Mem Inst Oswaldo Cruz, Rio de Janeiro, 96(4) May 2001
Original Article

Stomach Content Analyses of Simulium perflavum Roubaud 1906 (Diptera: Simuliidae) Larvae from Streams in Central Amazônia, Brazil

Yamile B Alencar
+, Thelma A Veiga LudwigI, Climéia C SoaresII, Neusa Hamada

Coordenação de Pesquisas em Entomologia
ILaboratório de Ficologia, Departamento de Botânica, Universidade Federal do Paraná, Curitiba, PR, Brasil
IICoordenação de Pesquisas em Biologia de Água Doce, Instituto Nacional de Pesquisas da Amazônia, Caixa Postal 478, 69011-970 Manaus, AM, Brasil

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Stomach contents ofu00a0Simulium perflavumu00a0Roubaud larvae were analyzed and compared with plankton and periphyton collected in five streams, in Central Amazonia (Manaus and Presidente Figueiredo counties), in Sep./Oct.1996 (dry season) and Feb./Mar. 1997 (rainy season). A total of 1,400 last-instar larvae were dissected; the stomach contents were analyzed using different methods: fresh and after oxidation. A total of 87 taxa (algae, diatoms and rotifers) were found in the stomach contents. In each stream, qualitative samples of plankton and periphyton were collected; these were mounted between slides and cover slips. A total of 94 taxa of plankton and 54 taxa of periphyton were collected. One species of Rotifera was present in the stomach contents, plankton and periphyton. Cluster analysis based on species composition of the organisms present in the stomach contents grouped the streams into two major groups, each belonging to a different drainage area. Correlations based on presence/absence of species of microalgae in the stomach contents, plankton and periphyton indicated significant associations (p<0.05) between stomach contents and plankton and between plankton and periphyton (z test); the Sorensen coefficient and cluster analysis corroborate the same associations.

Black fly larvae usually are abundant in running-water ecosystems and very important in the food chain because they process fine particulate organic matter making it available for other insects and fishes (predators). The size of particles ingested by black fly larvae is a factor influencing the effects of particulate insecticides on these larvae and on other organisms that live in the same habitat (Lacey & Lacey 1983). Knowing the minimum dimensions of particles ingested by the larvae can help in choosing the minimum dimensions of insecticides and microorganisms to be maintained in suspension in the water (Lozovei 1989).

Few studies of black fly larval feeding have been done in Brazil. Lozovei (1989) observed 118 species of diatoms (Bacillariophyta) in the larval stomach contents of three species of black fly, Dellome Filho (1989) reported 50 genera of Bacillariophyta, Chlorophyta, Cyanophyta and Euglenophyta in the stomach contents of Simulium incrustatum Lutz, 1910 larvae. Lacey and Lacey (1983) observed that food takes 29.2 min to pass through the intestine of Simulium fulvinotumCerqueira & Nunes de Mello(= Simulium rorotaense Floch & Abonnenc) in an Amazonian stream with a velocity of 1 m/s and 0.37 mg/l particles in suspension.

Some authors (e.g. Craig 1977, Kurtak 1979) studying the feeding behavior and the functional morphology of black fly larvae have suggested that they are grazing and filter feeding. Wotton (1980) and Fredeen (1964) observed large amounts of bacteria in the stomach contents of black fly larvae.

This is the first study on the stomach contents of black fly larvae in the northern region of Brazil. The study identifies the organisms in the stomach contents of S. perflavum larvae and compares them with the plankton and the periphyton present in the streams. The five streams are compared with each other based on stomach contents of black fly larvae.



This study was carried out in five streams in Central Amazônia, three of which were located in the area of the BR-174 highway, Manaus county and two in Presidente Figueiredo county (2°39'S 60°2'W; 2°23'S, 59°59'W; 2°29'S, 60°1'W; 2°2'S, 59°59'W; 2°2'S, 59°52'W, respectively) (Fig. 1). These streams have many characteristics in common: all flow through yellow latosol soil and are located in areas that have been disturbed due to the opening of highways.

In the Manaus area the year can be divided into two seasons: a rainy season from Dec. to May (Mar. being the rainiest month with an average of 281 mm), and a dry season from Jun. to Nov. (Aug. being the month with the lowest precipitation, with an average of 39 mm) (Araújo 1970). The samples were taken during two months of the dry season (Sep./Oct. 1996) and two months of the rainy season (Feb./Mar. 1997). In each stream, measurements were made of water conductivity (Cole-Parmer conductivimeter), temperature (mercury thermometer) and pH (Cole-Parmer pH Testr 2).

