Development and localization of microsatellite markers for the sibling species Chironomus riparius and Chironomus piger (Diptera: Chironomidae)

Five variable microsatellite loci are reported for the nonbiting midge species Chironomus riparius and Chironomus piger. All loci show considerable intraspecific variation and species‐specific alleles, which allow to discriminate among the two closely related species and their interspecific hybrids, and to estimate genetic diversity within and between populations. Additionally, the loci were localized on C. riparius polytene chromosomes to verify their single copy status and investigate possible chromosomal linkage. The described markers are used in different studies with regard to population and ecological genetics and evolutionary ecotoxicology of Chironomus.     

Polymorphism and Divergence of Multilocus DNA Markers in Sibling Species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera,Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group,Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius andC. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers—enzyme-coding genes and chromosomes (gene linkage groups)—showed that different components of the genome differed in their contribution to the genome divergence.     

Polymorphism and divergence of multilocus DNA markers in sibling species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera, Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group, Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius and C. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers--enzyme-coding genes and chromosomes (linked gene groups)--showed that different components of the genome differed in their contribution to the genome divergence.     

Reproductive isolation and ecological niche partition among larvae of the morphologically cryptic sister species Chironomus riparius and C. piger

Background

One of the central issues in ecology is the question what allows sympatric occurrence of closely related species in the same general area? The non-biting midges Chironomus riparius and C. piger, interbreeding in the laboratory, have been shown to coexist frequently despite of their close relatedness, similar ecology and high morphological similarity.

Methodology/Principal Findings

In order to investigate factors shaping niche partitioning of these cryptic sister species, we explored the actual degree of reproductive isolation in the field. Congruent results from nuclear microsatellite and mitochondrial haplotype analyses indicated complete absence of interspecific gene-flow. Autocorrelation analysis showed a non-random spatial distribution of the two species. Though not dispersal limited at the scale of the study area, the sister species occurred less often than expected at the same site, indicating past or present competition. Correlation and multiple regression analyses suggested the repartition of the available habitat along water chemistry gradients (nitrite, conductivity, CaCO₃), ultimately governed by differences in summer precipitation regime.

Conclusions

We show that these morphologically cryptic sister species partition their niches due to a certain degree of ecological distinctness and total reproductive isolation in the field. The coexistence of these species provides a suitable model system for the investigation of factors shaping the distribution of closely related, cryptic species.     

Different repetition frequencies of a 120 base-pair DNA-element and its arrangement in Chironomus thummi thummi and Chironomus thummi piger

The repetition frequency of a highly repetitive DNA sequence has been measured in the genomes of Ch. thummi thummi and Ch. th. piger. This sequence is known to be involved in the evolutionary duplication of defined chromosomal segments leading to a significant increase in the genome size of Ch. th. thummi. Reassociation of this highly repetitive DNA sequence which has a repeat length of 120 base-pairs, with total Ch. th. thummi and Ch. th. piger DNA has shown that the repetition frequency in the Ch. th. thummi DNA is 5.5 fold higher than in Ch. th. piger. In both genomes a 120 base-pair sequence is present as tandemly repeated sequence as shown by Southern analysis.     

Disturbance of macro- and microcirculation: relations with pulse pressure and cardiac organ damage

Whereas large arteries dampen oscillations resulting from intermittent ventricular ejection, small arteries steadily deliver optimal blood flow to various organs as the heart. The transition from pulsatile to steady pressure is influenced by several factors as wave travel, damping, and reflections, which are mainly determined by the impedance mismatch between large vessels and arteriolar bifurcations. The mechanism(s) behind the dampening of pressure wave in the periphery and the links between central and peripheral pulsatile pressure (PP) may determine cardiac damage. Active pathways participate to pulse widening and changes in pulse amplitude in microvessels. Steady and cyclic stresses operate through different transduction mechanisms, the former being focal adhesion kinase and the latter being free radicals and oxidative stress. Independently of mechanics, calcifications and attachment molecules contribute to enhance vessel wall stiffness through changes in collagen cross-links, proteoglycans, integrins, and fibronectin. Enhanced PP transmission may thus occur and precipitate organ damage at each time that autoregulatory mechanisms, normally protecting the heart from vascular injury, are blunted. Such circumstances, observed in old subjects with systolic hypertension and/or Type 2 diabetes mellitus, particularly under high-sodium diet, cause cardiac damage and explain why increased PP and arterial stiffness are significant predictors of morbidity and mortality in the elderly.     

