Peculiarities of structural organization of hemoglobin of Chironomus plumosus L. (Diptera: Chironomidae)

In Ch. plumosus and Ch. riparius there were revealed various structural hemoglobin variants including monomers, dimers, trimers, tetramers, hexamers, and octamers. The multitude of the protein organization ways seems to be based on diversity of the monomers with the approximately equal molecular weight from 12 to 16 kDa in Ch. plumosus and from 11.8 to 15.2 kDa in C. riparus. In PAGE with 8 M urea, the hemoglobins were aggregated into high molecular complexes with mol. weights about 260 and 180 kDa in Ch. plumosus and about 523 and 174 kDa in C. riparus.     

Composition and distribution of sibling species of <Emphasis Type="Italic">Chironomus</Emphasis> Meigen, 1803 (Diptera,Chironomidae) in Curonian Lagoon of the Baltic Sea

The species composition of Chironomus in the Curonian Lagoon has been investigated. It was shown that three sibling species of the plumosus group, namely, Ch. plumosus, Ch. balatonicus, and Ch. muratensis, and a first-generation interspecific hybrid Ch. muratensis × Ch. plumosus lived here. The occurrence frequency in the lagoon was 87, 19, 6, and 3% for Ch. plumosus, Ch. balatonicus, Ch. muratensis × Ch. plumosus, and Ch. muratensis, respectively. The species Ch. plumosus was recorded in all the areas (southern, central, and northern), while Ch. balatonicus was found only in the northern area in the village of Juodkrante near the city of Klaipeda. Presumably, the distribution of these sibling species is due to the presence of a salinity gradient in the lagoon.     

Comparison of Chironomus usenicus and Chironomus curabilis with species of the group plumosus (Diptera) inferred from the mitochondrial DNA Gene COI and by the polytene chromosomes banding pattern

The sequence of a 595-bp fragment of the mitochondrial COI gene was determined for the species Chironomus usenicus and Chironomus curabilis of the genus Chironomus. Phylogenetic reconstructions based on the analysis of the COI gene sequence coincide on the whole with cytogenetic data, permitting Ch. usenicus and Ch. curabilis to be regarded as members of the group plumosus. Chironomus usenicus and Ch. plumosus have identical COI gene sequences. Two hypotheses explaining this identity are considered: inheritance of mtDNA from one of the parental species during hybridogenesis and horizontal transfer of mitochondrial genes.     

Chromosomal and genomic mutations in <Emphasis Type="Italic">Chironomus plumosus</Emphasis> (L.) (Diptera,Chironomidae) from Novozybkov raion of Bryansk oblast

The data on karyopool analysis of Chironomus plumosus from Novozybkov raion of Bryansk oblast, radioactively contaminated as a result of the Chernobyl accident, are presented. In the karyopool of this population, four types of heterozygous inversions (A1.2, B1.2, C1.2, D1.2) and a structurally small rearrangement in arm D, which is thought to be also associated with inversion, were identified. For inversions A1.2, C1.2, and structurally small rearrangement in arm D the cases of somatic mosaicism were described. The mean number of inversions per individual constituted 0.78. The number of genotypic combinations was 13. In 5% of the individuals chromosome B was identified, while 15.6% were polyploid (3n). In 1.8% of triploids somatic mosaicism for the level of polytene chromosomes was observed. Most of the larvae were characterized by partial asynapsis of the homologs in different regions of chromosome III. Specific features of the Chironomus plumosus karyopool from Novozybkov are thought to be associated with the habitation of a number of generations of this population on radioactively polluted territory.     

Investigation of permolecular structure of lipase from <Emphasis Type="Italic">Rhizopus niveus</Emphasis>

It has been shown by classical biophysical and biochemical methods in combination with atomic microscopy that lipase from Rhizopus niveus exists in a water solution as a dimer with a molecular weight of 96 kDa. The rate of splitting of triglycerides by a dimeric molecules is twice that of monomers. The heat stability of the monomeric form of lipase at temperatures of 20–60°C is significantly higher than that of the native molecule.     

