The physiological response of larval <Emphasis Type="Italic">Chironomus riparius</Emphasis> (Meigen) to abrupt brackish water exposure

The physiological response of larval Chironomus riparius was examined following direct transfer from freshwater (FW) to brackish water (BW; 20% seawater). Endpoints of hydromineral status (hemolymph Na⁺, Cl⁻, and K⁺ levels, hemolymph pH, body water content, and whole body Na⁺/K⁺-ATPase and V-type H⁺-ATPase activity) were examined 1, 3, 5, 12 and 24 h following BW transfer. Larvae transferred from FW to FW served as a control. Hemolymph Na⁺ and Cl⁻ levels increased following BW transfer. Hemolymph pH was initially regulated, but significantly decreased after 24 h in BW. Changes in hemolymph ions were not caused by osmotic loss of water from the hemolymph, since larvae tightly regulated total body moisture content. Furthermore, salinity did not affect hemolymph K⁺. When larvae were transferred to BW, Na⁺/K⁺-ATPase (NKA) activity did not significantly alter relative to FW control animals. In contrast, V-type H⁺-ATPase (VA) activity in C. riparius significantly decreased in BW. In FW-reared C. riparius, whole body NKA and VA activities were equivalent. However, in the isolated gut with intact Malpighian tubules of FW-reared C. riparius, VA activity was significantly greater than whole body while NKA activity was equivalent. This suggested that gut and/or Malpighian tubule VA activity contributes significantly to whole body VA activity and that a decline in whole body VA activity in BW may be closely linked to alterations in the physiology of gut and Malpighian tubule tissue. Taken together, data indicate that VA is important for ion uptake in FW and that the NKA does not play a major role in regulating ion homeostasis when larvae are acutely exposed to BW.     

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.     

Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori

Abstract Two‐dimensional gel electrophoresis (2‐DE) followed by matrix‐assisted laser desorption ionization – time‐of‐flight/time‐of‐flight mass spectrometry (MS) analysis were used to charaterize the hemolymph proteomic profiles of the silkworm, Bombyx mori. At days 4 (V4) and 5 (V5) of the fifth (final) instar, when the larvae were at the fast‐growing stage, we found dramatic changes in spots representing proteins having an approximate molecular weight (MW) of 30 kDa. Of these spots, four 30K proteins were highly up‐regulated, implying a close association with the growth and development of B. mori larvae. To understand the molecular basis and underlying mechanisms involved in development and metamorphosis, the proteome of whole hemolymph at V5 was analyzed using shotgun liquid chromatography tandem mass spectrometry with an LTQ‐Orbitrap. A total of 108 proteins were identified without any false discovery hits. These proteins were involved in a variety of cellular functions, including metabolism, development, nutrient transport and reserve, and defense response. Gene ontology analysis showed that 3.4% of these proteins had nutrient reservoir activities and 5.7% were involved in the response to stimulus. Pathway analysis revealed that 22 proteins with common targets were involved in various cellular processes such as immunity, differentiation, proliferation and metamorphosis. These results suggested that some key factors such as the 30K proteins in hemolymph play important roles in B. mori growth and development. Moreover, the multiple functions of hemolymph may be operated by a complex biological network.     

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.     

Differences in feeding behaviour among Chironomus species revealed by measurements of sulphur stable isotopes and cadmium in larvae

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Hemoglobins of Kiefferulus,sister genus of Chironomus (Diptera: Insecta): Evolution of the Hb VIIB cluster

A genomic clone containing hemoglobin genes was isolated from a species of the chironomid genus Kiefferulus. Eight genes, including an apparent pseudogene, were sequenced and the amino acid sequences of the putative proteins were determined. By comparison to the previously described hemoglobins in the sister-genus Chironomus, they were identified as members of the dimeric Hb VIIB group. The results indicate that the existence of clusters of hemoglobin genes may be a common feature in chironomids and not just confined to Chironomus. The Kiefferulus genes show greatest similarity of amino acid sequence to Hb VIIB-7 from the Chironomus cluster. The results suggest that the ancestral cluster contained at least two gene types, one of which gave rise to VIIB-7 and the Kiefferulus genes while the other gave rise to the other Chironomus VIIB genes. Both clusters appear to have increased in size by duplication or unequal crossing over since the separation of the genera. It also appears that an unrelated gene present in the Chironomus cluster, Hb-Y, arose from a completely independent origin with no apparent equivalent gene anywhere in the genome of Kiefferulus or some other Chironomus species. Correspondence to: J. Martin     

Proteomics reveals new insights into the role of light in cadmium response in Arabidopsis cell suspension cultures

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light‐ and dark‐grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress‐related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light‐grown cells were more sensitive to heavy metal toxicity than dark‐grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism‐related proteins increased in relative abundance both in light‐ and dark‐grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti‐oxidative processes were decreased both in light‐ and dark‐grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light‐dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.     

