Diversity of larvae of littoral Chironomidae ( Diptera : Insecta ) and their role as bioindicators in urban reservoirs of different trophic levels

The Chironomidae (Diptera: Insecta) have a high species richness, with species adapted to live under widely different environmental conditions. The study of the taxonomic composition of chironomid larvae and the percentage of occurrence of deformities in mouthparts, mainly in the mentum, are used in biomonitoring programmes in order to obtain information on the levels of organic and chemical pollution of aquatic ecosystems. The objective of this study was to evaluate the abundance of chironomid larvae and to quantify the occurrence of mentum deformities in the specimens collected in three urban reservoirs with different trophic levels. The reservoirs are located in the hydrographic basin of the Paraopeba River, an affluent of the São Francisco River basin (Minas Gerais State, southeastern Brazil). The Serra Azul Reservoir is oligotrophic, the Vargem das Flores Reservoir is mesotrophic, and the Ibirité Reservoir is eutrophic. Along the littoral zone of each reservoir, 30 samples were collected during each sampling campaign. Sampling was carried out every three months for one year, with two sampling campaigns during the wet season and two during the dry season in 2008. Physical and chemical parameters measured in the water column included the water depth, Secchi depth, air and water temperature, electrical conductivity, total dissolved solids, redox potential, dissolved oxygen, pH, turbidity, Total-N, Total-P, P-ortho, and chlorophyll-a. The chironomid larvae were identified to the genus level. The structure of the chironomid assemblages was evaluated based on taxonomic richness (24 genera), density, equitability, and diversity. The potential indicator taxa for each reservoir were established through an Indicator Species Analysis. The values for taxonomic richness (20 taxa), equitability (0.737), and Shannon-Wiener diversity (2.215) were highest in the Serra Azul Reservoir. Fissimentum was the indicator taxon in Serra Azul, the oligotrophic reservoir; whereas Pelomus was the indicator taxon in Vargem das Flores, and Chironomus in Ibirité. The highest percentage of mentum deformities was found during the dry season in Serra Azul (6.9%), while the lowest percentage was found during the wet season in Vargem das Flores (0.8%). The results of this study evidenced significant differences in the taxonomic composition, richness, equitability, and diversity of the chironomid assemblages in these three reservoirs of different trophic levels.

Many studies have demonstrated that the physical and chemical parameters of the water influence chironomid composition and abundance (Oliver, 1971;Botts, 1997;Helson et al., 2006;Entrekin et al., 2007).The success of this family in exploiting a wide rage of trophic conditions in aquatic ecosystems is a consequence of its great capacity for physiological adaptation, which allows the individuals to live in environments where temperature, pH, dissolved oxygen concentration, pollution, salinity, depth, and productivity are variable (Helson et al., 2006;Entrekin et al., 2007).As a result, these organisms are able to colonise many types of substrates in high densities (Berg and Hellenthal, 1992;Tokeshi, 1995;Huryn and Wallace, 2000).These characteristics make chironomids efficient organisms for the evaluation of water quality in Neotropical reservoirs (Takahashi et al., 2008).
Reports on morphological abnormalities in the heads of chironomid larvae collected in polluted environments suggest a relationship between these deformities and pollution (Lenat, 1993;Vermeulen, 1995;Janssens de Bisthoven et al., 1998;Servia et al., 2000).The deformities are reported to be more frequent in more-polluted aquatic ecosystems, and some studies have used their frequency as an indicator of severe pollution (Servia et al., 2000;Martinez et al., 2002).These deformities occur at different intensities in the antenna and mouthparts, mainly in the mentum, when the larvae are exposed to heavy metals, agricultural pesticides and fertilisers, and industrial

Introduction
Reservoirs are artificial ecosystems, and their ecological functioning has intermediate characteristics between rivers and lakes (Tundisi et al., 1998).Reservoirs are constructed in order to provide water reserves for different purposes including the production of electricity, household and industrial supplies, transport, irrigation, and recreation (Branco and Rocha, 1977;Tundisi et al., 2008).Reservoirs are distinct landscape features, and in Brazil their surrounding areas are often the target of uncontrolled human occupation (Tundisi, 2006).Anthropogenic reservoir eutrophication leads to an increase in nutrient concentrations (nitrogen and phosphorus) and to the alteration of physical and chemical water parameters (temperature, dissolved oxygen, pH, electrical conductivity), causing reduction of the aquatic biodiversity (Camargo et al., 2005) and often cyanobacterial blooms (Costa et al., 2006;Conley et al., 2009).
Freshwater bioindicators are species, groups of species, or biological communities whose presence, density, and distribution indicate the magnitude of environmental impacts in an aquatic ecosystem and its catchment basin (Bonada et al., 2006).Biological communities reflect the ecological integrity of their ecosystems, integrating the effects of different impacting agents and providing an aggregate measure of the impact of these agents (Barbour et al., 1999).Biological indicators of water quality offer important advantages over physical and chemical parameters, since they represent environmental conditions obtained over periods of time, whereas physical and chemical data are instant measurements that reflect only the present conditions in the aquatic ecosystems (Callisto et al., 2005a).