Quantification of pumping rate of <Emphasis Type="Italic">Chironomus plumosus</Emphasis> larvae in natural burrows

This paper investigates and compares experimentally determined water velocity field above natural macrozoobenthos burrows generated by Chironomus Plumosus larva during their bio-irrigation activity. All experiments were carried out using particle image velocimetry and performed in mesocosms filled with sediment burrowed by larvae, and the water velocity fields near the inlets and outlets of the U-shaped burrows were measured. From water velocity data the average volumetric flow rates between 54.6 and 61.1 mm³/s were calculated. Assuming an average burrow diameter of 2.25 mm, the volumetric flow rates suggest the average flow velocities through burrows during the pumping period between 13.7 and 15.4 mm/s. Two additional interesting phenomena could also be shown by analyzing the flow field generated by the larva. The analysis of the amount of tracers used for visualizations revealed that some of the tracer particles added to the water must have been consumed along their path from the inlet toward the outlet, hinting clearly to the so-called filter-feeding action of C. plumosus. The second phenomenon is due to the form of motion C. plumosus generates. By careful flow visualizations it was found that unlike other organisms such as Urechis caupo that use peristaltic body contractions, C. plumosus worms its body sinusoidally catapulting the fluid far into the overlying water body. This action is of ecological advantage for it avoids generating short oxygen circuits for their respiration and filter feeding.     

B-Chromosomen beiChironomus

In the population “Herrenmühle” ofChironomus plumosus 11% of the individuals contain one supernumerary chromosome. This B-chromosome is present both in germ-line and somatic cells. — InChironomus melanotus 6% of the larvae of the population “Falkau” carry supernumerary chromosomes. These B-chromosomes cannot be found in all nuclei of testis and soma, their number varies between cells within the individual. In both species the B-chromosomes represent centromeric fragments of chromosome IV as can be shown by their structure and pairing behaviour. — The polytene B-chromosome ofCh. plumosus exhibits a banding pattern in the salivary gland nuclei. Furthermore it is able to form an additional nucleolus in the nuclei of the malpighian tubules. InCh. melanotus band structures can be seen only in the B-chromosome of malpighian tubules. The larvae ofCh. melanotus, carrying B-chromosomes, show heterochromatic bodies in the salivary gland nuclei, varying in number and size in the nuclei of the same gland. These bodies are interpreted to be polytenic B-chromosomes divided into subunits.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Chironomus species (Diptera: Chironomidae) in the profundal benthos of Spanish reservoirs and lakes: factors affecting distribution patterns

• 1 The taxonomy, distribution and habitat preferences of Chironomus larvae from profundal benthos of reservoirs and natural lakes in Spain are presented. The 114 water bodies studied were medium to large reservoirs and lakes throughout Spain, and were visited on at least two occasions (summer and winter). Out of the natural lakes investigated, Chironomus larvae were found only in one karstic lake (Banyoles), and to date, these have not been found in the high mountain lakes of the Spanish Pyrenees or Sierra Nevada, or in Spain's largest oligotrophic lake (Sanabria).
• 2 Multiple regression with environmental data from reservoirs showed that the density of Chironomus larvae was inversely correlated with water depth, temperature and sulphide content of the bottom water, but positively correlated with alkalinity and particulate organic nitrogen (PON) content of bottom water. Thus, the density of Chironomus larvae was higher in shallow reservoirs from the eastern part of Spain (calcareous water and higher PON), but markedly lower in southern reservoirs (higher temperature and higher sulphide).
• 3 According to morphological and cytological characteristics, three species of this genus have been identified in the samples examined: C. bernensis, C. plumosus and C. nuditarsis. Reasons for the absence of other widespread European species are discussed.
• 4 Using canonical correspondence analysis to relate species and environmental variables, C. bernensis showed a significant preference for reservoirs with higher oxygen concentrations in bottom water and lower total reactive phosphorus (TRP) content at the surface than those reservoirs where C. plumosus‐type were found. This suggests that the smaller C. bernensis prefers mesotrophic reservoirs and the larger C. plumosus‐type (C. plumosus + C. nuditarsis) prefers eutrophic reservoirs. Taking into account the TRP/depth index for those samples collected at depths below the thermocline, and thus, where oxygen can be a limiting factor, C. bernensis and C. nuditarsis were present in reservoirs in which the index was low (< 1:170 for C. bernensis and >1:800 for C. nuditarsis), while C. plumosus thrived in reservoirs with higher index values (<1:20). These differences may reflect the preference of C. bernensis and C. nuditarsis for detrital material, while C. plumosus prefers phytoplankton. These observations are in agreement with the depth distribution of each species: C. plumosus was restricted to reservoirs less than 46 m deep, whereas the other two species (i.e. C. bernensis and C. nuditarsis) were found up to depths of 65 and 80 m, respectively.
• 5 Data from a transect (5–20 m deep) in Lake Banyoles are consistent with the general pattern described in reservoirs. In this lake, a high density of C. bernensis was recorded from littoral and sublittoral areas with abundant oxygen (above or close to the thermocline), whereas C. plumosus was scarce in several parts of the lake, especially in oxygen‐deficient areas during the summer. This karstic, oligotrophic lake has a low TRP/depth index, and therefore, we expected C. bernensis to be dominant in the littoral, as is the case. Nevertheless, the severe depletion of oxygen caused by meromixis, and the high hydrogen sulphide concentration benefit C. plumosus in deeper zones. Chironomus nuditarsis was scarce in Lake Banyoles and only found at considerable depths.