Comparative proteomic analysis of Cd-responsive proteins in wheat roots

Cadmium (Cd) is a major environmental toxicant to plant cells due to its potential inhibitory effects on many physiological processes. To gain a comprehensive understanding of plant response to Cd, wheat seedlings were exposed to a range of Cd concentrations (10, 100 and 200 μM) for 1 week and a combination of physiological and proteomic approaches were used to evidence Cd effects and to access the plant response to Cd toxicity. Root and shoot elongation was decreased, whereas the H₂O₂ and malondialdehyde content in wheat seedlings was increased significantly at higher Cd concentration. Protein profiles analyzed by two-dimensional electrophoresis revealed that 46 protein spots showed 1.5-fold change in protein abundance following Cd exposure; 31 protein spots were up-regulated and 15 protein spots were down-regulated; 25 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As expected, most of the up-regulated proteins are involved in heavy metal detoxification and antioxidant processes. Enzyme activity analysis revealed that ascorbate peroxidase and glutathione S-transferase activity was stimulated by Cd treatment. Abundance changes of these proteins, together with their putative functions provide us a new insight that can lead to an integrated understanding of the molecular basis of Cd responses in plants.     

Isoform‐specific responses of metallothioneins in a marine pollution biomonitor,the green‐lipped mussel Perna viridis,towards different stress stimulations

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green‐lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non‐metal oxidant (hydrogen peroxide; H₂O₂) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10‐I and 72 for MT10‐II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two‐dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10‐I and MT10‐II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10‐I responded promptly to Cd but had a lagged induction to H₂O₂ treatments, while MT10‐II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H₂O₂, compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.     

Chronic toxicity of cadmium toChironomus riparius Meigen — effects upon larval development and adult emergence

Chronic cadmium toxicity tests were carried out withChironomus riparius Meigen using a static-with-replacement exposure procedure. Significant reductions in larval development, survival and production were detected at 0.15 mg Cd l^–1 a value approximately 5000 times lower than the 48 h LC50 for fourth instar larvae. The first instar appears to be the most critical stage in terms of sensitivity, with the number of adults eventually emerging reflecting first instar mortality. Male emergence was slightly delayed at 0.15 mg Cd l^–1. The results of this study illustrate the importance of chronic toxicity tests in identifying sensitive life cycle stages and explaining pollutant-induced stress.     

Differential efficacy of toxic pederin in deterring potential arthropod predators of Paederus (Coleoptera: Staphylinidae) offspring

This study investigates the effects of pederin, a hemolymph toxin that is accumulated in the eggs of most Paederus females, on potential arthropod predators of the offspring of P. fuscipes and P. riparius. Insects generally do not respond to pederin present in the prey. Paederus larvae are sufficiently agile to escape from these predators by running away, and the eggs are hidden by the females. Unlike insects, (wolf) spiders are deterred by prey with pederin. They turn away from larvae they have already captured and exhibit cleansing behavior. Larvae containing pederin survive the attacks of spiders without damage, whereas larvae descended from females that do not transfer pederin into their eggs are often killed and eaten. In the case of sudden attacks by spiders, the larvae have no chance of escape. Their survival thus depends on chemical defense. These investigations show for the first time why pederin might be of considerable importance for Paederus in the field.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Effects of water temperature on development and heavy metal toxicity change in two midge species of Chironomus riparius and C. yoshimatsui in an era of rapid climate change

Climate change is the most significant stressor that is anticipated increasingly to affect human and global ecosystems. Arthropods, including insects, are particularly vulnerable to global warming and this group is often used for various ecotoxicological tests. In addition, temperature is one of the most important toxicity‐modifying factors in ecotoxicology. Therefore, temperature dependent toxicological research is required to obtain ecologically relevant conclusions during the current era of rapid climate change. This study shows that two midge species (Chironomus riparius Meigen and C. yoshimatsui Martin et Sublette) exhibit different developmental characteristics and responses to cadmium and lead heavy metals with temperature. The former species is an internationally standardized test species in ecotoxicological studies, whereas the latter species is native to Korea. Hence, even though these two species belong to the same genus, Chironomus, their development differs with temperature, which leads to different responses to heavy metals. There was a decline in developmental time (from egg and larva to pupa) for both species with temperature; however, there was a species difference in the rate of decline. In the acute toxicity test, the 48‐hr LC₅₀ values for cadmium and lead decreased with temperature for both species. In the chronic toxicity test, emergence rates tended to decrease with temperature, except for when C. yoshimatsui was exposed to cadmium.     

