Chironomus Tentans Larvae Research Articles

Transcription regulation during stable elongation by a reversible halt of RNA polymerase II.

Regulation of RNA polymerase II (RNAPII) during transcription is essential for controlling gene expression. Here we report that the transcriptional activity of RNAPII at the Balbiani ring 2.1 gene could be halted during stable elongation in salivary gland cells of Chironomus tentans larvae for extended time periods in a regulated manner. The transcription halt was triggered by heat shock and affected all RNAPII independently of their position in the gene. During the halt, incomplete transcripts and RNAPII remained at the transcription site, the phosphorylation state of RNAPII was unaltered, and the transcription bubbles remained open. The transcription of halted transcripts was resumed upon relief of the heat shock. The observed mechanism allows cells to interrupt transcription for extended time periods and rapidly reactivate it without the need to reinitiate transcription of the complete gene. Our results suggest a so-far-unknown level of transcriptional control in eukaryotic cells.

Assessment of the effect of nanomaterials on sediment-dwelling invertebrate Chironomus tentans larvae

Studies were conducted to determine the effects of a panel of seven nanomaterials (NMs), namely: α-alumina, γ-alumina, precipitated silica; silica fume, calcined silica fume, colloidal antimony pentoxide (Sb2O5), and superfine amorphous ferric oxide (Fe2O3), on sediment dwelling invertebrates Chironomus tentans under controlled laboratory conditions. Percentage survival, enzyme activities, growth development, and DNA fragmentation parameters were studied as acute, biochemical, and physiological toxicities of NMs, respectively. Quantitation of catalase and peroxidase enzyme activity demonstrated that toxicant stress of the NMs increased enzyme activity in a concentration dependent fashion across all treatments. The percentage growth length of the test specimens exposed to different NMs was significantly reduced compared to the negative control while only five concentrations were not in the toxic range, namely; Fe2O3 (5μg/kg); silica fume (5μg/kg, 50μg/kg); Sb2O5 (5μg/kg) and calcined silica fume (5μg/kg). Genotoxic stress assessed by use of DNA laddering showed complementary findings to the other ecotoxicological endpoints tested in this study—the percentage survival and growth length inhibition.

Discontinous Movement of mRNPs in Nucleoplasmic Regions Devoid of Chromatin

Messenger RNP particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus through nuclear pore complexes. To further understand mRNP trafficking, we have studied the movement of a specific mRNP, the BR2 mRNP, in the nuclei of salivary gland cells in Chironomus tentans larvae. These polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled by hybridization with microinjected RNA or DNA oligonucleotides, and their movement was examined by single particle tracking.

EFFECTS OF DIETARY ESFENVALERATE EXPOSURES ON THREE AQUATIC INSECT SPECIES REPRESENTING DIFFERENT FUNCTIONAL FEEDING GROUPS

Given the chemical properties of synthetic pyrethroids, it is probable that compounds, including esfenvalerate, that enter surface waters may become incorporated into aquatic insect food sources. We examined the effect of dietary esfenvalerate uptake in aquatic insects representing different functional feeding groups. We used three field-collected aquatic insect species: A grazing scraper, Cinygmula reticulata McDunnough (Ephemeroptera: Heptageniidae); an omnivorous filter feeder, Brachycentrus americanus Banks (Trichoptera: Brachycentridae); and a predator, Hesperoperla pacifica Banks (Plecoptera: Perlidae). Laboratory-cultured algae were preexposed for 24 h to esfenvalerate concentrations of 0, 0.025, 0.05, and 0.1 microg/L and provided to two C. reticulata age classes (small and final-instar nymphs). Reduction in small nymph growth was observed following three weeks of feeding on algae exposed to 0.05 and 0.1 microg/L of esfenvalerate, and the highest dietary exposure reduced egg production in final-instar nymphs. The diet for B. americanus and H. pacifica consisted of dead third-instar Chironomus tentans larvae preexposed for 24 h to esfenvalerate concentrations ranging between 0.1 and 1.0 microg/L. Consumption of larvae exposed to 0.5 to 1.0 microg/L of esfenvalerate caused case abandonment and mortality in B. americanus caddisfly larvae. Although H. pacifica nymphs readily consumed esfenvalerate-exposed larvae, no adverse effects were observed during the present study. Furthermore, no evidence of esfenvalerate-induced feeding deterrence was found in any of the species tested, suggesting that aquatic insects may not be able to distinguish between pyrethroid-contaminated and uncontaminated food sources. These findings indicate that feeding deterrence is not a factor in regulating aquatic insect dietary exposures to synthetic pyrethroids.

