Chironomus Riparius Larvae Research Articles

Exposure to sediments from polluted rivers has limited phenotypic effects on larvae and adults of Chironomus riparius

Laboratory studies have sometimes failed to detect a relationship between toxic stress and morphological defects in invertebrates. Several hypotheses have been proposed to account for this lack of effect. (1) It was suggested that only a combination of stressful conditions – rather than a single one – would affect the phenotype. (2) Phenotypic defects should be detected on adult individuals, rather than on juveniles. (3) Phenotypic abnormalities might mostly affect the progeny of the exposed individuals, some contaminants exhibiting trans-generational effects. In the present study, we test those three hypotheses. We first examined the effects of a multiple exposure by using laboratory Chironomus riparius larvae cultured on two sediments sampled in contaminated rivers and those containing a mixture of mineral and organic compounds. On the larvae, we investigated mentum phenotypes: the frequency of phenodeviants, the shape fluctuating asymmetry and the mean shape. To test whether adult's morphology was more sensitive than the larval's, we also measured asymmetry and mean shape of the adult wings. Finally, to test for a trans-generational phenotypic effect, we measured mentum shape variations in the offspring derived from the measured adults. Overall, our results point out a very limited phenotypic response to contaminated sediments, suggesting that a multiple exposure is not necessarily sufficient to generate phenotypic defects. Adult traits were no more affected than larval traits, discarding the hypothesis that adult phenotypes would be more sensitive biomarkers. Finally, no effect was detected on the offspring generation, suggesting that no trans-generational effect occurs. This general lack of effect suggests that the use of phenotypic defects in C. riparius as an indicator of sediment contamination should be considered cautiously.

N-Bridged 1-deoxynojirimycin dimers as selective insect trehalase inhibitors

A small set of N-bridged 1-deoxynojirimycin dimers has been synthesized and evaluated as potential inhibitors of insect trehalase from midge larvae of Chironomus riparius, porcine trehalase as the mammalian counterpart and α-amylase from human saliva. All the tested compounds (2–4) proved to be active (micromolar range activity) against insect trehalase, showing selectivity toward the insect glycosidase. No activity was observed against α-amylase.

Characterization of the small heat shock protein Hsp27 gene in Chironomus riparius (Diptera) and its expression profile in response to temperature changes and xenobiotic exposures

Small heat shock proteins constitute the most diverse and least conserved group within the large family of heat shock proteins, which play a crucial role in cell response to environmental insults. Chironomus riparius larvae are widely used in environmental research for testing pollutant toxicity in sediments and freshwater environments. Different genes, such as Hsp70, Hsc70, Hsp90, and Hsp40, have been identified in this species as sensitive biomarkers for xenobiotics, but small Hsps genes remain largely unknown. In this study, the Hsp27 has been characterized in C. riparius and its transcriptional response evaluated under several environmental stimuli. The Hsp27 gene was mapped by FISH on polytene chromosomes at region I-C4 and was found to encode a 195 aa protein, which contains an α-crystallin domain bounded by three conserved regions. This protein shows homology with Drosophila melanogaster HSP27, Ceratitis capitata HSP27, and Sarcophaga crassipalpis HSP25. Real-time reverse transcriptase-polymerase chain reaction analysis showed that heat shock (35°C) and cadmium dramatically upregulate this gene. Moreover, exposures to triclosan and bisphenol A were able to significantly increase mRNA levels. However, neither nonylphenol nor tributyltin altered Hsp27 gene expression. The transcriptional activity of Hsp27 gene was modulated during cold stress. Interestingly, cold shock (4°C) significantly reduced Hsp27 transcripts, but this gene was significantly overexpressed during the recovery time at the normal growing temperature. These results show that the Hsp27 gene is sensitive to different environmental stimuli, including endocrine-disrupting pollutants, suggesting its potential as a suitable biomarker for ecotoxicological studies in aquatic systems.

