Australian Journal of Entomology (2004) 43, 359–365 Blackwell Science, LtdOxford, UKAENAustralian Journal of Entomology1326-67562004 Australian Entomological SocietyMay 2004434359365Original ArticleMolecular identification of ChironomusD Sharley et al. *Author to whom correspondence should be addressed (email: Y.Parsons@latrobe.edu.au). Molecular identification of Chironomus spp. (Diptera) for biomonitoring of aquatic ecosystems David J Sharley, 1 Vincent Pettigrove 1,2 and Yvonne M Parsons 1 * 1 Centre for Environmental Stress and Adaptation Research, La Trobe University, Vic. 3086, Australia. 2 Melbourne Water Corporation, 100 Wellington Parade, East Melbourne, Vic. 3001, Australia. Abstract Chironomidae larvae often represent a major component of the benthic fauna in inland water bodies and are used frequently as bioindicators of ecosystem health. The genus Chironomus is a recognised indicator of organic enrichment and has been used extensively in the northern hemisphere and New Zealand in ecotoxicological studies. However, similar use of Chironomus in Australia is limited due to the presence of cryptic species that restrict the collection of information on species-specific responses to environmental stress. To address the problems associated with species identification, we have used PCR-RFLP of the mitochondrial COI gene to develop DNA profiles for nine common Australian Chironomus species. Species-specific haplotypes were identified using reference taxa previously identified by cytological analysis, and verified with field specimens collected from seven wetlands around Melbourne. This research provides an effective tool for species identification of this ubiquitous and often abundant genus that will provide the basis of obtaining species-specific infor- mation to inform on the health of aquatic ecosystems. Key words biological monitoring, invertebrates, water pollution. INTRODUCTION Chironomidae (Diptera) are the most widely distributed and frequently the most abundant group of invertebrates in fresh- waters (Cranston 1995). They are a major component of bio- mass in lentic systems and the dominant food resource for many fish (Armitage 1995). There are few bodies of freshwa- ter anywhere in the world where chironomids do not occur, and their ubiquitous presence has led to their inclusion in nearly all aquatic monitoring programs and assessment schemes (Warwick 1985; Johnson et al. 1993; Pettigrove et al. 1994; Hardwick et al. 1994; Lindegaard 1995). Together with the Oligochaeta, chironomids represent the greater part of the sediment-dwelling fauna, which makes them especially useful for sediment quality assessment (Hynes 1960), and they have been used as freshwater bioindicators since the early 1900s (reviewed in Lindegaard 1995). Some species of chironomids are tolerant to heavy metals, whereas others are sensitive (Clements et al. 1988), highlighting the importance of identifying chironomids beyond the family level (Clements 1991). A positive correlation between deformities of chirono- mid larvae and sediment contamination has been proposed (Warwick 1985), and it has been suggested that the frequency of structural abnormalities could be an effective indicator of stress from pesticides in rice agriculture (Pettigrove et al. 1994). Chironomids have also been used extensively in ecotoxicological studies that involve exposing an organism to a toxic material, such as heavy metals or pesticides, and deter- mining the response (e.g., Chapman 1995; De Bisthoven et al. 1998). Within the Chironomidae, the genus Chironomus is recog- nised as a taxon characteristic of organic enrichment and has been widely used as an indicator of aquatic ecosystem health (Johnson et al. 1993; Lindegaard 1995). The widespread use of Chironomus in biomonitoring studies has probably arisen because of its known tolerance to many stressors and toxins (Lindegaard 1995). The variation in sensitivity to pollution that exists between species of Chironomus also makes them ideal indicators of water body degradation (Johnson et al. 1993; Lindegaard 1995). Studies using Chironomus in bio- assessment of freshwater aquatic ecosystems have been car- ried out predominantly in Europe and North America (Johnson et al. 1993), attributable to the extensive taxonomic research of this genus undertaken in these regions (e.g., Sublette & Sublette 1974; Wülker & Butler 1983; Sublette & Martin 1994). In contrast, no formal taxonomic keys for Australian Chironomus species are available (Cranston 1994) as many species are morphologically similar, particularly in the larval stage that is routinely used in biomonitoring studies. In his comprehensive revision of the Australian Chirono- midae, Freeman (1961) identified six Chironomus species based on adult morphology: C. nepeanensis Skuse, C. vitellinus Freeman, C. magnivalva Kieffer, C. tepperi Skuse, C. australis Macquart, and C. alterans Walker. More recent cytological studies have revealed that a number of these