Black fly stomach contents were analyzed using two different techniques. Seventy larvae from each stream were dissected monthly during the study period; from this total 20 larvae were maintained alive on ice, transported to the laboratory and dissected on the same day, two slides were mounted with the larval black fly stomach contents using glycerine jelly (Lozovei & Luz 1976), each slide contained the stomach contents of 10 larvae. The remaining 50 larvae were preserved in Carnoy (3 parts absolute ethanol: 1 part glacial acetic acid) or 80% ethanol for later dissection and treatment using the slow-oxidation technique (Moreira Filho & Valente Moreira 1981); permanent slides were mounted using Hyrax (I.R = 1.7) as the mounting medium.

Permanent and semi-permanent slides were examined under a Zeiss compound microscope. The organisms present on these slides were counted over the whole field of the coverslip at 40x and 100x.

Plankton were collected using a net with 20 µm mesh during 30 min. The samples were placed in 40 ml vials and preserved in Transeau solution (1:1 proportion). Periphyton were collected in the streams from submersed leaves used as substrate by larvae. The material was removed with a brush, placed in 120 ml plastic vials and preserved in Transeau solution.

Identifications of Cyanophyta, Euglenophyta, Dinophyta and Chlorophyta were based on Bourrely (1968, 1970, 1972). The identifications of Bacillariophyta (diatoms) were based on Husted (1930), Krammer and Lange-Bertalot (1985, 1986, 1988, 1991a,b), Kobayasi and Nagumo (1988), and Cox (1996).

Vouchers of S. perflavum (larval carcasses) were deposited in the invertebrate collection of the Instituto Nacional de Pesquisas da Amazônia (Inpa). Samples of plankton and periphyton and the permanent and semi-permanent slides with stomach contents were deposited in the Inpa and the Universidade Federal do Paraná (UFPR).

Similarity patterns based on species present in the three levels (i.e. plankton, periphyton and stomach contents of S. perflavum larvae) were verified using the Sorensen coefficient based on presence/absence data. Sorensen coefficient = 2A/2A + B + C, where A = number of species common to the two levels, B = number of species exclusive to level 1, and C = number of species exclusive to level 2. The significance of correlation among the three levels was tested using the z statistic, based on nominal data (Zar 1996). Similarity dendrograms from the similarity matrix with the Sorensen coefficient (binary data) were derived using UPGMA methods (non-balanced means) based on Pielou (1984).



The streams in the study area had pH values of 4.4 to 5.3, conductivities of 9.4 to 12.6S/cm2 and temperatures of 25° to 29°C, these being representative values of streams in the study, which are normally acidic and poor in mineral salts (Sioli 1965).

S. perflavum larvae feed on diatoms, algae and other organisms present in the plankton and periphyton (Figs 2-72). In the stomach-content analyses (permanent slides), during the dry and rainy seasons 40 species of diatoms were observed; the frequencies of occurrence are presented in Table I.

The Bueiro, ZF3 and Cemitério streams had Peronia sp. as the most frequent diatom species in the dry season (35%, 22% and 23%, respectively). In the rainy season, the most frequent diatom species were different in these three streams; in the Bueiro stream the most frequent species was Frustulia crassinervia (24%), in the ZF3 stream it wasEunotia gibbosa (66%) and in the Cemitério stream it was Actinella brasiliensis and Eunotia conversa (18% and 17.9%, respectively). The Escada stream had Egibbosa as the most frequent diatom species in the dry and rainy seasons (21% and 14%, respectively). The Ibama stream had F. crassinervia as the most frequent species in the dry season (24%) and E. bilunaris in the rainy season (44%).

Species of Actinella and Eunotia are indicators of acidic waters (Patrick & Reimer 1966). By the frequent presence of these two genera in the stomach contents of S. perflavum larvae we can characterize the breeding place of this black fly species as being tropical, oligotrophic, acidic waters.