Bioaccumulation kinetics and effects of sediment-bound contaminants on chironomids in deep waters: new insights using a low-disturbance in situ system

Effective assessment of sediment quality and associated risks requires further integration between laboratory-based studies and field observations. Tools for in situ exposure of laboratory-reared organisms are particularly suitable for this purpose. However, available tools suffer from technical drawbacks that limit their use in deep waters and their general ability to provide results linking laboratory and field observations. To overcome these limitations, we developed a new device (Sediment–Water Interface Study System – S-WISS1) for in situ exposure of fourth instar larvae of Chironomus riparius at depths up to 18 m. S-WISS1 allows minimum alteration of the sediment natural structure (upon both deployment and recovery) and, in a way analogous to laboratory tests, repeated delivery of food doses to the in situ exposed organisms. Results obtained in situ with S-WISS1 were compared with standard laboratory tests and simple caging systems (large-mouthed bottles partly filled with sand) used as field controlks. A first series of experiments (48 h, no food addition) showed that element bioavailability to chironomids was higher in laboratory settings than on-site or in situ. A second series of experiments (96 h, food addition every 24 h) confirmed that sediments rather than overlying waters acted as the principal source of contaminants and that exposure to field sediments reduced chironomids’ growth compared with on-site controls. S-WISS1 represents a new family of in situ tools capable of combining the environmental realism associated with field monitoring with the controlled experimental conditions typical of laboratory testing.

Genotoxic effects of environmental endocrine disruptors on the aquatic insect Chironomus riparius evaluated using the comet assay

Genotoxicity is one of the most important toxic endpoints in chemical toxicity testing and environmental risk assessment. The aim of this study was to evaluate the genotoxic potential of various environmental pollutants frequently found in aquatic environments and characterized by their endocrine disrupting activity. Monitoring of DNA damage was undertaken after in vivo exposures of the aquatic larvae of the midge Chironomus riparius, a model organism that represents an abundant and ecologically relevant macroinvertebrate, widely used in freshwater toxicology. DNA-induced damage, resulting in DNA fragmentation, was quantified by the comet assay after short (24h) and long (96h) exposures to different concentrations of the selected toxicants: bisphenol A (BPA), nonylphenol (NP), pentachlorophenol (PCP), tributyltin (TBT) and triclosan (TCS). All five compounds were found to have genotoxic activity as demonstrated by significant increases in all the comet parameters (%DNA in tail, tail length, tail moment and Olive tail moment) at all tested concentrations. Persistent exposure did not increase the extent of DNA damage, except for TCS at the highest concentration, but generally there was a reduction in DNA damage thought to be associated with the induction of the detoxification processes and repairing mechanisms. Comparative analysis showed differences in the genotoxic potential between the chemicals, as well as significant time and concentration-dependent variations, which most likely reflect differences in the ability to repair DNA damage under the different treatments. The present report demonstrates the sensitivity of the benthic larvae of C. riparius to these environmental genotoxins suggesting its potential as biomonitor organism in freshwater ecosystems. The results obtained about the DNA-damaging potential of these environmental pollutants reinforce the need for additional studies on the genotoxicity of endocrine active substances that, by linking genotoxic activity to other biological responses, could provide further understanding of adverse effects in aquatic environments.

Biochemical and morphological responses in Chironomus riparius (Diptera, Chironomidae) larvae exposed to lead‐spiked sediment

The aim of the present study was to assess the potential use of biochemical markers and mentum deformities as indicators of long-term exposure to lead (Pb) in Chironomus riparius larvae. To do this, the authors measured 3 biochemical markers (i.e., malondialdehyde level, metallothionein concentration, and energy reserve content) as well as larval growth and mentum deformities after 16-d exposure to sediment containing Pb. The concentrations studied ranged from 3.5 mg/kg to 505.5 mg/kg dry weight. Despite the bioaccumulation of Pb in C. riparius bodies, frequencies of both mentum deformities and the dry weight were not significantly different between the control and stressed groups. On the contrary, Pb exposure caused a significant increase of both malondialdehyde level and metallothionein concentration. The increase of body Pb concentrations did not significantly modify body copper and zinc concentrations. Moreover, we observed a decrease of total lipid content and an increase of glycogen content as a function of a dose-response relationship, while no variation in protein concentrations was observed. Despite the adverse effects observed at the biochemical level, larval development was not affected. These results suggested that measurements of malondialdehyde level, metallothionein concentration, and energy reserve content can be used as relevant biomarkers of long-term sublethal exposure to Pb in C. riparius larvae.