Expression of catalase and glutathione S-transferase genes in Chironomus riparius on exposure to cadmium and nonylphenol

Antioxidant enzymes play important roles in the protection against oxidative damage caused by environmental pollutants by scavenging high levels of reactive oxygen species and have been quantified as oxidative stress markers. However, combining mRNA expressions of genes coding for detoxification enzymes along with enzyme activities will be more useful biomarkers of stress. Therefore, in this study the cDNA of the catalase gene from the aquatic midge, Chironomus riparius (CrCAT) was sequenced using 454 pyrosequencing. The 2139 bp CrCAT cDNA included an open reading frame of 1503 bp encoding a putative protein of 500 amino acids with a predicted molecular mass of 56.72 kDa. There was an 18 bp 5’ and a long 618 bp 3' untranslated region with a polyadenylation signal site (AATAAA). The deduced amino acid sequence of CrCAT contained several highly conserved motifs including the proximal heme–ligand signature sequence RLFSYNDTX and the proximal active site signature FXRERIPERVVHAKGXGA. A comparative analysis showed the presence of conserved amino acid residues and all of the catalytic amino acids (His⁷⁰, Asn¹⁴³, and Tyr³⁵³) were conserved in all species. The CrCAT contained three potential glycosylation sites and a peroxisome targeting signal of ‘AKM’. The mRNA was detected using RT-PCR at all developmental stages. The time-course expression of CrCAT was measured using quantitative real-time PCR after exposure to different concentration and durations of Paraquat (PQ), cadmium chloride (Cd) and nonylphenol (NP). The expression of CrCAT was significantly up regulated on exposure to 50 and 100 mg/L PQ for 12 and 24 h. Among the different concentrations and durations of Cd tested, significantly highest level of expression for CrCAT mRNA and catalase enzyme activity was observed on exposure to 10 mg/L for 24 h. In the case of NP, the highest level of CrCAT expression was observed after exposure to 100 μg/L for 24 h. The expression profiles of three selected C. riparius glutathione S-transferase genes (CrGSTs) viz. CrGSTdelta3, CrGSTsigma4 and CrGSTepsilon1 was also studied on exposure to NP and were up or down regulated at different time points and concentrations. Significantly highest level of expression for CrGSTdelta3 was observed after 48 h and for CrGSTsigma4 and CrGSTepsilon1 after 24 h exposure to 100 μg/L of NP. The results show that CrGSTs and CrCAT could be used as potential biomarkers in C. riparius for aquatic ecotoxicological studies.     

Ecdysone-Related Biomarkers of Toxicity in the Model Organism Chironomus riparius: Stage and Sex-Dependent Variations in Gene Expression Profiles

Despite being considered a model organism in toxicity studies, particularly in assessing the environmental impact of endocrine disrupting compounds (EDCs) and other chemicals, the molecular basis of development is largely unknown in Chironomus riparius. We have characterized the expression patterns of important genes involved in the ecdysone pathway from embryos to pupa, but specially during the different phases of C. riparius fourth larval instar, according to the development of genital and thoracic imaginal discs. Real-Time PCR was used to analyze: EcR and usp, two genes encoding the two dimerizing partners of the functional ecdysone receptor; E74, an early response gene induced by ecdysteroids; vg (vitellogenin), an effector gene; hsp70 and hsc70, two heat-shock genes involved in the correct folding of the ecdysone receptor; and rpL13, as a part of the ribosomal machinery. Our results show for the first time stage and sex-dependent variations in ecdysone-responsive genes, specially during the late larval stage of C. riparius. The induction in the expression of EcR and usp during the VII-VIII phase of the fourth instar is concomitant with a coordinated response in the activity of the other genes analyzed, suggesting the moment where larvae prepare for pupation. This work is particularly relevant given that most of the analyzed genes have been proposed previously in this species as sensitive biomarkers for the toxicological evaluation of aquatic ecosystems. Identifying the natural regulation of these molecular endpoints throughout the Chironomus development will contribute to a more in-depth and accurate evaluation of the disrupting effects of EDCs in ecotoxicological studies.     

