI of 7.35 has been purified to homogeneity from chironomidae larvae. The activity of the enzyme reached maximum at pH 7.8 and decreased with the increase of pH. The enzyme activity varied with temperature and showed maximum activity around 37 degrees C. The purified enzyme was active towards protoporphyrin but inactive towards other porphyrins. The specific enzyme activity of ferrochelatase from chironomidae is about 10-fold higher than that of the rat. Electrophoresis of the purified fractions shows that the enzyme contains only one single polypeptide. The soluble ferrochelatase contained one mole of iron in each mole of the enzyme. The N-terminal sequence analysis of the enzyme shows a high percentage of conserved regions of the enzyme among other species. The enzyme properties are similar to those of the mammalian ferrochelatases except with slightly higher specific activity. Chironomidae ferrochelatase appeared to be more heat resistant and less susceptible than its mammalian equivalent to inhibition by lead.

Analysis of a novel DNA-binding protein kinase CKII-like enzyme of Chironomus cells.

We have previously described a Chironomus tentans nuclear 42 kDa phosphoprotein preferentially associated with transcriptionally active chromatin. In an attempt to purify and identify the kinase responsible for the phosphorylation of the 42 kDa protein, a casein-phosvitin affinity chromatography was used. Unexpectedly, in the eluted kinase fraction, a novel 42 kDa casein kinase, designated protein kinase CK42, with a kinase activity similar, but not identical, to protein kinase CKII, could be identified. In other studies, a nuclear protein that comigrates with protein kinase CK42 in electrophoresis and is capable to bind different gene promoters in single-stranded forms in a sequence-selective manner was found. The observations that both protein kinase and ssDNA-binding activities could be ascribed to a 42 kDa protein raised the possibility that the ssDNA-binding 42 kDa phosphoprotein is a protein kinase. By specific ssDNA-binding affinity chromatography, using a biotinylated oligodeoxyribonucleotide promoter probe and Streptavidine-agarose matrix, evidence that both activities arise from the same protein molecules was obtained. The similarity in the enzyme activities between protein kinase CK42 and CKII raised the question of whether the former was an alpha subunit of the latter. To provide an answer to this issue, CKII, isolated and purified from an epithelial cell line of C. tentans, was characterized and compared with protein kinase CK42 purified from the same cell system. Like other purified CKII preparations, CKII from Chironomus is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). However, the heparin and DRB sensitivities of protein kinase CKII were substantially higher than those of the protein kinase CK42. Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools of CKII could be detected. More than 80% of the nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta subunit were solubilized under the same conditions suggesting that a major portion of the nuclear CKII alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components, probably to chromatin. The biochemical and immunological data taken together strongly suggest that CK42 is a novel DNA-binding protein kinase that is not the alpha subunit of CKII.

Two secretory protein genes in Chironomus tentans have arisen by gene duplication and exhibit different developmental expression patterns.

The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 x 10(4) and 1 x 10(6). Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.