External Structures of the Antennae and Mouth Parts of the Fourth Instar of Chironomus flaviplumus and Chironomus salinarius (Diptera: Chironomidae)

Chironomidae is usually the most abundant macroinvetebrate in freshwater. They form an important part of the foodweb, and also have long been known as indicators of water quality. However, there is a great deal of difficulty in its identification. Such is due to the complexity of larval taxonomy. The external fine structures of the mouth parts and antennae of 4th instar larva C. flaviplumus and C. salinarius were examined using scanning electron microscopy (SEM). The antennae of the fourth instar larvae of C. flaviplumus and C. salinarius are short and composed of five segments. They consist of a scape, pedical, and three flagellar subsegments. The maxilla palps have 10 sensilla basiconica on its distal surface. They show the three morphological groups in the following numbers: six apical sensillae, three medial sensillae, and one lateral sensillae. The mandibles have strong apical and lateral teeth. These sensory structures are situated on the head capsule, each of which are represented with images to elucidate the identification of chironomidae.     

Ecological synopsis for Chironomus flaviplumus (Diptera: Chironomidae) in the Jung-rang stream

In this study, we conducted field sampling to assess the relative influences of water and substrate quality on benthic macroinvertebrate communities living in the Jung‐rang stream, Korea. We collected macroinvertebrates and assessed physicochemical variables from three sites in the stream between May 2001 and January 2002. Sites were located approximately within 20 km from the headwater. The structure of the benthic macroinvertebrate communities may be strongly affected by the physical conditions inherent to the environment in which they live. In this stream, we detected profound differences in water temperature (18 ～ 19.75°C), the concentrations of suspended solids (3.935 ～ 7.87 mg/L), and demand for chemical oxygen (10.575 ～ 14.425 mg/L). Nonylphenol concentrations ranging from 0.375 to 0.55 ng/mL were found in the water, and the sediments were measured to contain between 2.45 and 3.425 ng/mL. We identified a total of 20 macroinvertebrate species, including seven species of Chironomidae, the most abundant of which was Chironomus flaviplumus. At none of the sites did we find any significant differences in the structure of the communities. Using canonical correspondence analysis for the relation of species and environmental variables, chemical oxygen demand and suspended sediment gradients (SS) had significant preferences for site 1 with SS. The results of our study suggest that physico‐chemical variables exerted complex effects on the structure of the benthic community in the Jung‐rang stream. This study supports the contention that physico‐chemical analyses as well as community analysis are valuable tools to assess the effect of pollution on the ecological condition of a stream. Chironomids, in particular, showed a high degree of tolerance against contaminants.     

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

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

Succession of Chironomus Meigen Species (Diptera,Chironomidae) in Newly Filled Ponds

The colonization by Chironomus spp. of two ponds filled after a dry period was investigated. Field data were collected by means of quantitative benthic samples and emergence traps. C. melanescens. C. annularius, C. riparius, C. piger, C. luridus, C. obtusidens, C. plumosus, C. dorsalis and Camptochironomus tentans were determined cytotaxonomically in ponds investigated. C. melanescens and C. annularius appear to be the most characteristic species colonizing in high numbers in newly filled ponds. Larvae of C. annularius grew more slowly in ponds compared with the former one which resulted in a characteristic succession of both larval types. Most species mentioned disappeared completely from the ponds investigated after the metamorphosis of a single cohort. They were replaced by C. plumosus which was often attended by C. annularius in smaller ponds.     

Head capsule deformities in Chironomus spp. (Diptera: Chironomidae) as indicator of environmental stress in Sebeta River,Ethiopia

Urbanization and industrialization have placed most of the aquatic environments in developing countries at great risk. The absence of reliable biological monitoring programmes further complicates the situation. In this study, an attempt was made to assess incidence of deformities among Chironomus spp. response to the level of environmental degradation. For this purpose, chironomid larvae were sampled from Sebeta River (November, 2012) using Surber sampler and/or D‐frame dip net. Moreover, physico‐chemical analysis of the water and sediment heavy metal analysis were carried out at three study sites. The results indicate that most physico‐chemical variables were higher at the downstream sites (P < 0.05), together with high levels of Cr, Zn and Cu, which was attributed to untreated effluents that originated from garment and tannery industries. Several forms of deformities (mentum gap, notch, tooth missing and breakage) were encountered, and the majority occurred on mentum followed by pecten epipharynx. Taking 8% as a cut‐off value for normal deformities and the moderate rate of deformities detected (16.62% at Sb‐2), it is possible to conclude that the ecological state of Sebeta River is at critical condition. Therefore, corrective measures like designing of effective treatment plants and timely set environmental impact assessment must be in place prior to any developmental activity.     

