Morphological and molecular characterization of a new microsporidium, Janacekia tainanus n. sp. from the adipose tissue of Kiefferulus tainanus (Diptera: Chironomidae) in China.

We reported a new microsporidium Janacekia tainanus n. sp. from the adipose tissue of the midge Kiefferulus tainanus Kieffer, 1912 collected from a eutrophic pond in Daye city, Hubei Province, China. Infected chironomid larvae with hypertrophied adipose tissue exhibited porcelain-white. All developmental stages possessed large nuclei. The earliest stages observed were diplokaryotic meronts which were in direct contact with the host adipocyte cytoplasm. Diplokaryotic meronts developed into sporonts with the deposition of electron-dense coagulum on their surface. Multinucleate sporogonial plasmodia developed into uninucleate sporoblasts by the rosette-like division. Mature spores were oval and monokaryotic, measuring 6.14 ± 0.27 (5.65-6.67) µm long and 3.71 ± 0.12 (3.43-3.98) µm wide. Bipartite polaroplast consisted of a narrow anterior lamella and a wide posterior lamella. Isofilar polar filaments coiled 13-17 turns and arranged in one row. The exospore was thin and of no stratification, but remarkably covered with tubular secretions. The electron-lucent endospore was thick and measured 145-352 nm wide. Phylogenetic analysis based on the obtained SSU rDNA sequence indicated that the present species clustered closely with Jirovecia sinensis, a species with rod-shaped mature spores isolated from the coelomocytes of Branchiura sowerbyi. Consistent with the previous result, the monophyletic clade of Jirovecia-Bacillidium-Janacekia was sister to Pseudonosema clade and then collectively nested within Clade V of Class Aquasporidia sensu Vossbrinck and Debrunner-Vossbrinck (2005). The novel species did not form an independent monophyletic lineage with the congener, Janacekia debaisieuxi. Based on the morphological characters and ultrastructural features, as well as SSU rDNA-inferred phylogenetic relationships, a new species in the genus Janacekia, Janacekia tainanus n. sp. was designated. This is the first report of aquatic arthropod-infecting microsporidia in China.

Expression of aquaporins in response to distinct dehydration stresses that confer stress tolerance in the Antarctic midge Belgica antarctica.

Larvae of the Antarctic midge Belgica antarctica Jacobs (Diptera: Chironomidae) are highly tolerant of diverse environmental stresses, including freezing, severe desiccation, and osmotic extremes. Furthermore, dehydration confers subsequent desiccation and freeze tolerance. While a role for aquaporins-channels for water and other solutes-has been proposed in these dehydration processes, the types of aquaporins involved in dehydration-driven stress tolerance remain unknown. In the present study, we investigated expression of six aquaporins (Drip, Prip, Eglp1, Eglp2, Aqp12L, and Bib) in larvae of B. antarctica subjected to three different dehydration conditions: desiccation, cryoprotective dehydration, and osmotic dehydration. The expression of Drip and Prip was up-regulated under desiccation and cryoprotective dehydration, suggesting a role for these aquaporins in efficient water loss under these dehydration conditions. Conversely, expression of Drip and Prip was down-regulated under osmotic dehydration, suggesting that their expression is suppressed in larvae to combat dehydration. Larval water content was similarly decreased under all three dehydration conditions. Differences in responses of the aquaporins to the three forms of dehydration suggests distinct water management strategies associated with different forms of dehydration stress.

Impact of 2,4-D and fipronil on the tropical midge Chironomus sancticaroli (Diptera: Chironomidae).