Field methods and laboratory analyses
The reservoirs were sampled every three months in 2008, during the dry season (June and September) and during the wet season (March and December).Along the littoral zone of each reservoir, 30 samples were collected, using an Eckman-Birge (0.0225 m 2 ) sampler.The samples were deposited in plastic bags and transported to the laboratory, where they were washed on sieves of 1 mm and 0.5 mm meshes (Larsen et al., 1991).Sub-surface water samples were collected using Van Dorn bottles, for the measurement of the physical and chemical parameters.The physical and chemical parameters of the surface water (pH, temperature, dissolved oxygen, electrical conductivity, and turbidity) were measured in situ, using a multi-analyser and portable apparatus (YSI).The Secchi disc was used to evaluate the depth of the trophic zone.For the measurements of total nitrogen, total phosphorus, and orthophosphate, 30 water samples were collected from each reservoir and transported to the laboratory in refrigerated polyethylene bottles.These measurements were performed according to the Standard Methods for the Examination of Water and Wastewater (APHA, 1992).In order to analyse the chlorophyll-a content, 500 mL of reservoir water was filtered through Millipore AP40 filters.After filtration, the filters were manually macerated and extracted with 90% acetone following the procedure described by Golterman et al. (1978).The trophic state index (Carlson, 1977), which uses the Total-P values, the chlorophyll-a values, and the Secchi disc values, were used to assess the trophic level of the reservoirs.Values of this index equal to or less than 20 indicate ultra-oligotrophy, values between 21 and 40 indicate oligotrophy, values between 41 and 50 evidence mesotrophy, values between 51 and 60 evidence eutrophy, and values equal or greater than 61 indicate hyper-eutrophy.

Chironomid larvae
The chironomid larvae were treated with a 10% lactophenol solution and identified under a microscope (400x) with the aid of taxonomic keys (Trivinho-Strixino and Strixino, 1995;Epler, 2001).The occurrence of morphological deformities in the mentum was recorded and counted.All chironomids collected were analysed.The lack or excess of teeth, asymmetry, fusion, tooth malformation, and combinations of these characteristics were considered deformities.We considered the presence or absence of deformities, but did not calculate their frequencies (Dickman et al., 1992).
Sampling stations that provided samples with deformity frequencies equal to or less than 3% were considered natural, between 3 and 6% were considered altered, and impacted when the frequency of deformities exceeded 6% (Burt et al., 2003).
The objective of this study was to evaluate the taxonomic composition, distribution, and abundance of chironomid larvae, as well as to quantify the occurrence of mentum deformities in specimens collected in three reservoirs of different trophic levels.The hypothesis was that human activities in the catchment basin of a reservoir alter the composition and structure (richness, equitability, and diversity) of the chironomid assemblages in the littoral zone of reservoirs, and can cause morphological deformities in the mouthpart structures of these organisms.
Because the level of degradation of a reservoir is usually related to its trophic status, we predicted that: i) the oligotrophic reservoir, well preserved and relatively unimpacted, will show higher richness and equitability than the mesotrophic and eutrophic reservoirs, which are impacted by industrial activities and by the disposal of domestic sewage; ii) a higher frequency of occurrence of morphological deformities in the mentum of chironomid larvae will be observed in the mesotrophic and eutrophic reservoirs; and iii) the mesotrophic and eutrophic reservoirs will have high nutrient contents in the water, and pollutiontolerant taxa; whereas the oligotrophic reservoir will have low nutrient contents and pollution-sensitive taxa.