     

C-banding in the polytene chromosomes of species of the plumosus group (Diptera,Chironomidae) and their experimental hybrids

The localization and amount of heterochromatin in the plumosus group were studied, including the species Chironomus plumosus L., C. vancouveri and C. balatonicus. The appearance of C bands of Chironomus plumosus in several European populations is traced. The role of the C heterochromatin in the differentiation of this species is discussed. From the evolutionary point of view the Swiss populations, in which large centromere heterochromatin blocks have been discovered, are more varied as to the amount of heterochromatin. The importance of duplications for this process is pointed out. The chromosomes of the individuals from C. vancouveri and C. balatonicus have centromeric, telomeric and interstitial heterochromatin. The centromeric heterochromatin is represented by thin C-bands. The particularities in the appearance of C heterochromatin in C. vancouveri and C. balatonicus reflect the structural peculiarities of their chromosomes. The change in the euchromatin regions in these forms is discussed in the light of transformation of euchromatin to heterochromatin in the process of evolution.The appearance of heterochromatin in hybrids (between populations and between species) created experimentally is traced. A change has been discovered in the appearance of heterochromatin in the hybrids compared to the initial parent forms. This difference is expressed more strongly in inter-species hybrids than in interpopulation hybrids of C. plumosus.     

Porin Isolated from the Outer Membrane of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> and Its Encoding Gene

A major Erwinia amylovora outer-membrane protein (Omp-EA) and the gene encoding for this protein (omp-EA) were isolated and characterized. The native Omp-EA protein forms a trimeric structure of approximately 114 kDa. This protein demonstrated high resistance to detergents such as SDS and octyl-glucopyranoside, but disaggregated to monomers with a molecular weight (MW) of approximately 39 kDa after heating at 95°C for 10 minutes in sample buffer. The pore-forming ability of the oligomeric Omp-EA was determined by the liposome swelling assay, demonstrating that the oligomeric protein formed nonspecific channels with an exclusion limit of approximately 660 Da. On dissociation, the monomers did not exhibit pore-forming ability. The omp-EA gene was cloned and sequenced (GenBank Accession No. DQ184680). Sequence analysis revealed an open reading frame of 1152 bases. The deduced amino-acid sequence had 383 amino acids. The mature protein consisted of 362 amino acids and had a calculated MW of 39,210 Da. Multiple-sequence alignment of Omp-EA with other porins from the Enterobacteriaceae family revealed 51% to 63% identity. The first 16 amino acids from the N-terminal exhibited the highest identity (100%) to the porins OmpC, OmpF, and PhoE of Escherichia coli. Two methods were used to predict the secondary structure: APSSP2 and Hidden and Markov’s model. The monomers of Omp-EA porin presented a topology of 16 transmembranal β-strands. The area of the loops between the β -strands was proposed. It is suggested that further research on the porin and its loops may be important for understanding the mechanism of E. amylovor to invade plant tissues.     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997     

Molecular properties of the partially SDS-resistant lectin from the albumen gland of Helix pomatia and demonstration of lectin-related molecules on the surface of H. pomatia haemocytes

Lectins (agglutinins) are components of the immunobiologicalrecognition system of vertebrates and invertebrates. The presentstudy focused on the molecular properties of the agglutininfrom the albumen gland of Helix pomatia (HPA) and on the occurrenceof lectin-related molecules on the surface of H. pomatia haemocytes.According to the current model (Hammarström et al., 1972,Scandinavian Journal of Immunology, 1: 259–301), the hexamericHPA of about 79 kDa is composed of three non-covalently associateddimers (26 kDa), each consisting of two disulphide-bridged 13kDa monomers. However, on native-gradient polyacrylamide gelelectrophoresis (PAGE), we obtained high molecular weight bandsrepresenting lectin polymers. The stepwise dissociation of thesewas achieved by incubation with SDS at temperatures from 20to 40°C (1 h) and at 100°C (10 min). The results obtainedon SDS–PAGE included the occurrence of partially SDS-resistanthexamers of about 66 kDa, of two dimer bands of 22 and 19 kDa,and of two minor heteromonomer fractions. Complete dissociationinto heteromonomers of 13 and 11 kDa was achieved by boilingthe lectin (10 min) with SDS under reducing conditions. Fornative lectin molecules, both monomers occurred as disulphide-linkedhomodimers. Monomers or dimers electroeluted from an SDS–gel,reassociated to SDS-resistant oligomers upon re-electrophoresis.Finally, molecules antigenetically related to the lectin wereextracted from the membrane of H. pomatia haemocytes. Anti-HPAantibodies recognized peptides with an apparent molecular weightof about 30 and 56 kDa, which were shown to represent cell-surfacemolecules. (Received 4 March 2008; accepted 9 September 2008)     