Seasonal influence on head capsule deformities in Chironomus zealandicus (Hudson) (Diptera: Chironomidae)

The natural incidence of deformities in the head capsules of Chironomus zealandicus was investigated at four lake sites in the central North Island (New Zealand) in summer (December) of 1994, 1995 and 1996, and winter (June) of 1995 and 1996. Significant differences were observed in deformities between sites and seasons. Individuals from Hamurana Stream, a relatively clean site, had the lowest incidence of deformities. However, there were still significant numbers of deformed chironomids. The incidence of deformity increased in summer in larvae from all sites except Hamurana Stream. No seasonal differences were observed in larvae from Hamurana Stream. There are indications that substrate type, season and genetic factors, as well as sediment chemistry may have collectively contributed to the incidence of head capsule abnormalities in C. zealandicus.     

Chromosomal variability in natural populations of Chironomus cingulatus meigen (Diptera, Chironomidae)

Chromosomal polymorphism has been studied in seven natural populations of Chironomus cingulatus from Western Europe, Western Siberia, and the Republic of Sakha (Yakutia). The banding sequences pool of the species includes 15 banding sequences. Chromosomal polymorphism was revealed in five out of seven chromosomal arms. Arm B is the most polymorphic with four banding sequences. There are three banding sequences in arm A. Arms D, E, and G have two banding sequences. None of the chromosome rearrangements were revealed in arms C and F. The populations of C. cingulatus differ clearly in their number and frequency of banding sequences, which indicates that different gene sequences are adaptive in different populations.     

广东省侵染摇蚊的库蚊虫霉研究

在中国首次报道了侵染摇蚊的库蚊虫霉(Entomophtora culichs)的形态特征、侵染症状以及发生动态等。库蚊虫霉初生分生孢子,钟罩形,顶部有一明显小尖突,(14.6±1.5)μm×(10.8±1.2)μm。次生分生孢子形似初生分生孢子,但略小,(11.0±1.1)μm×(8.0±0.5)μm。休眠孢子,球形至亚球形,直径(29.3±1.9)μm。被侵染摇蚊尸体以假根附着于基物上,通体覆盖着绿色的真菌子实层。库蚊虫霉侵染率在不同植物上明显不同。库蚊虫霉侵染率随季节不同波动很大,春季最高达90.2%,其次为秋季和冬季,夏季最低为0。2006—2009年库蚊虫霉侵染率为93.5%～95.7%,年际之间变动不大。     

The role of glycogen during the ontogenesis of Chironomus anthracinus (Chironomidae, Diptera)

Large fluctuations in glycogen content were found in larvae, pupae and adults of Chironomus anthracinus (Zetterstedt) from the profundal zone of Lake Esrom, Denmark. In 2nd, 3rd and 4th instar larvae the glycogen concentration (expressed as percentage of dry weight) increased during periods of aerobic conditions to a maximum of 25%, but decreased in periods of hypoxia longer than two months to 10–12% in young larvae. A further decrease to about 5% took place, when moulting from 2nd to 3rd or from 3rd to 4th instar occurred after overturn. Prior to pupation the glycogen concentration was restored to 26–28%. The glycogen concentration approximated 22% in young pupae, but decreased during the pupal stage and newly hatched adults contained 12–15%. Finally, the glycogen store of both males and females was further reduced during the swarming period. Thus, glycogen seems to be an important energy source (1) during periods with hypoxic conditions, (2) during periods with high internal energy requirement such as ecdyses and metamorphosis, and (3) during the non-feeding adult life stage.     

Chironomus species (Diptera: Chironomidae) in the profundal benthos of Spanish reservoirs and lakes: factors affecting distribution patterns