Complex interactions between native and invasive fish: the simultaneous effects of multiple negative interactions

We suggest that the ultimate outcome of interactions between native species and invasive species (extinction or coexistence) depends on the number of simultaneous negative interactions (competition and predation), which depends on relative body sizes of the species. Multiple simultaneous interactions may constrain the ability of native species to trade fitness components (i.e., reduced growth for reduced risk of predation) causing a spiral to extinction. We found evidence for five types of interactions between the adults and juveniles of introduced western mosquitofish (Gambusia affinis) and the juveniles of native least chub (Iotichthys phlegethontis). We added ten large (23–28 mm) and seven small (9–13 mm) young-of-the-year (YOY) least chub to replicate enclosures with zero, low, and high densities of mosquitofish in a desert spring ecosystem. Treatments with mosquitofish reduced the average survival of least chub by one-third. No small YOY least chub survived in enclosures with high mosquitofish densities. We also performed two laboratory experiments to determine mortality to predation, aggressiveness, and habitat selection of least chub in the presence of mosquitofish. Mean mortality of least chub due to predation by large mosquitofish was 69.7% over a 3-h trial. Least chub were less aggressive, selected protected habitats (Potamogeton spp.), and were more stationary in the presence of mosquitofish where the dominance hierarchy was large mosquitofish>>large least chubsmall mosquitofish>>small least chub. Least chub juveniles appear to be figuratively caught in a vice. Rapid growth to a size refuge could reduce the risk of predation, but the simultaneous effects of competition decreased least chub growth and prolonged the period when juveniles were vulnerable to mosquitofish predation.     

Competition for dissolved glucose between meiobenthos and sediment microbes

Meiobenthos, small invertebrates inhabiting the surface layers of marine sediments, can absorb dissolved organic matter (DOM). Experiments were performed to test if meiobenthos can compete with sediment microbes for uptake of small amounts of [¹⁴C]glucose. Meiofaunal glucose uptake rates were measured by themselves and in the presence of sediment microbes. Glucose uptake by meiofauna was not inhibited by the presence of bacteria, nor did it appear that bacterial uptake was inhibited by meiofauna. Thus, there was no direct or interference competition. Uptake rates by 1 cm³ of sediment (bacteria) were four orders of magnitude greater than those of individual meiofauna, but on a biomass specific basis, meiofaunal uptake was in the same range if not higher than that of sediment bacteria. Thus, the potential for indirect or resource competition exists. Since bacterial biomass dominated the system studied, uptake of glucose was dominated by bacteria. The results support the hypothesis that in natural sediments, where the biomass of bacteria is higher than that of meiofauna, heterotrophic uptake is primarily a microbial process. However, resource competition between meiofauna and bacteria for DOM in sediments probably exists where bacterial biomass is low relative to meiofaunal biomass.     

The life-history,distribution, and production of Chironomus riparius and Glyptotendipes paripes in a prairie pond

C. riparius and G. paripes exhibited univoltine life-cycles in Stephenson Pond; pupation, emergence and oviposition occurred mainly during May, and both species overwintered as mature fourth instar larvae. The marjority of larval growth for both species took place during the fourth instar stage (August–October), and growth and production were very low during late May to mid July when only young instars were present. Low production occurred during an interval when sestonic chlorophyll a concentration was very low, and the high production period corresponded to the Aphanizomenon bloom (August) and the autumn diatom pulse. None of the growth and production parameters investigated were correlated with temperature at the mud-water interface. Tube structure and behavior of the larvae indicate that G. paripes larvae are filter-feeders, whereas, C. riparius larvae are deposit-feeders.     

A new method for establishing a permanent laboratory culture of Chironomus riparius Meigen (Diptera: Chironomidae)

A new technique for the establishment of a permanent laboratory culture of Chirono-mus riparius is described. Larvae and pupae were reared in plastic tanks containing tap water, filamentous algae and a commercially prepared fishfood. The tanks were aerated, maintained at a constant temperature of 24°C and subjected to a constant lighting regime of 13 h of light followed by 11 h of darkness. Adults flew freely in a modified handling box, mated, and oviposited on strips of filter paper placed in the plastic tanks containing the aquatic stages. It has proved unnecessary to change the water or medium, which has saved both time and effort. Moreover, larvae established themselves in ail parts ofthe alga, and therefore more could be kept in each tank than when, as in previous methods, they are provided only with a substratum on the floor of the container.     