Gene expression profiling reveals decreased expression of two hemoglobin genes associated with increased consumption of oxygen in Chironomus tentans exposed to atrazine: A possible mechanism for adapting to oxygen deficiency

Atrazine is an extensively used triazine herbicide in agricultural and residential areas and has been routinely detected in many surface and ground waters. This study reveals various up- and down-regulated genes associated with hypoxic stress in atrazine-treated fourth-instar Chironomus tentans larvae (midges) by using restriction fragment differential display-PCR. Two down-regulated hemoglobin cDNAs were isolated from the midges. Northern blot analysis indicated CteHb-IIβ and CteHb-III mRNA expressions decreased by 36 and 21%, respectively, in midges exposed to atrazine at 1 μg/L for 96 h. Decreased hemoglobin gene expression was associated with elevated oxygen consumption in atrazine-treated midges. Midges exposed to atrazine at 1 μg/L increased their oxygen consumption by 47%, whereas midges exposed to atrazine at 1000 μg/L for 48 h increased their oxygen consumption by 66%. Our study demonstrates for the first time that atrazine, at environmentally relevant concentrations, can elevate respiration, possibly eliciting counteractive measures at the transcriptional level to adapt to oxygen deficiency in an ecologically important aquatic insect. Our results further suggest that the ability to modulate both the quantity and quality of Hb serves as an adaptive response to counteract the initial onset of oxygen deficiency induced by atrazine in midges.

Morphologic and Growth Responses in Chironomus tentans to Arsenic Exposure

Laboratory bioassays consisting of sediments spiked with three concentrations (30, 130, and 260 microg g-1 As dry wt) of arsenic (As+3) were used to assess morphologic responses and growth in Chironomus tentans larvae. Chironomid larvae were raised in contaminated sediments from egg stage to emergence, and mouthpart abnormalities, larval length, and larval head width were used as end points to determine differences between metal-spiked and control specimens. C. tentans exhibited significantly higher mouthpart deformity proportions, smaller body sizes, smaller head widths, and slower development than control larvae. Our results demonstrate a dose-response relationship between As and mentum deformities in C. tentans. However, the proportion of deformed larvae did not increase with time in the treatment tanks. Results demonstrate that As induces mouthpart abnormalities at various concentrations. This research provides more support for the use of chironomid abnormalities as a tool for the assessment of heavy-metal pollution in aquatic systems.

Expression of heat shock protein and hemoglobin genes in Chironomus tentans (Diptera, chironomidae) larvae exposed to various environmental pollutants: A potential biomarker of freshwater monitoring

To identify a sensitive biomarker of freshwater monitoring, we evaluated pollutant-induced expression of heat shock proteins (HSPs) and hemoglobins (Hbs) genes in the larvae of the aquatic midge Chironomus tentans (Diptera, Chironomidae). As pollutants, we examined nonylphenol, bisphenol-A, 17α-ethynyl estradiol, bis(2-ethylhexyl) phthalate, endosulfan, paraquat dichloride, chloropyriphos, fenitrothion, cadmium chloride, lead nitrate, potassium dichromate, benzo[ a]pyrene and carbon tetrachloride. We also investigated larval growth as a physiological descriptor by measuring changes in the body fresh weight and dry weight after chemical exposure. The response of the HSPs gene expression by chemical exposure was rapid and sensitive to low chemical concentrations but it was not stressor specific. Interestingly, an increase in the expression of HSPs genes was observed not only in a stress inducible form (HSP70), but also in a constitutively (HSC70) expressed form. The expression of Hb genes showed chemical-specific responses: that is, alkyl phenolic compounds increased the expression of hemoglobin genes, whereas pesticides decreased the expression. As expected, molecular-level markers were more sensitive than physiological endpoints, suggesting that gene expression could be developed as an early warning biomarker in this animal. The overall results suggest that the expression of HSP and Hb genes in Chironomus could give useful information for diagnosing general health conditions in fresh water ecosystem. The expression of Hb genes, in particular, seems to be a promising biomarker, especially in view of the potential of Chironomus larvae as a biomonitoring species and of the physiological particularities of their respiratory pigments.