Increased use of pesticides in conventional agriculture implies potential risks to the environment. In aquatic ecosystems, benthic organisms may be exposed to pesticides via contaminated water and sediment, leading to several potential cascading effects on the food web. The aim of this study was to assess the functional implications of environmental realistic concentrations of the herbicide 2,4-D and the insecticide fipronil (alone and in combination) to the native tropical chironomid Chironomus sancticaroli. These two pesticides are widely applied to different crops and have frequently been detected (together) in surface water bodies in Brazil and elsewhere. Commercial products containing fipronil (Regent® 800WG) and 2,4-D (DMA® 806BR) were evaluated in 8-day toxicity tests for their effects on larval survival, growth (body length and biomass), head capsule width, development, and mentum deformities. Fipronil decreased the larval survival at the highest test concentration and the effective concentrations (EC) after eight days of exposure were: EC10 = 0.48 µg L-1 (0.395-0.565), EC20 = 1.06 µg L-1 (0.607-1.513), and EC50 = 3.70 µg L-1 (1.664-5.736). All sublethal test concentrations of fipronil decreased the larval growth, causing reductions in biomass up to 72%. The two highest test concentrations of fipronil decreased the head capsule width and after exposure to 3.7 µg fipronil L-1, only half of the larvae reached the fourth instar. The incidence of deformities was increased by fipronil in a concentration dependent manner with an increase ranging from 23% to 75%. The highest test concentration of 2.4-D (426 µg L-1) decreased the head capsule width, but larval development was unaffected at all concentrations evaluated. In the mixture tests, antagonism was observed at lower fipronil concentrations and synergism at higher fipronil concentrations for growth. The incidence of deformities rose with increasing fipronil concentrations. The results showed that environmental realistic concentrations of fipronil may have serious ecological implications for C. sancticaroli populations and that a mixture with the herbicide 2,4-D can have synergistic effects, potentiating the risks to the aquatic ecosystem.

Cadmium-induced developmental alteration and upregulation of serine-type endopeptidase transcripts in wild freshwater populations of Chironomus plumosus.

Cadmium, a toxic heavy metal, is a persistent environmental contaminant with irreversible toxicity to aquatic organisms. Chironomus plumosus, a natural species, is the largest sediment-burrowing aquatic midge in freshwater environments. In this study, we evaluated developmental defects in C. plumosus resulting from Cd exposure. In C. plumosus larvae, Cd exposure induced decreased survival and growth rates, reduction of emergence rate and sex ratio, and delayed emergence, as well as elevating the incidence of split tooth deformities. To identify potential biomarker genes to assess environmental pollutants such as Cd, we identified differentially expressed genes (DEGs) in C. plumosus exposed to various Cd concentrations. Among fourteen characterized DEGs, serine-type endopeptidase (SP) and heat shock protein 70 (HSP70) genes exhibited significant upregulation in C. plumosus larvae after Cd exposure. Therefore, we evaluated SP and HSP70 responses in natural C. plumosus populations collected from three sites of a Korean river and analyzed their correlations with eighteen environmental quality characteristics using principal component analysis. The highest expression of SP and HSP70 transcripts was observed in C. plumosus populations from Yeosu in Korea, which has high concentrations of polluting heavy metals. SP transcript expression was positively correlated with concentrations of Cd, Pb, Al, Fe, NO2, and NO3. These results suggested that environmental pollutants such as Cd can impair proteolytic activity in the digestive system of C. plumosus and may ultimately induce developmental alterations. We therefore suggest SP as a potential biomarker to assess the effects of environmental pollutants in aquatic ecosystems.

Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect.

Rapid cold hardening (RCH) is a type of beneficial phenotypic plasticity that occurs on extremely short time scales (minutes to hours) to enhance insects' ability to cope with cold snaps and diurnal temperature fluctuations. RCH has a well-established role in extending lower lethal limits, but its ability to prevent sublethal cold injury has received less attention. The Antarctic midge, Belgica antarctica, is Antarctica's only endemic insect and has a well-studied RCH response that extends freeze tolerance in laboratory conditions. However, the discriminating temperatures used in previous studies of RCH are far below those ever experienced in the field. Here, we tested the hypothesis that RCH protects against non-lethal freezing injury. Larvae of B. antarctica were exposed to control (2°C), direct freezing (-9°C for 24 h) or RCH (-5°C for 2 h followed by -9°C for 24 h). All larvae survived both freezing treatments, but RCH larvae recovered more quickly from freezing stress and had a significantly higher metabolic rate during recovery. RCH larvae also sustained less damage to fat body and midgut tissue and had lower expression of two heat shock protein transcripts (hsp60 and hsp90), which is consistent with RCH protecting against protein denaturation. The protection afforded by RCH resulted in energy savings; directly frozen larvae experienced a significant depletion in glycogen energy stores that was not observed in RCH larvae. Together, these results provide strong evidence that RCH protects against a variety of sublethal freezing injuries and allows insects to rapidly fine-tune their performance in thermally variable environments.