Study area
The study was carried out in three reservoirs located in the catchment of the Paraopeba River, an affluent of the São Francisco River basin (Minas Gerais State, Brazil) (Figure 1).
The Ibirité Reservoir (19° 07' 00"-20° 02' 30" S and 44° 07' 30-44° 05' 00" W) is formed by the influx of the Pintado and Retiro do Onça rivers.It is affected by intense human impacts such as the disposal of domestic sewage and the presence of unorganised human settlements in its surroundings.As a consequence, this ecosystem shows advanced artificial eutrophication (Callisto et al., 2005b;Moreno and Callisto, 2006).The reservoir has a surface area of 2.8 km 2 , a volume of 15,423,000 m 3 and a mean depth of 16 m (Rodrigues, 2004).
The Vargem das Flores Reservoir (19° 53' 30''-19° 55' 25" S and 44° 07' 22" and 44° 10' 59" W) is fed by Betim Creek.It has moderate human occupancy in its surroundings and is, together with the Serra Azul Reservoir, one of the main sources of water supply for the Belo Horizonte metropolitan region.It is a mesotrophic ecosystem, with a surface of 5.5 km 2 , a volume of 44,000,000 m 3 , a mean depth of 6 m, and a maximum depth of 18 m (COPASA, 2004).
The Serra Azul Reservoir (19° 54' 09" -20º 00' 52'' S and 44° 23' 16"-44° 30' 20" W) is fed by Juatuba Creek.It is located on the boundary between the Juatuba and the Mateus Leme municipalities.It is an oligotrophic ecosystem, with a surface of 8.9 km 2 , a volume of 93,000,000 m 3 and a chironomid assemblages was used to evaluate if there were significant differences among the three reservoirs.
A cluster analysis (Software Primer 6 Beta 2004) was performed in order to assess the similarity in the taxonomic composition of the assemblages found in the three reservoirs.The Bray-Curtis index and an UPGMA (Unweighted Pair Group Method with Arithmetic Mean) were used as the amalgamation method.