Comparison of a partial life‐cycle bioassay in artificial streams to a standard beaker bioassay to assess effects of metal mine effluent on Chironomus tentans

A novel, partial life-cycle bioassay using Chironomus tentans in artificial streams was developed for evaluating the effects of metal mine effluent. The utility of this bioassay was compared to an existing beaker life-cycle bioassay under laboratory conditions. Chironomus tentans larvae were exposed to 45% (v/v) treated metal mine effluent from day 11 through hatching of the second generation. Response patterns were consistent between the two bioassays for hatching success and time to emergence but inconsistent for other endpoints. Significant effects of effluent were obtained for growth, survival, number of adults emerged, and number of eggs per egg case in the artificial stream bioassay but not in the beaker bioassay. Conversely, significant effects on sex ratio and number of egg cases per female were observed in the beaker bioassay but not in the artificial stream bioassay. These differences are believed to be a consequence of the number of organisms per replicate used in each bioassay, which results in a difference in statistical power. As a result, higher coefficients of variation and effects sizes were observed in the beaker bioassay relative to the artificial stream bioassay for almost all endpoints. These results provide evidence that the bioassay in artificial streams can be an effective tool for evaluating the effects of metal mine effluent on life-cycle endpoints in C. tentans.

Lethal and sublethal effects of low dissolved oxygen condition on two aquatic invertebrates, Chironomus tentans and Hyalella azteca.

Low dissolved oxygen (DO) conditions occur frequently during sediment toxicity testing, with potentially adverse effects on test organisms. The present study addressed the current lack of good information regarding low DO thresholds for toxicity tests using two common test species, juvenile Hyalella azteca and Chironomus tentans larvae. Results indicated that H. azteca was less tolerant of hypoxia than C. tentans. The 10-d highest- and no-observed-effect concentrations (HOEC and NOEC, respectively) for H. azteca were 1.2 +/- 0.1 and 2.9 +/- 0.1 mg/L DO, respectively. The 10-d NOEC for C. tentans was 1.2 +/- 0.1 mg/L DO, the lowest test concentration. Mortality was the predominant response of H. azteca to low DO exposure, with changes in growth and positioning behavior only evident at lethal DO concentrations. Although exposure to 1.2 +/- 0.1 mg/L DO for 10 d did not affect C. tentans survival or growth, significant behavioral changes were evident at 2.0 +/- 0.1 mg/L DO or less. Overall, the present results indicate that the North American guidelines for low DO thresholds during 10-d toxicity tests seem reasonable for juvenile H. azteca. However, the Environment Canada Guideline (3.4 mg/L DO at 23 degrees C) may be considered to be conservative for 10-d toxicity testing with C. tentans if only short-term effects on survival and growth are considered.

Morphological abnormalities in Chironomus tentans exposed to cadmium—and copper-spiked sediments

The induction of mouthpart deformities and the developmental response with exposure to sediments spiked with three concentrations (9, 39, and 61 μg g −1 Cd dry wt.) of cadmium (Cd) and three concentrations (30, 125, and 215 μg g −1 Cu dry wt.) of copper (Cu) were investigated. Mouthpart deformity proportions in Chironomus tentans larvae were compared between metal-spiked and control populations and between parent and offspring (F1) populations. Cd- and Cu-treated sediments induced deformities (low Cd=13%, medium Cd=7%, high Cd=4%, low Cu=6%, medium Cu=9%, high Cu=6%) at significantly higher proportions than control (3%) sediments. No negative developmental response was determined. Larval sizes in metal-treated aquaria and control aquaria were not significantly different. F1 larvae from parents reared in medium and high Cu had significantly lower deformity rates than their parents. Our research adds to the growing evidence implicating heavy metals in general, and Cd and Cu specifically, as teratogenic agents.