Comparison of insect hemoglobins (Erythrocruorins) from Chironomus thummi thummi and Chironomus thummi piger (Diptera). The primary structure of the monomeric hemoglobin CTP III

The monomeric hemoglobin fractions of Chironomus thummi thummi (CTT) and Chironomus thummi piger (CTP) differ in the ratio of their components. The determination of the primary structure of the component CTP III was achieved by automatic Edman degradation of the native chain, the tryptic peptides and the C-terminal fragment, obtained by cleavage at the single tryptophan residue. It revealed two chains in the ratio 1:1 which share the ambiguity threonine/isoleucine in position 57 with CTT III. Whereas one chain is identical to the CTT III hemoglobin, the other differs in having isoleucine in position 105 and alanine in position 134. The CTP monomeric hemoglobin fraction comprises 8% of a component (CTP IV A) with a more negative charge than CTT IV but with an identical sequence up to position 44. This study reveals a very high polymorphism within Chironomus species and points out the need for more data at the gene level in order to provide better understanding of this striking phenomenon.     

A structural study of the anal papillae of the midge Chironomus riparius Meigen (Diptera: Chironomidae)

Larvae of the midge, Chironomus riparius Mg., have four anal papillae arranged as two pairs, one dorsal to and the other ventral to the anus. Structural study with light and electron microscopes has revealed that their integument consists of a thin cuticle overlaying a thick, syncytial epidermis which is specialised to facilitate ion transport. There is a distinct neck or collar region at the junction of each papilla with the rest of the body wall. Although in many respects these structures resemble those in mosquitoes, there are both morphological and physiological features which suggest that differences exist in the means by which ion exchange with the environment is controlled and effected in the two families.     

Water quality and the occurrence of Chironomus riparius Meigen (Diptera: Chironomidae) in a stream receiving sewage effluent

SUMMARY. The effects of a sewage effluent in Moat Brook, Staffordshire, were determined from regular collections of water and biological samples from directly comparable stations above and below the sewage outfall.
The eutrophic conditions below the outfall result in the replacement of Polypedilum laetum by Chironomus riparius (except in the spring) as the dominant member of the Chironominae. Continuous recruitment of first instar larvae to the population of C. riparius in summer and autumn results in much overlap of generations, but emergence trap captures suggest at least five generations in the year.
The usefulness of C. riparius as an indicator of enriched conditions in lotic ecosystems should be considered in relation to oviposition behaviour and the occurrence and seasonal variation in density of the adults and aquatic stages (including the more conspicuous third and fourth instar larvae).     

Characterization and expression analysis of phospholipid hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to cadmium

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is an antioxidant enzyme in the glutathione peroxidases (GPx) family that reduces hydroperoxides of phospholipids and maintains the integrity of biomembranes. Here, we report the identification and characterization of a full length cDNA of PHGPx from the ecotoxicologically important aquatic midge Chironomus riparius (CrPHGPx1) from the Expressed Sequence Tags (ESTs) database generated through pyrosequencing. The 837 base pair (bp) cDNA contained an open reading frame of 597 bp, and a 75 bp 5' and a 159 bp 3'untranslated region. The theoretical molecular mass of the deduced amino acid (aa) sequence (197 aa) was 22.40 kDa with an estimated pI of 8.77. The Cys-codon was present at residue 74 and also the active site residues Gln(91) and Trp(164). The active-site motifs and GPx family signature motifs LAFPCNQF(101-108) and WNFTK(163-168) were also found. Phylogenetic analysis showed that CrPHGPx1 is grouped with PHGPx1 from other species and is more closely related to insects belonging to the dipteran order. The mRNA of CrPHGPx1 was detected in larvae, pupae and adults. The expression of CrPHGPx1 is induced by cadmium exposure indicating that the mRNA expression of CrPHGPx1 is differently regulated in response to oxidative stress caused by environmental stressors.