Bisphenol P activates hormonal genes and introduces developmental outcomes in Chironomus tentans.

The endocrine disrupting properties of bisphenol A (BPA) discharged to the environment have been newly identified by the European Chemicals Agency, increasing the need to assess the environmental endocrine disrupting potentials of its alternatives with which it shares close structural features. However, few investigations of the environmental endocrine disrupting functions of BPA analogs have been conducted to date. In this study, the endocrine disrupting effects of a BPA analog of bisphenol P (BPP) were investigated in the nonbiting midge (Chironomus tentans), a model organism in ecotoxicology. An initial ex vivo test using salivary gland cells explanted from the larvae and a subsequent in vivo test using embryos and larvae revealed the upregulatory effects of BPP on ecdysone receptor genes encoding the ecdysone receptor (EcR) and the early responsive gene E74, with a similar temporal pattern of gene activation. Partial life cycle and full life cycle toxicity tests demonstrated BPP altered embryo hatching, larval emergence, and adult sex ratio at concentrations close to the effective concentrations for hormonal genetic endpoints in embryos and larvae after 48 h of exposure. Although embryos appeared to be more sensitive to BPP than the fourth instar larvae, the impact on neither life stage seemed enough to estimate the developmental impairment of the insects. These results demonstrate the ecdysone pathway is a target of BPP, and that long-term exposure could cause apical effects on the development of C. tentans. The endocrine disrupting effects towards aquatic organisms, as well as the high persistence and bioconcentration potential, indicate an urgent need to assess the environmental risks associated with BPP.

Contribution of trace metallic elements to weakly contaminated lacustrine sediments: effects on benthic and pelagic organisms through multi-species laboratory bioassays.

Surficial sediments exhibit higher levels of contamination than overlying water, especially from persistent contaminants such as trace metallic elements (TMEs). While sediments could in turn act as sources of contamination for the water column, their ecotoxicology is yet rarely assessed in a multi-compartments perspective. This study aims at assessing the response of benthic and pelagic organisms exposed to weakly contaminated sediments using a multi-species laboratory assay by focusing on TMEs (Cd, Cr, Cu, Ni, Pb, and Zn) contamination. Chironomus riparius larvae, Daphnia magna, and Lemna minor were simultaneously exposed for 10 days to six sediments sampled from the littoral of a large French lake (Lake Bourget). The endpoints consisted in the survival and growth rates and the bioconcentration factor (BCF). Significant negative relationships between sediment TME concentrations and survival rates of C. riparius and growth rates of C. riparius and D. magna suggested that both benthic and pelagic macro-invertebrates were impacted by sediment contamination, which was not observed in L. minor. Significant relationships of the sediment with the internal TME concentrations were positive while negative with the BCFs, suggesting an increase in biological regulation processes in all organisms with the increase of sediment TME concentrations. These results underline the importance of including both benthic and pelagic organisms in ecotoxicological assessment of low contaminated sediments and the relevance of the relationship BCFs/sediment contamination as prior biomarkers than higher life history traits.

Effects of phenol on glutathione S-transferase expression and enzyme activity in Chironomus kiiensis larvae.

Detoxifying enzyme mRNAs are potentially useful stress biomarkers. Glutathione S-transferase (GST) metabolises lipophilic organic contaminants and mitigates oxidative damage caused by environmental pollutants. Herein, 12 Chironomus kiiensis GSTs (CkGSTs1-6, CkGSTt1-2, CkGSTd1-2, CkGSTm1-2) were cloned and grouped into sigma, theta, delta and microsomal subclasses. Open reading frames (450-699 bp) encode 170-232 amino acid proteins with predicted molecular masses of 17.31-26.84 kDa and isoelectric points from 4.94 to 9.58. All 12 GSTs were expressed during all tested developmental stages, and 11 displayed higher expression in fourth-instar larvae than eggs. GST activity after 24 h of phenol exposure was used to estimate environmental phenol contamination. After exposure to sublethal concentrations of phenol for 48 h, expression and activity of CkGSTs were inhibited in C. kiiensis larvae. Expression of CkGSTd1-2 and CkGSTs1-2 varied with phenol concentration, indicating potential use as biomarkers for monitoring environmental phenol contamination.