Induction of morphological deformities in Chironomus tentans exposed to zinc‐ and lead‐spiked sediments

AbstractLaboratory experiments were used to assess morphological responses of Chironomus tentans larvae exposed to three levels of zinc and lead. Chironomus tentans egg masses were placed into triplicate control and metal‐spiked aquaria containing the measured concentrations 1,442, 3,383, and 5,562 μg/g Pb dry weight and 1,723, 3,743, and 5,252 μg/g Zn dry weight. Larvae were collected at 10‐d intervals after egg masses were placed in aquaria until final emergence. Larvae were screened formouthpart deformities and metal body burdens. Deformities increased with time of exposure in both Zn and Pb tanks. Deformity rates between the three Zn concentrations differed statistically, with low and medium Zn levels containing the highest overall deformity rates of 12%. Deformity rates for larvae held in the Pb aquaria were found to differ significantly. Larvae in the low‐Pb tanks had a deformity rate of 9%. Larvae and water from both the Zn and Pb aquaria had increasing metal concentrations with increasing sediment metal concentration. Results demonstrate that Zn and Pb each induce chironomid mouthpart deformities at various concentrations. However, a clear dose‐related response was not demonstrated. Our research provides more support for the potential use of chironomid deformities as a tool for the assessment of heavy metal pollution in aquatic systems.

Induction of morphological deformities in Chironomus tentans exposed to zinc- and lead-spiked sediments.

Laboratory experiments were used to assess morphological responses of Chironomus tentans larvae exposed to three levels of zinc and lead. Chironomus tentans egg masses were placed into triplicate control and metal-spiked aquaria containing the measured concentrations 1,442, 3,383, and 5,562 microg/g Pb dry weight and 1,723, 3,743, and 5,252 microg/g Zn dry weight. Larvae were collected at 10-d intervals after egg masses were placed in aquaria until final emergence. Larvae were screened for mouthpart deformities and metal body burdens. Deformities increased with time of exposure in both Zn and Pb tanks. Deformity rates between the three Zn concentrations differed statistically, with low and medium Zn levels containing the highest overall deformity rates of 12%. Deformity rates for larvae held in the Pb aquaria were found to differ significantly. Larvae in the low-Pb tanks had a deformity rate of 9%. Larvae and water from both the Zn and Pb aquaria had increasing metal concentrations with increasing sediment metal concentration. Results demonstrate that Zn and Pb each induce chironomid mouthpart deformities at various concentrations. However, a clear dose-related response was not demonstrated. Our research provides more support for the potential use of chironomid deformities as a tool for the assessment of heavy metal pollution in aquatic systems.

Atrazine induction of cytochrome P450 in Chironomus tentans larvae

Cytochrome P450-dependent aldrin epoxidation was characterized in third instar larvae of the aquatic midge, Chironomus tentans. Optimal in vitro assay conditions for the epoxidase were pH 7.6 and 31°C. Activity was linear up to 40 min of incubation time and 0.5 mg microsomal protein per incubation. The activity was concentrated in the microsomal fraction of whole body homogenates and was NADPH-dependent. The effect of atrazine exposure on aldrin epoxidase was measured to determine if this herbicide induces cytochrome P450-dependent activity. Comparisons of control and atrazine-exposed midges indicated increased epoxidase activity as a result of atrazine exposure, and a 45 kDa protein of increased intensity was observed after SDS-PAGE of microsomal protein. The molecular weight of this protein was similar in size to cytochrome P450 enzymes reported for other insects. Heme staining of SDS-PAGE gels and immunochemical studies using a Drosophila melanogaster anti-P450 polyclonal antiserum, further support the cytochrome P450 nature of this inducible 45 kDa protein.