Transcriptional responses, metabolic activity and mouthpart deformities in natural populations of chironomus riparius larvae exposed to environmental pollutants

Biomarkers are an important tool in laboratory assays that link exposure or effect of specific toxicants to key molecular and cellular events, but they have not been widely used in invertebrate populations exposed to complex mixtures of environmental contaminants in their natural habitats. The present study focused on a battery of biomarkers and their comparative analysis in natural populations of the benthic larvae of Chironomus riparius (Diptera), sampled in three differentially polluted rivers (the Con, Sar, and Louro in Galicia, Spain). In our study, some parameters were identified, such as hsp70 gene activity, GST enzymatic activity, total glycogen content and mouthpart deformities, which showed significant differences among populations from the three rivers that differed in the levels and types of sedimentary contaminants analyzed (metals, organic-chlorine pesticides, alkylphenols, pharmaceutical, and personal care products). In contrast to these sensitive biomarkers, other parameters showed no significant differences (hsc70 gene, EcR gene, P450 gene, RNA:DNA ratio, total protein content), and were stable even when comparing field and nonexposed laboratory populations. The hsp70 gene seems to be particularly sensitive to conditions of pollutant exposure, while its constitutive counterpart hsc70 showed invariable expression, suggesting that the hsc70/hsp70 ratio may be a potential indicator of polluted environments. Although further studies are required to understand the correlation between molecular responses and the ecological effects of pollutants on natural populations, the results provide new data about the biological responses to multiple-stressor environments. This field study adds new molecular endpoints, including gene expression, as suitable tools that, complementing other ecotoxicological parameters, may help to improve the methodologies of freshwater monitoring under the increasing burden of xenobiotics.

DNA damage and transcriptional changes induced by tributyltin (TBT) after short in vivo exposures of Chironomus riparius (Diptera) larvae

Tributyltin (TBT) is a widespread environmental contaminant in aquatic systems whose adverse effects in development and reproduction are related to its well-known endocrine-disrupting activity. In this work, the early molecular effects of TBT in Chironomus riparius (Diptera) were evaluated by analyzing its DNA damaging potential and the transcriptional response of different endocrine-related genes. Twenty-four-hour in vivo exposures of the aquatic larvae, at environmentally relevant doses of TBT, revealed genotoxic activity as shown by significant increases in DNA strand breaks quantified with the comet assay. TBT was also able to induce significant increases in transcripts from the ecdysone receptor gene (EcR), the ultraspiracle gene (usp) (insect ortholog of the retinoid X receptor), the estrogen-related receptor (ERR) gene and the E74 early ecdysone-inducible gene, as measured by real-time RT-PCR. In contrast, the expression of the vitellogenin (vg) gene remained unaltered, while the hsp70 gene appeared to be down-regulated. The ability of TBT to up-regulate hormonal target genes provides the first evidence, at genomic level, of its endocrine disruptive effects and also suggests a mechanism of action that mimics ecdysteroid hormones in insects. These data reveal for the first time the early genomic effects of TBT on an insect genome.

Bioconcentration of benzo[a]pyrene in Chironomus riparius and Lumbriculus variegatus in relation to dissolved organic matter and biotransformation

The relative health of benthic invertebrate populations is threatened by both dissolved and strongly-bound contaminants. Sediment-bound contaminants can be mobilized and metabolized in the guts of deposit-feeders and may lead to even more potent derivatives. For this reason, measures of parent substance concentrations in organisms are not a suitable exposure indicator when substantial biotransformation can occur. In this study, we assessed the concentration of waterborne 14C-benzo[a]pyrene (BaP) in Chironomus riparius larvae and in the oligochaete Lumbriculus variegatus at different levels of dissolved organic carbon (DOC). These two species have different biotransformation capabilities. Polydimethylsiloxane (PDMS) fiber was used to assess the freely-dissolved concentrations for the prediction of uptake and elimination kinetics. Exposure concentrations decreased due to the presence of DOC. Radioactivity that corresponded to the metabolites exceeded 90% of total extractable fractions in C. riparius, and suggested that BaP metabolism was faster than uptake from solution. However, only 10% of the extracted activity was attributed to metabolites in L. variegatus. Measured tissue concentrations and model-predicted uptake and elimination rates showed that L. variegatus mainly accumulated parent compound, whereas C. riparius accumulated mainly metabolites. No effect of DOC was observed on the biotransformation of BaP in either species.