Chironomus sancticaroli (Diptera, Chironomidae) as a Sensitive Test Species: Can We Rely on Its Use After Repeated Generations, Under Laboratory Conditions?

In ecotoxicological assays, previously selected and standardized organism tests are exposed to an environmental sample. Some species of the Chironomus genus have been extensively used in ecotoxicological assays. Among these, Chironomus tentans is usually utilized in the USA and Chironomus sancticaroli in Brazil. We conducted ecotoxicological bioassays to compare a population of C. sancticaroli, kept for 6 years under laboratory conditions, with a sylvatic population of the same species, collected in the field. The aim was to test the hypothesis that populations of C. sancticaroli, maintained in the laboratory for long periods, could have a different response to stressors/substances. We analyzed the responses of C. sancticaroli for potassium chloride, zinc chloride, potassium dichromate, linear alkylbenzene sulphonate (LAS) and caffeine. The results showed no significant differences between the two populations in the analyses and seems to indicate the possible use of C. sancticaroli from populations kept in the laboratory for long periods.

The genomic footprint of climate adaptation in Chironomus riparius.

The gradual heterogeneity of climatic factors poses varying selection pressures across geographic distances that leave signatures of clinal variation in the genome. Separating signatures of clinal adaptation from signatures of other evolutionary forces, such as demographic processes, genetic drift and adaptation, to nonclinal conditions of the immediate local environment is a major challenge. Here, we examine climate adaptation in five natural populations of the harlequin fly Chironomus riparius sampled along a climatic gradient across Europe. Our study integrates experimental data, individual genome resequencing, Pool-Seq data and population genetic modelling. Common-garden experiments revealed significantly different population growth rates at test temperatures corresponding to the population origin along the climate gradient, suggesting thermal adaptation on the phenotypic level. Based on a population genomic analysis, we derived empirical estimates of historical demography and migration. We used an FST outlier approach to infer positive selection across the climate gradient, in combination with an environmental association analysis. In total, we identified 162 candidate genes as genomic basis of climate adaptation. Enriched functions among these candidate genes involved the apoptotic process and molecular response to heat, as well as functions identified in studies of climate adaptation in other insects. Our results show that local climate conditions impose strong selection pressures and lead to genomic adaptation despite strong gene flow. Moreover, these results imply that selection to different climatic conditions seems to converge on a functional level, at least between different insect species.

Lethal and sublethal effects of metal-polluted sediments on Chironomus sancticaroli Strixino and Strixino, 1981.

The Cantareira Complex is one of the most important water supplies of the metropolitan region of São Paulo, Brazil. Previously, it was demonstrated that the sediments in this complex were polluted with metals and that Paiva Castro Reservoir-the last reservoir in the sequence, which receives water from the five previous reservoirs-was the reservoir with the greatest concentration of pollutants. Based on field data, it was noticed that copper concentrations in sediments were related to morphological alterations in chironomids. The present study provides novel monitoring methods and results for the complex by isolating the environmental and biological sources of variation. An adaptation of the in situ assay proposed by Soares et al. (Arch Environ Contam Toxicol 49:163-172, 2005), which uses a native tropical Chironomus species and low-cost materials, is also provided. The aim of this study was to isolate the effects of sediments from Paiva Castro on controlled populations of C. sancticaroli larvae using an in situ assay. A seven-day experiment was performed in triplicate. Third instar larvae were inoculated in chambers containing sediments from two distinct regions of Paiva Castro reservoir and a control site with sand. Five biological responses were considered: mouthpart alterations, larval length, width of cephalic capsule, mortality and total damage. The results suggest the effects of sediment toxicity on larvae include a reduction in length and a higher occurrence of total damage.

Combined effects of insecticide exposure and predation risk on freshwater detritivores.

