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.     

基于环境DNA-宏条形码技术的水生生态系统入侵生物的早期监测与预警

外来生物入侵是继生境破坏后造成生物多样性丧失的第二大威胁因素, 已对入侵地的生态安全、经济和社会发展及人类健康等造成严重负面影响, 成为21世纪五大全球性环境问题之一。作为水产养殖、航运和水生宠物交易大国, 我国水生生态系统的生物入侵问题尤为严重。研究表明, 系统地构建并应用早期监测预警技术是防控水生生态系统生物入侵最有效的途径。和陆生生物相比, 水生生物群落的物种繁多、群落结构复杂、生物形体微小且在入侵初期群体规模极小、隐匿于水下、可用于物种鉴定的外部形态缺乏, 使得在水生生态系统中构建并应用早期监测和预警体系在技术层面更具挑战。随着高通量测序技术的快速发展, 环境DNA-宏条形码技术成为构建水生生态系统入侵生物早期监测与预警技术的首选。本文主要综述了基于环境DNA-宏条形码技术的水生生态系统入侵生物的早期监测与预警技术方法; 解析了环境DNA-宏条形码监测系统的应用现状、技术优势; 着重探讨了影响监测结果准确性的I型和II型错误及其产生原因, 并为避免两类错误提供了可行的优化/改进方案; 最后对该方法在水生入侵生物监测中的应用前景进行了展望。     

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.     

Design of molecular markers for identification of species of the genus Chironomus (Diptera,Chironomidae)

Accurate identification and differentiation of species of the genus Chironomus based on their morphological features is a difficult problem. Unambiguous species identification by means of molecular markers is possible at any stage of the life cycle. Polymerase chain reaction (PCR) with species-specific primers was used to develop molecular markers (amplicons) for identification of Chironomus piger, Ch. dorsalis, and Ch. pseudothummi. Nucleotide sequences of the internal transcribed spacer region (ITS) of the locus coding for ribosomal RNA were used to design species-specific primers for these target species. Each of the species-specific primer pairs yielded species-specific amplicons (molecular markers) only with the DNA of target species: Ch. piger, Ch. dorsalis, and Ch. pseudothummi. Test PCRs with the DNA of eighteen Chironomus species confirmed the specificity of the primers obtained. The molecular markers produced in PCR with the designed species-specific primers permit reliable identification of Ch. piger, Ch. dorsalis, and Ch. pseudothummi and their differentiation from other species of the genus Chironomus.     

Linking interdecadal changes in British river ecosystems to water quality and climate dynamics

Macroinvertebrate communities in Western European rivers have changed substantially in recent decades. Understanding the causes is challenging because improvements in water quality have coincided with climatic variations over this period. Using data covering >2300 rivers and 21 years (1991–2011) across England and Wales, we analysed family‐level distributions and nationwide trends in prevalence (proportion of sampling locations where an organism was present) to diagnose the causes of ecological change. Our aims were to: (i) reveal the taxa driving assemblage‐level trends; (ii) identify the main changes in family‐level prevalence and distribution patterns; and (iii) test whether changes were accounted for by improving water quality, increasing temperatures or variations in discharge. While previous analyses revealed increasing richness among British river invertebrates, a partial turnover of taxa is now evident. Two distinct components of temporal trend have comprised: (i) overall increases or decreases in taxon prevalence over 21 years, which correlated with pollution sensitivity and discharge; and (ii) short‐term variations in prevalence that correlated primarily with temperature and nutrient concentrations. The longer‐term changes in prevalence were reflected in expansions or contractions in families' distributions linked to water quality, with little evidence of shifts consistent with increasing temperatures. Although these monitoring data had limitations (e.g., family‐level data, few headwaters), they provide no clear evidence of long‐term climate effects on invertebrates; the one feature consistent with climate warming – a small northward expansion of the range of many taxa – was accounted for by large improvements in water quality in northern England. Nevertheless, changes linked to discharge and temperature over the shorter‐term (<2 years) point to the climatic sensitivity of invertebrate communities. It is therefore likely that any long‐term climatic changes since 1990 have been outweighed by the strength and geographical extent of the recovery from poor water quality.     

Behavioural,developmental and morphological responses of Chironomus gr. thummi larvae (Diptera,Nematocera) to aquatic pollution

Populations of Chironomus gr. thummi larvae from two differently polluted lowland streams (Dommei, high cadmium and zinc; Ijse, medium copper and organic xenobiotics) were screened for behavioural and morphological responses to pollution. Behaviours such as locomotion (swimming and looping), respiration movements (ventilation) and inactivity were quantified with impedance conversion technique. Chironomids from the Dommel were more active than larvae from Ijse. In Ijse, deformed larvae showed less emergence, less locomotion and more ventilation than non-deformed larvae. In Dommel, deformed and normal larvae were equally fit (behaviour, emergence).     

Resistance to anoxia of Chironomus plumosus and Chironomus anthracinus (Diptera) larvae

Survival of the midge larvae Chironomus plumosus and C. anthracinus in anoxia at 4°C was investigated. C. plumosus survived about twice as long as C. anthracinus . The corresponding LT 50 values were ca 205 and 100 d. There was no statistically significant difference between the survival in anoxia and in aerated water, which indicates that the main reason for death in anoxia is not the absence of oxygen. This main reason is presumed to be starvation. The presence of undissociated H₂S in low concentrations (ca 1.2 mg 1⁻¹) did not influence the survival. In anoxia the larvae were usually motionless and did not feed but they increased in weight due to uptake of water. During 43 d C. plumosus and C. anthracinus increased ca. 10%.     