The effect of gut contents on dry weight estimates of Chironomus tentans larvae: Implications for interpreting toxicity in freshwater sediment toxicity tests

AbstractGut contents have long been recognized as a potentially important source of bias in studies concerned with estimating contaminant body burdens in benthic macroinvertebrates. In this study, we show that gut‐associated, inorganic material may also lead to significant overestimates of weight in Chironomus tentans larvae in sediment toxicity tests and thus influence statistical interpretation of effects on growth with this organism. Two methods were evaluated regarding their potential to correct for the proportion of larval dry weight contributed by gut contents: purging and determination of ash‐free dry weight (afdw). Each method was assessed in a series of 10‐d growth tests using (1) clean sand and clay substrates with a restricted particle size distribution (mean particle size: 8, 146, 218, 306, 448, and 592 μm), and (2) sediments collected from both clean and contaminated field sites encompassing a wide range in particle size distribution. Both purging and afdw determination resulted in significant reductions in the dry weight of larvae in most substrates <306 μm and in the field‐collected sediments. In these treatments, 7–59% of the dry weight of fourth instar larvae was comprised of inorganic material contained within the digestive tract. Sediment organic carbon was also important; the contribution of gut contents to larval dry weight in a highly organic (23.6%) sediment was only 7.9% despite a small particle size distribution (mean = 149 μm). Correction for ingested material changed the rank order of treatment means in all tests and reduced the number of statistically significant outcomes for many treatment comparisons. Both methods reduced variability (lower standard deviation) associated with mean dry weight estimates, although variability was generally greater with purging than with the afdw method. Based on the results of this study, we recommend that afdw, not dry weight, be used to determine the weight of C. tentans larvae in 10‐d and life‐cycle tests with this organism.

Deformities in cultured Chironomus tentans larvae and the influence of substrate on growth, survival and mentum wear

Effects of four substrates (shredded papertowelling, coarse sand, fine sand, and silty clay)on growth, survival and mentum wear of Chironomustentans larvae were investigated in thelaboratory. Three categories of mentum wear were defined:minimal, moderate and substantial. Thenumber of larvae with worn mentum teeth did not differ in anyof the four substrates. However, thenumber of larvae with minimal wear of their teeth wassignificantly different among the substrates andwas in the order coarse sand > shredded paper towelling >clay/silt > fine sand. Mentumdeformities were observed in the cultured larvae, presumablybecause of inbreeding and not the effectof treatment. Differences in biomass and survival of larvaeamong the substrates were not significant(p<0.05), however, there was a trend for lower survival inthe sand substrates. These findingssuggest that shredded paper towelling is the most suitablereference substrate. The occurrence ofnatural abnormalities must be considered when using chironomidmouthpart deformities as an index of environmental degradation.

The effect of 210Pb and stable lead on the induction of menta deformities in chironomus tentans larvae and on their growth and survival

AbstractTo determine whether menta deformities in chironomid larvae collected from Port Hope Harbour, Ontario, were the result of exposure to ionizing radiation or to heavy metals in the sediment, Chironomus tentans larvae were exposed in the laboratory to concentrations of 210Pb and stable lead representative of contaminant levels in the harbour. Exposure to 100, 1,000, and 2,000 Bq 210Pb g−1 dry sediment had no effect on larval survival, growth, or frequency of deformities. Likewise, exposure to 0.5 and 5.0 mg lead g−1 dry sediment had no effect on the larvae, but exposure to 35.0 mg lead g−1 dry sediment (the maximum levels observed in Port Hope Harbour) resulted in 100% mortality of the larvae. These results indicate that the observed mouthpart deformities in chironomid larvae of Port Hope Harbour are not the result of radiation exposure. They also indicate that heavy metals (lead) may be having an impact on the Port Hope Harbour population. Deformities were observed in all treatments and were confined mainly to the median tooth, with cleft or forked median teeth being the most common deformity. Restriction of the deformities to the median tooth is consistent with an inherited genetic effect, and we consider them to be natural abnormalities. These natural abnormalities must be considered when using chironomid mouthpart deformities as an index of environmental degradation.