Insecticides usually present in low concentrations in streams are known to impair behaviour and development of non-target freshwater invertebrates. Moreover, there is growing awareness that the presence of natural stressors, such as predation risk may magnify the negative effects of pesticides. This is because perception of predation risk can by itself lead to changes on behaviour and physiology of prey species. To evaluate the potential combined effects of both stressors on freshwater detritivores we studied the behavioural and developmental responses of Chironomus riparius to chlorantraniliprole (CAP) exposure under predation risk. Also, we tested whether the presence of a shredder species would alter collector responses under stress. Trials were conducted using a simplified trophic chain: Alnus glutinosa leaves as food resource, the shredder Sericostoma vittatum and the collector C. riparius. CAP toxicity was thus tested under two conditions, presence/absence of the dragonfly predator Cordulegaster boltonii. CAP exposure decreased leaf decomposition. Despite the lack of significance for interactive effects, predation risk marginally modified shredder effect on leaf decomposition, decreasing this ecosystem process. Shredders presence increased leaf decomposition, but impaired chironomids performance, suggesting interspecific competition rather than facilitation. C. riparius growth rate was decreased independently by CAP exposure, presence of predator and shredder species. A marginal interaction between CAP and predation risk was observed regarding chironomids development. To better understand the effects of chemical pollution to natural freshwater populations, natural stressors and species interactions must be taken into consideration, since both vertical and horizontal species interactions play their role on response to stress.

Cytochrome P450 CYP6EV11 in Chironomus kiiensis Larvae Involved in Phenol Stress.

Phenol is one of the organic pollutants which can cause water environment pollution. It is not only enriched in aquatic organisms but is also a serious threat to human health. Chironomus kiiensis is very sensitive to the contaminants in water and its cytochrome P450s are usually chosen as biomarkers for water pollution. To examine whether CYP6EV11 plays a role in the oxidative metabolism of phenol, we measured the silencing efficiency of CYP6EV11 and evaluated larval susceptibility to sublethal phenol levels by RNA interference (RNAi) technology. The results showed that the transcription of CYP6EV11 was found significantly up-regulated when the 4th instar C.kiiensis larvae were exposed to three doses of phenol. However, the transcriptional levels of CYP6EV11 were significantly suppressed by 92.7% in the 4th instar C. kiiensis larvae soaked in dsCYP6EV11 compared with those soaked in dsGFP for 6 h. The CYP6EV11 expression and mortality of the 4th instar C. kiiensis larvae with CYP6EV11 silencing were mostly decreased under phenol stress. Therefore, the CYP6EV11 gene may be used as a molecular biomarker for earlier warning and monitoring for water pollution.

A multi-year study following BACI design reveals no short-term impact of Bti on chironomids (Diptera) in a floodplain in Eastern Austria.

Short-term impacts of aerial application of Bacillus thuringiensis israelensis (Bti) on Culicidae and Chironomidae were investigated over several years in temporary waters of the Dyje and Morava floodplains in Eastern Austria. The sampling followed a Before-After-Control-Impact (BACI) approach with sampling dates immediately before and shortly after the application and was repeated for 3 years. To test for effects of the Bti treatment on the two Diptera families, linear mixed-effects models were used. Data analysis included the factors Before-After and Control-Impact as fixed effects, while general temporal and spatial variables were random effects. One hundred sixteen taxa of chironomids were identified. Abundance varied between 2 and 1125 larvae per m2, while culicid densities reached values of several 100 ind. per liter. Total culicid abundance significantly decreased after the Bti treatment, whereas no significant effects were found on the abundance of total chironomids and dominant chironomid subfamilies, tribes, and genera, on relative proportions of chironomid feeding guilds, diversity, and species composition. Further studies from this area are needed to extend the investigation over a period of several weeks in order to reveal possible delayed effects of the larvicide application.

Habitat modification by invasive crayfish can facilitate its growth through enhanced food accessibility.