Effects of bioturbation by tube-dwelling chironomid larvae on oxygen uptake and denitrification in eutrophic lake sediments

1. Oxygen uptake and denitrification were determined in two bioturbated sediments from a eutrophic lake in southern Sweden. In laboratory mesocosms, an organic profundal sediment was incubated with Chironomus plumosus L. and a sandy littoral sediment with an organic-rich top layer was incubated with Polypedilum sp. Both species of chironomid are sediment tube-dwelling. 2. Oxygen consumption, expressed per gram of larval dry weight, was enhanced to the same extent by the larvae in both sediments. Measurements of the respiration rate of individual larvae revealed that the respiration per gram dry weight of the smaller Polypedilum sp. was more than three times higher than that of C. plumosus. 3. Denitrification was measured using the ‘nitrogen isotope pairing’ technique. In the organic sediment, denitrification of nitrate from the water phase (dw) and denitrification of nitrate from coupled nitrification (dn) were each correlated with the biomass of C. plumosus. In the sandy sediment, dw was correlated with the biomass of Polypedilum sp., while dn did not show any correlation with Polypedilum sp. 4. Oxygen uptake in the organic sediment was increased by a factor of 2.5, dw 5-fold and dn 2.5-fold at a biomass of 10 g m^–2 dry weight of C. plumosus. The same biomass of Polypedilum sp. in the sandy sediment resulted in a 2-fold stimulation of oxygen uptake and a 3-fold stimulation of dw, while dn was not affected. These differences in stimulation between oxygen uptake and denitrification by the larvae in the sediments suggest that the stimulation pattern cannot be explained by simple extension of the sediment surface. The burrows evidently reduce the distance between the nitrate source in the water column and the denitrifiers in the anoxic zones. 5. This study indicates that bioturbation by macrofauna elements can have a great impact on denitrification in lake sediments, and that different organisms can influence nitrogen turnover in specific ways.     

Fatty acid biomarkers show dietary differences between dominant chironomid taxa in Lake Erken

1. In this field study, diatom-specific and bacteria-specific fatty acids were used as biomarkers to evaluate the differences in diet between Chironomus plumosus (a spring-emerging cohort) and C. anthracinus (an autumn-emerging cohort), and Procladius spp. Furthermore, total lipid content of Chironomus larvae was analysed. 2. Individual dry mass of the spring cohort of C. plumosus rapidly increased during spring and early summer. Surprisingly, the autumn-emerging cohort showed remarkably little growth over the same time interval. The individual dry mass of C. anthracinus initially declined in early spring, but then increased during April and May. 3. Accumulation of the diatom-specific fatty acid palmitoleic acid (16 : 1ω7) during spring was much higher in C. plumosus (> 3-fold increase) than in C. anthracinus (1.5-fold). Conversely, the bacterial indicating iso form of septadecanoic acid (17 : 0i) was higher in C. anthracinus than in the spring-emerging cohort of C. plumosus. This shows that C. plumosus assimilates energy from the spring diatom bloom to a greater degree, whereas C. anthracinus feeds more exclusively on detritus in the sediment. 4. Concentrations of 17 : 0i in Procladius larvae were 0.54 ± 0.13 mg g^–1, i.e. consistently higher than for both Chironomus taxa, indicating that this predator gains a relatively high fraction of its energy through detrital pathways (from bacteria). 5. These results show that fatty acid biomarkers can be an appropriate tool to detect differences in larval diet between coexisting chironomid species, between two closely related Chironomus species and between different cohorts. 6. The different feeding modes of both Chironomus species may be important for among-lake distribution patterns, with filter-feeding C. plumosus being dominant in relatively shallow lakes and deposit-feeding C. anthracinus more common in deeper lakes with stable stratifications. This conjecture was supported by a compilation of data from Lake Mälaren.     