Prey switching in Rhyacophila dorsalis (Trichoptera) alters with larval instar

1. Ontogenetic shifts in predator behaviour can affect the assessment of food‐web structure and the development of predator–prey models. Previous studies have shown that the diel activity pattern and functional response differed between larval instars of the carnivorous caddis, Rhyacophila dorsalis. The present study examines switching by larvae of R. dorsalis presented with different proportions of two prey types; either small (length 2–4 mm) and large (5–8 mm) Chironomus larvae for second, third, fourth and fifth instars of R. dorsalis; or Baetis rhodani (9–12 mm) and large Chironomus larvae for fourth and fifth instars. Experiments were performed in stream tanks with one Rhyacophila larva per tank and 200 prey arranged in nine different combinations of the two prey types (20 : 180, 40 : 160, 60 : 140, 80 : 120, 100 : 100, 120 : 80, 140 : 60, 160 : 40 and 180 : 20). Prey were replaced as they were eaten. A model predicted the functional response in the absence of switching and provided a null hypothesis against which any tendency to switch could be tested. 2. There was no prey switching in the second and third instars, with both instars always showing a preference for small over large Chironomus larvae. Prey switching occurred in the fourth and fifth instars. As the relative abundance of one prey type increased in relation to the alternative, the proportion eaten of the former prey changed from less to more than expected from its availability, the relationship being described by an S‐shaped curve. In the experiments with small and large Chironomus, the two instars switched to large larvae when their percentage of the total available prey exceeded 29% and 37% for fourth and fifth instars, respectively. In the experiments with Baetis and large Chironomus, both instars switched to Baetis larvae when their percentage of the total available prey exceeded 36%. 3. Non‐switching in second and third instars was related to their feeding strategies, both instars preferring smaller prey items. When the fourth and fifth instars foraged actively at night, they preferred larger over small Chironomus larvae, but when they behaved as ambush predators at dusk, they captured the more active Baetis larvae in preference to the more sedentary large Chironomus larvae and only switched to the latter when they were >64% of the available prey.     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

Response to nickel in the proteome of the metal accumulator plant Brassica juncea

To gain a comprehensive understanding of the molecular mechanism of heavy metal accumulation in Brassica juncea, comparative proteomic approaches were used to analysis protein profiles in leaf tissues of 6-week-old B. juncea after exposure to 100 µM Ni. Proteomic analysis revealed that 61 protein spots showed 1.5-fold change in protein abundance after Ni exposure as compared to that of corresponding spots in control. Out of the 61 differentially expressed protein spots, 37 protein spots were ambiguously identified by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). The majority of these identified proteins were found to be involved in sulphur metabolism, protection against oxidative stress, clearly indicated that heavy metal sequestration and antioxidant system were activated by Ni treatment. The induced expression of photosynthesis and ATP generation-related proteins were also observed in plants exposed to metals, suggesting the tolerance and accumulation is an energy-demanding process. The identification of these proteins in response to Ni can lead a deep understanding of heavy metal accumulation and tolerance in B. juncea.     

Cadmium,zinc, lead and copper inChironomus riparius (Meigen) larvae (Diptera,Chironomidae): uptake and effects

Cadmium, Zn, Pb and Cu uptake and effects in larvae ofChironomus riparius (Meigen) were studied in an integrated laboratory investigation, in which metal analyses in different instar stages, uptake kinetics and effects on development and growth were considered in three separate experiments.In short-term experiments with fourth instar larvae, it was demonstrated that only a minor portion of metals was adsorbed on the larval exoskeletons. No conclusive evidence on the uptake mechanism was found, but active uptake of trace metals seemed highly unlikely.In partial life cycle experiments, all four metals studied were readily accumulated in chironomid larvae. Uptake could be described satisfactorily utilizing a first-order one-compartment uptake model which incorporated growth. In all cases steady state conditions were approached and high uptake and elimination rate constants were estimated. Distinct differences between essential (Zn and Cu) and non-essential (Cd and Pb) metals were noted. Larval growth was significantly impaired upon exposure.Finally, long-term exposure experiments with low Cd concentrations (0.010 and 0.025 mg 1^{–1) resulted initially in growth impairment and high mortality in first instar stages, but surviving larvae restored growth and adults emerged even before control adults. In similar experiments with Zn (0.1 and 1.0 mg 1–1}), development of larvae was significantly retarded. High Zn concentrations in larvae were noted and almost no adult midges emerged.     

Effect of cold acclimation on the antioxidant defense system of two larval lepidoptera (noctuidae)

Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione-S-transferase (GST), as well as total glutathione (tGSH) concentration were analyzed in the hemolymph and fat body of the European corn borer Ostrinia nubilalis Hubn. and the Mediterranean borer Sesamia cretica Led. (Lepidoptera, Noctuidae). Controls were maintained at 8°C while experimental groups of larvae were exposed to –3°C for ten days and then to –12°C for 23 days (only for Ostrinia). Cold exposure significantly increased fat body SOD, GR, and GST activities of Ostrinia larvae. Only GST activity and tGSH levels increased significantly in Ostrinia larval hemolymph on cold exposure. In Sesamia larvae after cold exposure, hemolymph CAT activity was significantly lower, while fat body tGSH increased. The antioxidant defense systems of these two species show differences, probably influenced by their respective cold-hardiness metabolism. According to its antioxidant profile, the response of Ostrinia suggests a significant physiological alteration in its metabolism during cold exposure, indicating a compensatory mechanism. By contrast this is not evident in Sesamia.Arch. Insect Biochem. Physiol. 36:1–10, 1997. © 1997 Wiley-Liss, Inc.