• 1 The taxonomy, distribution and habitat preferences of Chironomus larvae from profundal benthos of reservoirs and natural lakes in Spain are presented. The 114 water bodies studied were medium to large reservoirs and lakes throughout Spain, and were visited on at least two occasions (summer and winter). Out of the natural lakes investigated, Chironomus larvae were found only in one karstic lake (Banyoles), and to date, these have not been found in the high mountain lakes of the Spanish Pyrenees or Sierra Nevada, or in Spain's largest oligotrophic lake (Sanabria).
• 2 Multiple regression with environmental data from reservoirs showed that the density of Chironomus larvae was inversely correlated with water depth, temperature and sulphide content of the bottom water, but positively correlated with alkalinity and particulate organic nitrogen (PON) content of bottom water. Thus, the density of Chironomus larvae was higher in shallow reservoirs from the eastern part of Spain (calcareous water and higher PON), but markedly lower in southern reservoirs (higher temperature and higher sulphide).
• 3 According to morphological and cytological characteristics, three species of this genus have been identified in the samples examined: C. bernensis, C. plumosus and C. nuditarsis. Reasons for the absence of other widespread European species are discussed.
• 4 Using canonical correspondence analysis to relate species and environmental variables, C. bernensis showed a significant preference for reservoirs with higher oxygen concentrations in bottom water and lower total reactive phosphorus (TRP) content at the surface than those reservoirs where C. plumosus‐type were found. This suggests that the smaller C. bernensis prefers mesotrophic reservoirs and the larger C. plumosus‐type (C. plumosus + C. nuditarsis) prefers eutrophic reservoirs. Taking into account the TRP/depth index for those samples collected at depths below the thermocline, and thus, where oxygen can be a limiting factor, C. bernensis and C. nuditarsis were present in reservoirs in which the index was low (< 1:170 for C. bernensis and >1:800 for C. nuditarsis), while C. plumosus thrived in reservoirs with higher index values (<1:20). These differences may reflect the preference of C. bernensis and C. nuditarsis for detrital material, while C. plumosus prefers phytoplankton. These observations are in agreement with the depth distribution of each species: C. plumosus was restricted to reservoirs less than 46 m deep, whereas the other two species (i.e. C. bernensis and C. nuditarsis) were found up to depths of 65 and 80 m, respectively.
• 5 Data from a transect (5–20 m deep) in Lake Banyoles are consistent with the general pattern described in reservoirs. In this lake, a high density of C. bernensis was recorded from littoral and sublittoral areas with abundant oxygen (above or close to the thermocline), whereas C. plumosus was scarce in several parts of the lake, especially in oxygen‐deficient areas during the summer. This karstic, oligotrophic lake has a low TRP/depth index, and therefore, we expected C. bernensis to be dominant in the littoral, as is the case. Nevertheless, the severe depletion of oxygen caused by meromixis, and the high hydrogen sulphide concentration benefit C. plumosus in deeper zones. Chironomus nuditarsis was scarce in Lake Banyoles and only found at considerable depths.

     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Site specific differentiation in metal tolerance in the midge Chironomus riparius (Diptera,Chironomidae)

Metal tolerance in Chironomus riparius (Diptera) populations from contaminated streams was studied by comparing the effects of cadmium, zinc and iron on first generation laboratory reared midges. First instar larvae were exposed for four days, after which surviving larvae were counted and their length measured. Larvae from two highly polluted sites, kept under control conditions, grew substantially slower than those from other populations. All populations showed the same growth responses to increased zinc concentrations, but differences were found in the responses to both cadmium and iron. Since population differentiation was demonstrated in first generation laboratory animals, it is suggested that the differences between populations of C. riparius have a genetic basis.     

Effect of cadmium exposure on the globin protein expression in 4th instar larvae of Chironomus riparius Mg. (Diptera: Chironomidae): An ecotoxicoproteomics approach

In order to identify Chironomus hemoglobin (Hb) as a biomarker of ecotoxicity monitoring; herein, the effects of cadmium chloride (Cd) on Hb parameters were investigated in the 4th instar larvae of Chironomus riparius. The expressions of globin mRNA and hemolymph protein, using ecotoxicoproteomic approach, were investigated. Conventional ecotoxicity tests were also conducted to validate the ecotoxicological relevance of the response of Chironomus Hb as a biomarker. The proteomic analysis indicated that exposure to Cd lead alteration in the expression of hemolymph protein, with the total expressions of 12 hemolymph protein spots decreasing in response to treatment, with that of two increasing in response to Cd exposure. In addition, all of the spots differentially expressed in response to Cd treatment were identified as globin proteins. The decreased total Hb content observed in the hemolymph of larvae exposed to Cd suggested that the decreased expression of selected globin proteins in response to Cd exposure impacted on Hb synthesis. The overall results suggested that Hb could be a target molecule for exposure to Cd in C. riparius, with a proteomic approach appearing to be an ideal tool for the discovery of biomarkers in ecotoxicological research.     