Function of Ventral and Lateral Processes in Larvae of Chironomus (Diptera, Chironomidae)

Oxygen consumption by larvae of five Chironomus species from the Plumosus group belonging to different larval forms was studied. Larvae of C. agilis ( f. l. reductus) were unable to utilize oxygen at its concentrations lower than 1 mg/l. Larvae of C. plumosus ( f. l. plumosus) used oxygen from water at concentrations higher than 0.7 mg/l; pupas of this species and larvae of C. borokensis ( f. l. plumosus) utilized oxygen at concentrations lower than 0.4 mg/l. C. entis and C. muratensis (both-- f. l. semireductus) utilized oxygen from water at its concentrations up to 0.05 mg/l. The lack of correlation between the rate and ability of larvae to use oxygen at low concentrations and the degree of development of processes allows suggesting that these structures are not additional respiratory organs, as is commonly accepted. At the same time, ability of species to inhabit under conditions of oxygen deficiency correlates directly with the size of the processes. Therefore, ventral and lateral processes are suggested to perform a function of excretion of anaerobic metabolism metabolites.     

Feeding efficiencies of Chironomus plumosus (L.) and C. anthracinus Zett. (Diptera: Chironomidae) in mesotrophic Lake Erken

SUMMARY.

• 1 Absorption efficiencies of nitrogen and carbon in two Chironomus species found dominating in the profundal zone of mesotrophic Lake Erken were determined gravimetrically.
• 2 Absorption efficiencies for C. plumosus showed greater seasonal variation than those of C. anthracinus, with low efficiencies coinciding with the summer dominance of flagellate phytoplankton and with high C:N ratios.
• 3 Overall mean absorption efficiencies (±SE) for carbon and nitrogen, respectively, were 26.8% (±1.9) and 29.3% (±1.9) for C. plumosus, 24.6% (±1.7) and 28.1% (±1.8) for C. anthracinus.
• 4 Significant differences were found to exist between the C: N ratios of the superficial 2 cm sediment layers and those of Chironomus anterior midgut contents.
• 5 C. anthracinus appears to be a deposit feeder ingesting particulate matter scraped from the recently deposited surface sediments. The greater seasonal variation found in the absorption efficiencies of C. plumosus, together with the lower C:N ratios, support the contention that this species is a filter feeder with the nutritional quality of ingested matter depending primarily on pelagic inputs.

     

Tissue specific gene activities and proteins in the Chironomus salivary gland

Summary The secretory proteins of the larval salivary gland of some Chironomus species were analysed according to a method developed by Grossbach (1969). After reduction of the disulphide bonds and alkylation the secretion of Chironomus thummi was separated by disc electrophoresis at acrylamide concentrations of 4.8 to 9.5% into seven main and some minor fractions. By increasing the acrylamide concentration up to 20% nine main fractions could be resolved. In addition to these high molecular weight structural proteins a number of proteins soluble in simple buffer solutions and separable at pH 8.8 in a 15% acrylamide gel are present in the secretion but only to a very small amount.The electrophoretic pattern of the secretory proteins of Chironomus strenzkei, Ch. luridus and Ch. obtusidens are qualitatively the same as the Ch. thummi pattern. Some quantitative differences may exist. In the secretion of Chironomus plumosus at least eleven main fractions were detected in 20% acrylamide gels. A comparison between the number of Balbiani rings of the salivary gland chromosomes and the number of main protein fractions in the salivary gland secretion yielded no direct correlation.The results are discussed in respect to the question wether the major puffs of the gland, the Balbiani rings, encode the major products of the gland, the secretory proteins.Abbreviations used BR Balbiani ring - bis N,N-Methylenebisacrylamide     

Characterization of two soil metagenome-derived lipases with high specificity for <Emphasis Type="Italic">p</Emphasis>-nitrophenyl palmitate

Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters. P. Wei and L. Bai contributed equally to this work.     

Study of the oligomeric structure and some physicochemical properties of inulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> Y-303

It has been found using a combination of atomic force microscopy with infrared spectroscopy, gel chromatography, and electrophoresis that inulinase from Kluyveromyces marxianus Y-303 has oligomeric structure, which includes two subunits differing in size, molecular mass, and catalytic activity. It has been shown that the division of the inulinase dimer into monomers leads to an increase in the number of irregular regions by 6% for subunit 1 (54.8 kDa) and by 10% for subunit 2 (8.4 kDa) compared with the native enzyme.