Data analyses
The Shannon-Wiener diversity index, Pielou's equitability index (Magurran, 1988), organism density (individuals/ m 2 ), and taxonomic richness (total number of taxa in each sample) were calculated in order to evaluate the structure of the chironomid assemblages.
A variance analysis (ANOVA) (Software Statistica for Windows 5.1) of the data on the composition of the The analysis of indicator species showed Chironomus as the indicator taxon for Ibirité Reservoir, with an indicator value of 81; Pelomus as the indicator taxon for Vargem das Flores Reservoir, with an indicator value of 31; and Fissimentum as the indicator taxon of Serra Azul Reservoir, and an indicator value of 75.The cluster analysis showed a higher similarity between Ibirité and Vargem das Flores reservoirs, which were separated from Serra Azul Reservoir (Figure 2).
Despite the importance of identification of genera belonging to the subfamily Tanypodinae for the study of the taxonomic composition of the chironomid assemblages, deformities in mouthparts were found only among larvae belonging to the subfamily Chironominae.During the dry season in Serra Azul Reservoir, the occurrence of morphological deformities exceeded 6% (6.9%).In this reservoir during the wet season, and in Ibirité and Vargem das Flores reservoirs, the percentages of morphological deformities were less than 6% (Table 3).
Deformities in the mentum of chironomid larvae were found in the genera Aedokritus, Chironomus, Fissimentum, and Polypedilum.Aedokritus individuals with deformities were found in Ibirité and Vargem das Flores.Chironomus larvae with mentum deformities were found in all three reservoirs.Individuals belonging to the genera Fissimentum and Polypedilum that displayed morphological deformities were only found in Serra Azul.
The results of the physical and chemical analyses (Table 2) showed that the highest value for Total-P was found in Ibirité during the dry season (229.19 mg/L), and the lowest value was observed in Serra Azul during the wet season (19.40 mg/L).Intermediate values were found in Vargem das Flores (21.41 mg/L during the dry season and 24.90 mg/L during the wet season).The highest chlorophyll-a concentration was found in Ibirité during the dry season (90.08 μg/L), while Serra Azul showed low concentrations (2.13 μg/L) throughout the year, and Vargem das Flores showed intermediate concentrations Using the three reservoirs as the groups to be indicated, an indicator species analysis (Dufrêne and Legendre, 1997) using the PC-Ord software (version 3.11, 1997) was carried out in order to establish the indicator taxa for each reservoir.The taxa that showed p-values below <0.05 in a randomisation Monte Carlo test (10,000 randomisations) were considered to be indicators for one or two reservoirs.
The values for chironomid taxonomic richness (F 2.87 = 4.60, p = 0.01), Pielou's equitability (F 2.86 = 4.73, p = 0.01), and Shannon-Wiener diversity (F 2.87 = 3.728, p = 0.02) among the three reservoirs were significantly different.On the other hand, the total density values for the three reservoirs were not significantly different (F 2.87 = 1.63, p = 0.20).The taxonomic composition was significantly different among the reservoirs when the dry and the wet seasons were compared (F 153.38 = 3.177, p = 0.0016).
Serra Azul Reservoir was the most diverse, in terms of both taxonomic richness (20 taxa) and Shannon-Wiener diversity (2.215).This reservoir had the genus Fissimentum, which is common in good-quality freshwaters (Leal et al., 2004), as its indicator taxon.In contrast, the chironomid Seasonal differences in the taxonomic composition and density could be explained by the different amounts of allochthonous material entering these systems.Increased input of allochthonous matter produces a decrease in organism density (Higuti and Takeda, 2002).
The high percentage of deformities found in individuals of the subfamily Chironominae in Serra Azul (>6%) may be a response to the high concentrations of manganese (>0.1 mg/L) present in the sediment of this reservoir, due to the geological nature of the underlying matrix (Martins, 1996).The manganese content in this reservoir exceeds the acceptable level (CONAMA Resolution No. 357 of March  17 th , Brasil, 2005) for special-class aquatic ecosystems.High concentrations of this heavy metal in the sediment of aquatic ecosystems can cause the development of morphological deformities in chironomid larvae (Janssens de Bisthoven et al., 2005).Toxic contaminants can influence the presence of chironomids because these insects depend on microhabitat structure and physico-chemical conditions; and contaminants may also cause malformations in the larvae (Nazarova et al., 2004).assemblage in Ibirité Reservoir had the genus Chironomus as the indicator taxon, and showed low levels of taxonomic richness (11 taxa) and diversity (1.473) compared to Serra Azul Reservoir.The genus Chironomus is characterised by its tolerance to pollution and high organic-matter concentrations, thus being typical of impacted ecosystems (Devái, 1988;Marques et al., 1999;Helson et al., 2006).Vargem das Flores Reservoir showed intermediate levels of taxonomic richness (16 taxa) and diversity (1.817) compared to the other two reservoirs, and had as an indicator taxon the genus Pelomus, which is typical of clean environments (Simpson and Bode, 1980) and lives on sand substrata of lentic littoral zones (Strixino and Trivinho-Strixino, 1998).
The high abundance of individuals of the genus Fissimentum in Serra Azul is due to the fact that members of this genus are common in aquatic ecosystems where there are fluctuations of the water level (Cranston and Nolte, 1996), and are typical of lentic ecosystems that have good ecological potential (Leal et al., 2004).The low taxonomic richness in the sediment of Ibirité Reservoir (11 taxa) could be due to the high concentrations of chlorophyll-a, Total-P, and Total-N observed in the water column, which are characteristic of eutrophic ecosystems (Camargo et al., 2005).In theory, an oligotrophication of the reservoir with the consequent reduction of these parameters would favour an increase of its taxonomic richness, allowing the number of taxa to reach the levels found in Vargem das Flores (16 taxa) and Serra Azul (20 taxa).
The hypothesis of the study was partially corroborated.As expected, the results evidenced significant differences in taxonomic composition, richness, equitability, and diversity among the three reservoirs.Pollution-tolerant taxa were recognised as indicators in Ibirité and Vargem das Flores reservoirs, whereas a pollution-sensitive taxon was recognised as an indicator in Serra Azul.In addition, a low similarity in composition and distribution of the chironomid larvae was observed for Serra Azul compared to the other two reservoirs.High percentages of morphological deformities were found only in the oligotrophic reservoir, and were probably due to the high manganese content in the sediments.

Figure 1 .
Figure 1.Map of the Serra Azul, Vargem das Flores, and Ibirité reservoirs, located in the basin of the Paraopeba River, Minas Gerais.

Figure 2 .
Figure 2. Similarity dendrogram for the taxonomic composition of Chironomidae found in Serra Azul, Vargem das Flores, and Ibirité reservoirs.

Table 1 .
Means and standard deviations of Chironomidae larvae collected in Serra Azul, Vargem das Flores, and Ibirité reservoirs during 2008.

Table 2 .
Means and standard deviations of physical and chemical parameters in Serra Azul, Vargem das Flores, and Ibirité reservoirs during the dry and rainy seasons in 2008.

Table 3 .
Percentage of the total number of mentum deformities found in larvae of the subfamily Chironominae, and total percentage of deformities found in each genus in Serra Azul, Vargem das Flores, and Ibirité reservoirs during the dry and rainy seasons in 2008.