Effects of inbreeding on mouthpart deformities of Chironomus riparius under sublethal pesticide exposure

Mouthpart deformities in chironomids have been reported to indicate adverse effects of environmental pollutants. The authors assessed rates of mouthpart deformities in tributyltin-exposed, inbred, and outcrossed Chironomus riparius larvae over multiple generations. The authors found that the occurrence of mouthpart deformities was significantly correlated with inbreeding, whereas no correlation was found with the tributyltin exposure. The present study confirms the strong effect of high inbreeding rates on developmental deformities in chironomids.

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

DECOTAB: a multipurpose standard substrate to assess effects of litter quality on microbial decomposition and invertebrate consumption

Currently available tools for studying plant litter decomposition and invertebrate consumption in aquatic ecosystems have at least 2 major limitations: 1) the difficulty of manipulating litter chemical composition to provide mechanistic insights into attributes of litter quality controlling decomposition rate, and 2) lack of a standardized litter that hampers comparisons of results among studies. These limitations point to a need for a standard litter surrogate with adjustable chemical composition. We propose using a decomposition and consumption tablet (DECOTAB) consisting of cellulose powder embedded in an agar matrix to evaluate decomposition and consumption rates in aquatic environments. We describe the preparation of DECOTABs and demonstrate some applications in laboratory microcosms and outdoor mesocosms. A leaf shredder, the isopod Asellus aquaticus, and a collector-gatherer, the nonbiting midge larva Chironomus riparius, readily consumed DECOTABs, leading to massive mass loss of the tablets within 21 d (∼90%). The isopod also consumed DECOTABs amended with extracts of riparian plants and soil to create a chemically complex source of organic matter. Our results highlight the potential utility of DECOTABs to assess invertebrate contributions to organic matter decomposition in aquatic systems. In the absence of invertebrates, exposure of basic and complex DECOTABs to microorganisms resulted in significant mass loss within 21 d (10–25%), and addition of an antibiotic and fungicide suppressed microbial decomposition, suggesting that the tablets are useful for studying microbial processes. Complex tablets decomposed faster than the basic tablets, a result illustrating the importance of chemical composition of organic material for microbial decomposers. DECOTABs are a novel, versatile tool for addressing long-standing questions in aquatic ecology and environmental assessment.

MOLECULAR CLONING OF SOLUBLE TREHALASE FROM Chironomus riparius LARVAE, ITS HETEROLOGOUS EXPRESSION IN Escherichia coli AND BIOINFORMATIC ANALYSIS

Trehalase is involved in the control of trehalose concentration, the main blood sugar in insects. Here, we describe the molecular cloning of the cDNA encoding for the soluble form of the trehalase from the midge larvae of Chironomus riparius, a well-known bioindicator of the quality of freshwater environments. Molecular cloning was achieved through multiple alignment of Diptera trehalase sequences, allowing the synthesis of internal homology-based primers; the complete open reading frame(ORF) was subsequently obtained through RACE-PCR(where RACE is rapid amplification of cDNA ends). The cDNA contained the 5' untranslated region (UTR), the 3' UTR including a poly(A) tail and the ORF of 1,725 bp consisting of 574 amino acid residues with a predicted molecular mass of 65,778 Da. Recombinant trehalase was successfully expressed in Escherichia coli as a His-tagged protein and purified on Ni-NTA affinity chromatography. Primary structure analysis showed a series of characteristic features shared by all insect trehalases, while three-dimensional structure prediction yielded the typical glucosidase fold, the two key residues involved in the catalytic mechanism being conserved. Production of recombinant insect trehalases opens the way to structural characterizations of the catalytic site, which might represent, among others, an element for reconsidering the enzyme as a target in pest insects' control.