BACKGROUND: Invasive ecosystem engineers can facilitate their invasions by modifying the physical environment to improve their own performance, but this positive feedback process has rarely been tested empirically except in sessile organisms. The invasive crayfish Procambarus clarkii is an ecosystem engineer that destroys aquatic macrophytes, which provide a physical refuge for animal prey, and this destruction is likely to enhance vulnerability to predators. Using two series of mesocosm experiments, we tested the hypothesis that the invasive crayfish increases its feeding efficiency on animal prey by reducing submerged macrophytes, thus increasing its individual growth rate in a positive density-dependent manner. RESULTS: In the first experiment, increasing crayfish density reduced both macrophytes and animal prey (dragonfly and chironomid larvae) and, importantly, increased the growth rate of individual crayfish, in accordance with our expectation. In the second experiment, we used artificial macrophytes to clarify whether the physical architecture of macrophytes itself protects animal prey and limits crayfish growth rate. Increasing the artificial macrophyte quantity not only increased the survival of animal prey, but also retarded the crayfish growth rate. CONCLUSIONS: We conclude that macrophytes strengthen bottom-up control of crayfish, but this effect can be relaxed by increasing the density of crayfish via reduction in macrophytes. This positive feedback process may explain the crayfish outbreaks and regime shifts occasionally observed in invaded freshwater ecosystems.

Assessment of thiamethoxam toxicity to Chironomus riparius.

The insecticide thiamethoxam (TMX) is a systemic neonicotinoid widely used for pest control in several agricultural crops. TMX mimics the action of acetylcholine causing uncontrolled muscular contraction eventually leading to insect death. TMX is being found in freshwater ecosystems at concentrations of up to 225µg/L. Still, chronic toxicity data for freshwater invertebrates is limited. Therefore, the aim of this study was to evaluate the acute and chronic effects (at organismal and biochemical levels) of TMX on the freshwater insect Chironomus riparius. C. riparius life history responses were significantly affected by TMX exposure, namely with a decrease in growth and delay in emergence. Concerning the biochemical responses, after a short exposure (48h) to TMX, our results showed that low concentrations of TMX significantly reduced CAT activity and LPO levels of C. riparius. No effects were observed in AChE, GST and ETS activities. Effects in terms of survival, development rates and biochemical responses of C. riparius exposed to low concentrations of TMX observed in this study suggest potential deleterious effects of this neonicotinoid on aquatic insects inhabiting freshwaters environments near agricultural areas.

Field assessment of reproduction-related traits of chironomids using a newly developed emergence platform (E-Board).

Further progress in the development of reliable biomonitoring strategies requires to better link effects in aquatic ecological systems to ambient concentrations of chemical contaminants. Among existing tools, in situ bioassays using caging method represent an interesting way to achieve this challenge. However, elaboration of adapted exposure chambers and suitable operating procedures is still required, particularly to assess ecological relevant traits such as those related to the reproduction. In such context, we developed a new device (Emergence board - E-Board) which allows assessing in rivers the development of the Chironomus riparius species from the early fourth instar larvae to the adult stage. The system acts as a suspended matter trap floating in the subsurface of the water equipped of an emergence trap for catching adults. The system was tested in actual field conditions. Its easy handling allowed obtaining data which demonstrated its applicability for assessing the development of the chironomids. Moreover, by adapting energy-based models (DEB) specifically developed in the laboratory for the species C. riparius, we were able to predict the growth pattern and the emergence of chironomids in real environmental conditions. The E-Board represents thus a promising new in situ tool in perspective of evaluation of the quality of the ecosystems.

FLUID AND ION SECRETION BY MALPIGHIAN TUBULES OF LARVAL CHIRONOMIDS, Chironomus riparius: EFFECTS OF REARING SALINITY, TRANSPORT INHIBITORS, AND SEROTONIN.