Cell-free synthesis of Chironomus thummi (Diptera) globins

RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi.     

Seasonal dynamics and larval drift of Chironomus riparius (Diptera) in a metal contaminated lowland river

Chironomus riparius is one of the insect species which inhabit polluted rivers in large densities, indicating a high adaptive capacity. Previous studies showed that this capacity is expressed by the occurrence of adapted strains in metal polluted rivers. Differences in life history between metal-exposed and non-exposed midges have been demonstrated in laboratory experiments, and therefore a comparative field study of seasonal dynamics was carried out at two metal polluted sites and one reference site. Just downstream from a massive metal discharge, seasonal dynamics were almost identical to the upstream reference site. Circa four generations per year were produced. Further downstream, lower larval densities were recorded, especially during the second half of the sampling period. The influx of upstream C. riparius larvae into polluted sites was estimated by measuring larval drift just upstream from the point source of metal contamination and indicated a massive input to the standing stock downstream. It is concluded that drift of non-tolerant larvae is dominating the seasonal dynamics of midges downstream. Accordingly, genetic uniformity of chironomids inhabiting upstream and downstream sites is expected most of the time. However, research performed during the last decade, demonstrated that genetically adapted strains of C. riparius may develop at certain stages in the seasonal cycle. However, a stable metal-adapted C. riparius population at the first downstream site, is most likely present on rare occasions only.     

Molecular identification of Fasciola spp. (Digenea: Fasciolidae) in Egypt

A total of 134 Egyptian liver flukes were collected from different definitive hosts (cattle, sheep, and buffaloes) to identify them via the use of PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS1). Specimens of F. hepatica from France, as well as F. gigantica from Cameroon were included in the study for comparison. PCR products of ITS1 were subjected for digestion by RsaI restriction enzyme and visualized on agarose gel. According to RFLP pattern, Egyptian flukes were allocated into two categories. The first was identical to that of French hepatica flukes to have a pattern of 360, 100, and 60 (bp) band size, whereas the second resembled to that of Cameroonian gigantica worms to have a profile of 360, 170, and 60 bp in size. Results of RFLP analysis were confirmed by sequence analysis of representative ITS1 amplicons. No hybrid forms were detected in the present study. Taken together, this study concluded that both species of Fasciola are present in Egypt, whereas the hybrid form may be not very common.     

An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (Pleuronectes vetulus): development, validation and cross-reactivity with other pleuronectids

Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E₂) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E₂-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml⁻¹ (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E₂-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.     

Some carotenoids in Chironomus annularius Meig. larvae (Diptera : Chironomidae)

Summary The author investigated the presence of various carotenoids in the body of the larvae of the Chironomus annularius MEIG. moquito (Diptera: Chironomidae) by means of columnar and thin-layer chromatography.The investigations revealed the presence of the following carotenoids in the larvae: -carotene, -carotene, canthaxanthin, cryptoxanthin, lutein, astacene and a kind of xanthophyl which was not possible to identify more closely.     

Molecular identification of symbiotic dinoflagellates (Symbiodinium spp.) from Palythoa spp. (Anthozoa: Hexacorallia) in Japan

In Japan, zooxanthellate Palythoa tuberculosa Klunzinger and Palythoa mutuki Verrill (Anthozoa: Hexacorallia: Zoantharia) are found over a 1,000 + km latitudinal range, often in environments where most other zooxanthellate anthozoans are not found (i.e. tidal lagoon pools, around shallow water hydrothermal vents, subtropical rocky shorelines). Sequences of internal transcribed spacer of ribosomal DNA (ITS-rDNA) of the symbiotic dinoflagellate genus Symbiodinium (zooxanthellae) Freudenthal (Order Suessiales) from P. tuberculosa and P. mutuki from several locations in Japan (34°11′N–24°16′N) were analysed. Unexpectedly, despite the ability of the genus Palythoa to be flexible in association with different Symbiodinium subclades, most (35 of 36) Palythoa investigated here specifically associate with subclade C1 and closely related types. Symbiodinium subclade C1 has been characterized as a “generalist” in terms of the ability to associate with a range of hosts, but present results suggest that subclade C1 may also be a “generalist” in terms of being able to live in a variety of environments over a latitudinal range. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

1. Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification.
2. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub-sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically.
3. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification.
4. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.

     

Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p‐distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the ‘Mosquitoes of Australia–Victoria’ project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.     

Immunological characterization of the haemoglobins of Chironomus thummi (Diptera)

The major fourth instar-specific haemoglobin (Hb) of Chironomus thummi was purified to homogeneity as assessed by five analytical systems. This Hb was further resolved by isoelectric focussing into two variants, differing only slightly in their isoelectric points. These variants proved to be immunologically identical in each of three antigenically distinct components. Cross-reactivity studies indicated the presence of two or more antigenic components in other Hbs of C. thummi and of C. tentans, a related species. The data exclude the possibilities that non-haeme protein contaminants or that Hb (globin) fragments were present in our preparations. Therefore, multiple precipitin lines on double diffusion plates must have arisen from differentially antigenic sub-populations which are not separable by routine purification procedures.     

A review of the genus Chironomus (Diptera,Chironomidae)

Analysis of the banding pattern of the salivary gland chromosomes of Chironomus tepperi indicates that, despite a somewhat modified male hypopygium, the relationships of this species are close to the other Australian species of the genus, particularly to Ch. oppositus. No inversion polymorphism has been found in Ch. tepperi and this, together with the relatively high chiasma frequency as measured at metaphase I, would appear to be an adaptation to provide genetic variability necessary for its colonizing ability.