Characterization and expression of superoxide dismutase genes in Chironomus riparius (Diptera, Chironomidae) larvae as a potential biomarker of ecotoxicity

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme involved in the scavenging of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In this study, a copper–zinc superoxide dismutase (Cu–ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene in aquatic midge, Chironomus riparius (CrSODs) was identified using an Expressed Sequence Tag (EST) database generated by 454 pyrosequencing. A multiple sequence alignment of C. riparius sequences revealed high homology with other insect sequences in terms of the amino acid level. Phylogenetic analysis of the CrSODs revealed that they were grouped with SODs of other organisms, such as Polypedilum vanderplanki, Drosophila melanogaster, Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus and Bombyx mori. Expression of the corresponding CrSODs was analyzed during different developmental stages and following exposure to various environmental contaminants with different mode of actions i.e., paraquat, cadmium, benzo[a]pyrene, and chloropyrifos. CrSOD gene expression was significantly up or down regulated in response to exposure to the chemicals tested. The overall results suggested that SOD gene expression provided a platform for the understanding of oxidative stress responses caused by exposure to various environmental contaminants, and the SOD genes could be used as biomarkers for environmental disturbances such as oxidative stress initiated by xenobiotics.

Induction of mouthpart deformities in chironomid larvae exposed to contaminated sediments

The aim of the present study was to improve the cause–effect relationship between toxicant exposure and chironomid mouthpart deformities, by linking induction of mouthpart deformities to contaminated field sediments, metal mixtures and a mutagenic polycyclic aromatic compound metabolite (acridone). Mouthpart deformities in Chironomus riparius larvae were induced by both the heavy metal mixture and by acridone. A clear correlation between metal concentrations in the sediment and deformities incidence was only observed when the contaminated field sediments were left out of the analysis, probably because these natural sediments contained other toxic compounds, which could be responsible for a higher incidence of deformities than predicted by the measured metal concentrations only. The present study clearly improved the cause–effect relationship between toxicant exposure and the induction of mouthpart deformities. It is concluded that the incidence of mouthpart deformities may better reflect the potential toxicity of contaminated sediments than chemical analysis.

Survival, reproduction, and arsenic body burdens in Chironomus riparius exposed to arsenate and phosphate

Despite the increasing awareness of arsenic (As) contamination in surface waters worldwide, little is known about how As alone and in the presence of other chemicals affects aquatic insects. Larvae of Chironomus riparius were exposed in a laboratory investigation to factorial combinations of 0, 0.13, 2.0, 5.3, and 13μmolAsl−1 and 0, 0.15, and 15μmolPO4l−1 throughout development from first instar to pupal emergence. The time between male and female emergence increased from 1.8±0.17days to 2.9±0.34days with exposure at higher As levels. The highest As exposure also decreased the number of eggs per egg mass, which may affect population maintenance. For these parameters, there was no effect from PO4, and no interaction between As and PO4. Total As determination of larval and adult tissues was conducted using Hydride Generated Atomic Absorption Spectroscopy (HGAAS) and revealed concentrations ranging from 2.48±0.363 to 30.5±0.473μg/g and 1.03±0.286 to 8.97±0.662μg/g, respectively, indicating elimination of approximately 72% of total As body burdens between the fourth instar and adult stages. There was no effect of PO4, indicating PO4 does not alter uptake of As in C. riparius. The potential for movement of As to terrestrial systems exists, though trophic transfer may be more likely during the aquatic larval stage.

Structural changes in freshwater fish and chironomids exposed to bacterial exotoxins

Mass fish mortalities have been reported in the past decade from British waters, often coinciding with blooms of filamentous actinobacteria, particularly strains of Streptomyces griseus. The present study has shown that some fractions of the exudate of S. griseus, prepared after a series of high pressure liquid chromatography (HPLC) separations, and analysed with liquid chromatography mass spectrometry (LC–MS), induced pathological changes to the gills of carp and/or tench fry following exposure under laboratory conditions up to 96h Similar changes were induced by streptomycin, a secondary metabolite of S. griseus, and these included loss of microridging and fusion of secondary lamellae, with carp fry in the case of the exudate of S. griseus being more sensitive than tench fry, especially with exposure to fraction 9 and selected sub-fractions of 9. Some deformities using a severity index were also observed in the head capsule of larvae of the non-biting midge Chironomus riparius, including loss and splitting of teeth on the mentum. The results are discussed in relation to further identification of metabolites derived from samples of the organic filtrate of S. griseus and implications for the functioning of freshwater ecosystems.