Larvae of Chironomus riparius respond to ion-poor and brackish water (IPW, BW) conditions by activating ion uptake mechanisms in the anal papillae and reducing ion absorption at the rectum, respectively. The role that the Malpighian tubules play in ion and osmoregulation under these conditions is not known in this species. This study examines rates of fluid secretion and major cation composition of secreted fluid from tubules of C. riparius reared in IPW, freshwater (FW) and BW. Fluid secretion of tubules from FW and BW larvae was similar but tubules from IPW larvae secrete fluid at higher rates, are more sensitive to serotonin stimulation, and the secreted fluid contains less Na(+) . Therefore in IPW, tubules work in concert with anal papillae to eliminate excess water while conserving Na(+) in the hemolymph. Tubules do not appear to play a significant role in ion/osmoregulation under BW. Serotonin immunoreactivity in the nervous system and gastrointestinal tract of larval C. riparius was similar to that seen in mosquito larvae with the exception that the hindgut was devoid of staining. Hemolymph serotonin titer was similar in FW and IPW; hence, serotonin is not responsible for the observed high rates of fluid secretion in IPW. Instead, it is suggested that serotonin may work in a synergistic manner with an unidentified hormonal factor in IPW. Ion transport mechanisms in the tubules of C. riparius are pharmacologically similar to those of other insects.

Morphological identification and COI barcodes of adult flies help determine species identities of chironomid larvae (Diptera, Chironomidae).

Establishing reliable methods for the identification of benthic chironomid communities is important due to their significant contribution to biomass, ecology and the aquatic food web. Immature larval specimens are more difficult to identify to species level by traditional morphological methods than their fully developed adult counterparts, and few keys are available to identify the larval species. In order to develop molecular criteria to identify species of chironomid larvae, larval and adult chironomids from Western Lake Erie were subjected to both molecular and morphological taxonomic analysis. Mitochondrial cytochrome c oxidase I (COI) barcode sequences of 33 adults that were identified to species level by morphological methods were grouped with COI sequences of 189 larvae in a neighbor-joining taxon-ID tree. Most of these larvae could be identified only to genus level by morphological taxonomy (only 22 of the 189 sequenced larvae could be identified to species level). The taxon-ID tree of larval sequences had 45 operational taxonomic units (OTUs, defined as clusters with >97% identity or individual sequences differing from nearest neighbors by >3%; supported by analysis of all larval pairwise differences), of which seven could be identified to species or 'species group' level by larval morphology. Reference sequences from the GenBank and BOLD databases assigned six larval OTUs with presumptive species level identifications and confirmed one previously assigned species level identification. Sequences from morphologically identified adults in the present study grouped with and further classified the identity of 13 larval OTUs. The use of morphological identification and subsequent DNA barcoding of adult chironomids proved to be beneficial in revealing possible species level identifications of larval specimens. Sequence data from this study also contribute to currently inadequate public databases relevant to the Great Lakes region, while the neighbor-joining analysis reported here describes the application and confirmation of a useful tool that can accelerate identification and bioassessment of chironomid communities.

Changes in carbon sources fueling benthic secondary production over depth and time: coupling Chironomidae stable carbon isotopes to larval abundance.

Stable C isotope ratio (δ(13)C) values of chironomid remains (head capsules; HC) were used to infer changes in benthic C sources over the last 150 years for two French sub-Alpine lakes. The HCs were retrieved from a series of sediment cores from different depths. The HC δ(13)C values started to decrease with the onset of eutrophication. The HC δ(13)C temporal patterns varied among depths, which revealed spatial differences in the contribution of methanotrophic bacteria to the benthic secondary production. The estimates of the methane (CH4)-derived C contribution to chironomid biomass ranged from a few percent prior to the 1930s to up to 30 % in recent times. The chironomid fluxes increased concomitantly with changes in HC δ(13)C values before a drastic decrease due to the development of hypoxic conditions. The hypoxia reinforced the implication for CH4-derived C transfer to chironomid production. In Lake Annecy, the HC δ(13)C values were negatively correlated to total organic C (TOC) content in the sediment (Corg), whereas no relationship was found in Lake Bourget. In Lake Bourget, chironomid abundances reached their maximum with TOC contents between 1 and 1.5 % Corg, which could constitute a threshold for change in chironomid abundance and consequently for the integration of CH4-derived C into the lake food webs. Our results indicated that the CH4-derived C contribution to the benthic food webs occurred at different depths in these two large, deep lakes (deep waters and sublittoral zone), and that the trophic transfer of this C was promoted in sublittoral zones where O2 gradients were dynamic.