Seasonal Dynamics of a Chironomus plumosus (L.) (Diptera,Chironomidae) Population from a Fish Pond in Southern Bohemia

A population of Chironomus plumosus (L.) from a fish pond has been investigated for two years. Quantitative benthic samples and emergence traps were used to study the seasonal dynamics. Effect of some factors on the duration of development was studied in laboratory experiments. Data on larval abundance, length frequency analysis and emergence of adults suggest a bivoltine life cycle. The population of C. plumosus investigated showed a pronounced tendency to dormancy, even under long-day conditions. Factors regulating the bivoltine life cycle are discussed.     

Nonrandom chromosomal distribution of spontaneous breakpoints and satellite DNA clusters in two geographically distant populations of Chironomus riparius (Diptera: Chironomidae)

Two geographically distant populations of Chironomus riparius (syn. C. thummi) from two environmentally polluted sites (Santena, Italy and Varna, Bulgaria) show numerous somatic and inherited chromosomal aberrations (inversions, deletions and deficiencies). Fifty-five percent of the observed breakpoints occurred in at least two larvae from both populations. Breakpoints occurring twice or more were considered as common structural chromosomal breakpoints. We tested whether such common breakpoints in larvae of the two polluted populations had a random chromosomal distribution or occurred preferentially in specific heterochromatic regions. Distribution of common breakpoints was not random, and proximal regions of first and third chromosome had significantly more common breakpoints than distal ones. By FISH we identified and mapped 56 chromosomal sections containing clusters of two tandem-repetitive satellite DNA families called Hinf and Alu elements. Like the common breakpoints, these repetitive DNA clusters appeared to be significantly more abundant in regions of constitutive heterochromatin such as the pericentromeric regions, while in distal sections of chromosomal arms they were rare or absent. Twenty-four out of 45 common breakpoints (i.e., 53.3%) occurred in cytogenetic sections where Alu and Hinf satellite DNA probes hybridized. The frequency of co-localization between common breakpoints and repetitive DNA hybridization signals was significantly higher than expected by chance. We hypothesize that spontaneous or induced breaks occur more frequently in sections containing blocks of repetitive DNA.     

A new species of Chironomus Meigen, 1803 (Diptera,Chironomidae) from the southeast of Brazil

The article describes the larva, pupa, adults and karyotype of Chironomus reissi, sp. n., which live on leaf detritus beds of small temporary waters in the southeast of Brazil. The species belongs to the pseudothummi cytocomplex with the chromosome arm combination AE, BF, CD, G.     

The acute toxicity of cadmium to different larval stages of Chironomus riparius (Diptera: Chironomidae) and its ecological significance for pollution regulation

Summary Acute toxicity tests using a static-with-replacement testing procedure were carried out with the four larval instars of the freshwater detritivore Chironomus riparius (Meigen). Median lethal concentrations (10, 24, 48 and 96 h LC50's) indicated great differences in sensitivity to cadmium between instars. Larvae became more tolerant with increasing age, the most resistant stage (fourth instar) having a 24 h LC50 of 2,400 mg Cd l^-1, approximately 950 times greater than the corresponding value of 2.1 mg Cd l^-1 recorded for the most sensitive (first instar) stage. These observations are discussed in relation to the establishment of water quality standards.     

Two-dimensional gel electrophoresis of proteins for genetic studies in douglas fir (Pseudotsuga menziesii)

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100°C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.This work was supported in part by Grant ATP PIRDES 508 444 from the INRA-CNRS.     

Response of larval Chironomus tepperi (Diptera: Chironomidae) to individual Bacillus thuringiensis var. israelensis toxins and toxin mixtures

The biopesticide Bacillus thuringiensis israelensis (B.t.i.) is highly toxic to the larvae of Chironomus tepperi, an important pest of aerially sown rice in southern Australia. In this study, all of the known Cry genes and the Cyt1A gene from B.t.i. were expressed and tested for individual toxicity against fourth instar C. tepperi larvae. Possible synergism between toxins in two component mixtures involving all toxins except Cry10A was also evaluated. Of the Cry toxins, only Cry11A and Cry4B displayed substantial toxicity; however, both were 10- to 20-fold less toxic than the parental B.t.i. strain. The only detected synergy was between the mildly toxic Cry4A and Cyt1A toxins. In direct contrast to previous studies with mosquitoes, mixtures of Cry11A/Cry4B and Cry11A/Cyt1A were mildly antagonistic. The activity of Cry11A and Cry4B is sufficient to justify investigation as to whether their expression in transgenic rice plants could provide control of C. tepperi larvae.