Comparative effects of butyl benzyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP) on the aquatic larvae of Chironomus riparius based on gene expression assays related to the endocrine system, the stress response and ribosomes

In this work, the effects of butyl benzyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP), two of the most extensively used phthalates, were studied in Chironomus riparius under acute short-term treatments, to compare their relative toxicities and identify genes sensitive to exposure. The ecotoxicity of these phthalates was assessed by analysis of the alterations in gene expression profiles of selected inducible and constitutive genes related to the endocrine system, the cellular stress response and the ribosomal machinery. Fourth instar larvae, a model system in aquatic toxicology, were experimentally exposed to five increasing concentrations (0.01, 0.1, 1, 10, and 100 mg/L) of DEHP and BBP for 24 h. Gene expression was analysed by the changes in levels of transcripts, using RT-PCR techniques with specific gene probes. The exposures to DEHP or BBP were able to rapidly induce the hsp70 gene in a concentration-dependent manner, whereas the cognate form hsc70 was not altered by either of these chemicals. Transcription of ribosomal RNA as a measure of cell viability, quantified by the levels of ITS2, was not affected by DEHP, but was slightly, yet significantly, downregulated by BBP at the highest concentrations tested. Finally, as these phthalates are classified as endocrine disruptor chemicals (EDCs), their potential effect on the ecdysone endocrine system was studied by analysing the two genes, EcR and usp, of the heterodimeric ecdysone receptor complex. It was found that BBP provoked the overexpression of the EcR gene, with significant increases from exposures of 0.1 mg/L and above, while DEHP significantly decreased the activity of this gene at the highest concentration. These data are relevant as they show for the first time the ability of phthalates to interfere with endocrine marker genes in invertebrates, demonstrating their potential capacity to alter the ecdysone signalling pathway. Overall, the study clearly shows a differential gene–toxin interaction for these two phthalates and adds novel genomic tools for biomonitoring environmental xenobiotics in insects.

Evaluation of the effects of nonylphenol in fourth-instar larvae of Chironomus riparius (Diptera)

Evaluation of the effects of nonylphenol in fourth-instar larvae of Chironomus riparius (Diptera)

Larvae of the midge Chironomus riparius possess two distinct mechanisms for ionoregulation in response to ion-poor conditions

This study examined the role of the anal papillae of the freshwater (FW) chironomid larva Chironomus riparius in ionoregulation under ion-poor conditions. The scanning ion-selective electrode technique (SIET) was utilized to characterize the species, direction, and rates of inorganic ion transport by the anal papillae following acute and long-term exposure to ion-poor water (IPW). The major inorganic ions in the hemolymph of larvae treated as above were measured using standard ion-selective microelectrodes. The anal papillae of C. riparius are sites of net NaCl uptake and H(+) secretion under FW and IPW conditions and are not likely to be a major contributor of K(+) exchange. Acute and long-term exposure to IPW increased total net transport of Na(+), Cl(-), and H(+) by the anal papillae, but the mechanisms underlying the increase under the two conditions were different. Acute IPW exposure increased the magnitude of net ion fluxes at sites along the anal papillae, while long-term IPW exposure resulted in increased size of the anal papillae with no change in the magnitude of net ion fluxes. The contribution of the anal papillae to observed alterations of hemolymph ion activities upon exposure to IPW is discussed. Inhibitors of the Na(+)/H(+) exchangers (EIPA) and carbonic anhydrase (methazolamide) provide evidence for Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange mechanisms in the anal papillae. This study demonstrates that C. riparius larvae employ two different mechanisms to upregulate the total net transport of ions by the anal papillae, and these mechanisms are at least partially responsible for regulating hemolymph ion activity.