DNA cytochemistry in polytene chromosomes: electron contrasting agents for the ultrastructural detection of chromatin DNA after alkaline hydrolysis/methylation-acetylation.

Salivary glands from Chironomus tentans larvae were fixed in glutaraldehyde and either subjected to alkaline hydrolysis followed by methylation-acetylation, or dehydrated without these treatments as controls. Ultrathin sections from Durcupan-embedded samples were contrasted by means of uranyl acetate, ruthenium red, indium trichloride, or the complex indium (III)-hematoxylin. Electron microscopic observations revealed a general contrasting pattern in control sections, while after the hydrolytic and blocking procedure only chromatin from polytene chromosomes appeared selectively contrasted. The nucleolus, Balbiani ring granules and puff materials showed weak or no electron opacity. After toluidine blue staining of semithin sections, an orthochromatic blue colour was found in chromatin bands from treated samples. These results indicate that alkaline hydrolysis/methylation-acetylation followed by contrasting with cationic heavy compounds is a valuable procedure to visualize chromatin DNA in polytene chromosomes.

Genetic variation in laboratory and field populations of the midge, Chironomus tentans fab.: Implications for toxicology

AbstractChironomus tentans larvae are used extensively in toxicoiogical bioassays. Gel electrophoresis was performed on seven laboratory populations and a wild population of C. tentans. Direct count heterozygosity was significantly different between wild and laboratory populations, as well as among laboratory populations. Percent polymorphic loci demonstrated the same trends but were marginally nonsignificant. The wild midge population had seven unique alleles and a significantly higher level of heterozygosity. Despite these differences the wild population was genetically similar to laboratory populations. The impact of significant differences in Chironomus genetic variability on toxicoiogical bioassays is discussed.

Developmental changes in the responsiveness to ecdysterone of chromosome region I-18C of Chironomus tentans

The presence of nascent RNA in chromosome region I-18C was revealed by the method of induced hybrid formation (IEH) and by use of anti-RNA:DNA antibodies. IEH signals were quantitated, and used as a measure of transcriptional activity in the region. The expansion of the I-18C region was also determined and used as an indication of local chromatin decondensation. Chironomus tentans larvae may undergo oligopause or they may develop subitaneously. It was found that the course of basal activity in I-18C differs substantially in these two modes of development. Most interestingly they also differ in activation of I-18C in response to in vitro ecdysterone treatment. Changes in hormonal responsiveness of I-18C parallel changes in the preexisting state of I-18C decondensation. Oligopause stage 5 animals, being most refractory in this respect, exhibit the most condensed I-18C chromosome region.

Microcloning of a long segment of a gene from the primary ecdysterone-controlled puff region I-18C of Chironomus tentans: Molecular characterization and effect of hormone on its transcription

Transcribed gene sequences from the ecdysterone-inducible puff site I-18C of salivary gland chromosomes of Chironomus tentans larvae have been microcloned. Two adjacent genomic fragments (pCteI-18C 1 and pCteI-18C 2) were isolated which encompass approximately 7 kbp DNA. The I-18C gene gives rise to a cytoplasmic poly(A) +RNA of 4.6 kb. The 3′end of the gene is located in the pCteI-18C 2 fragment. Toward the 5′end there is at least one intron. This gene is relatively inactive in salivary gland chromosomes of oligopausing larvae (OL) but it is highly active in glands of prepupae (PP); in isolated OL glands it is activated by ecdysterone within 1 hr. This was demonstrated by an in situ hybridization method which indirectly reveals nascent RNA of the gene. Furthermore, in cultured epithelial cells ecdysterone starts stimulating transcription of the I-18C gene within the first hour. This leads eventually to an approximately 10-fold accumulation of I-18C-specific RNA.