The semi-permanent slides with the stomach contents of S. perflavum larvae had a predominance of non-silicose algae. A total of 42 species of non-silicose microalgae were identified, six species of diatoms and one species of Rotifera,Lecane (Monostylaquadridentata Ehrenberg (Table II). Oedogonium sp. was the most frequent species in the Bueiro stream in the dry and rainy seasons (70.8% and 74.3%, respectively). In the ZF3 stream, the most frequent species in the dry and rainy seasons was Microspora sp. (44.6% and 63.1%, respectively). In the Cemitério stream, Pleurotaenium minutum Ralfs Delp. was the most frequent species in the dry season (34.6%) and Pleurotaenium sp. in the rainy season (27.8%). In the Escada stream, Microspora sp. was the most frequent species (47%) in the dry season and in the rainy season Closterium sp. was the most frequent (28.6%). In the Ibama stream, Hyalotheca sp. was the most frequent species in the dry and rainy season (53.5% and 37.6%, respectively).

The dimensions of diatoms observed in the stomach contents of S. perflavum in the five studied streams were between 67.8 and 133.57 µm for E. serra Ehrenberg and Stenopterobia curvula (W. Smith) Krammer, respectively. For the other divisions of microalgae, the sizes range from 150 to 608 µm (Closterium sp., Mougeotia sp.) and from 65 to 1000 µm (Oedogonium sp.) (Burton 1973).

Depending on the classification of the phytoplankton, the size can vary from 2 to 500 µm. Kurtak (1978) conducted detailed studies on the size and proportion of the ingested particles of black fly larvae and observed a predominance of large particles (150 µm) compared to the smaller ones (5-10 µm).

A great variety of food items is reported in black fly larval diets: fungal spores and mycelia, silt, detritus, Rotifera, several species of algae and diatoms (Peterson 1956, Kuznetsov 1981). The diatoms may be important food items because they are reported to be the main components of autoctonous periphyton and are constantly removed from the substrate by the water current, becoming abundant in the plankton (Dudleyet al. 1986, Thompson 1987).

In this study, the qualitative analysis of the plankton found 94 infrageneric taxa: 53 Chlorophyta, 29 Bacillariophyta, 7 Cyanophyta, 2 Euglenophyta, 2 Rhodophyta and 1 Dinophyta (Table III). In the qualitative periphyton samples taken during the dry and rainy seasons we found 33 species of microalgae, 21 species of diatoms and one species of Rotifera,L. quadridentata (Table III).

The similarity patterns based on qualitative data on items in the larval black fly stomach contents, plankton and periphyton were verified with the Sorensen coefficient. The results indicated 58% similarity between organisms present in the stomach contents and plankton in the streams, with 41.4% of the species in common. The similarity between the periphyton and the larval stomach contents was 54%, with 36.8% of the species in common. The Sorensen coefficient also indicated a 63% similarity among organisms in the plankton and periphyton with 45.6% of the species in common.

The hierarchical grouping analyses based on presence and absence with the Sorensen coefficient resulted in 72% similarity between food items in the larval stomach contents and plankton in the streams, and 67% similarity between the larval stomach contents and the periphyton (Fig. 73). Therefore, the stomach content items were more similar to the plankton than to the periphyton. However, organisms found in the periphyton also were similar to the plankton (45.6%), possibly because the periphyton detached from the substrate and entered the water column.

Correlations based on qualitative data on items in the stomach contents, plankton and periphyton agree with the Sorensen coefficient. The correlations between the stomach content organisms and the plankton and between the plankton and the periphyton were significant (z test, p<0.05), while the correlation between stomach contents and periphyton was not significant (z test, p>0.05) (Table IV).

Based on the organisms found in the stomach contents of black fly larvae, the sampled streams can be divided into two groups with 77% similarity. The first group was composed of the Bueiro and ZF3 streams, and the second of the Cemitério, Escada and Ibama streams, being the latter group less similar to the other streams (Fig. 74).

Considering the geographical proximity of the studied streams we observed that they have geological, pedological and climatic characteristics in common. The Bueiro and Ibama streams belong to the Tarumã River Basin, ZF3 stream belong to the Rio Preto da Eva River Basin while Cemitério and Escada streams belong to the Urubu River Basin. This grouping result may be an indication that, even though the studied streams were disturbed by anthropogenic influences, they maintained characteristics of the hydrographic basins to which they belong, reflected in the composition of the phycoflora. Many workers have found that the classes of food items in the stomach contents of black fly larvae reflect, in general, the items available in the environment (e.g. Wotton 1977, Kurtak 1979).

Knowledge of black fly food items can provide important information on larval nutrition and help to clarify differences observed in population productivity of larvae in different habitats (Colbo 1982). More studies will be necessary, not only on larval feeding, but also on the ecological associations between breeding places, black fly larvae, plankton and periphyton, in order to improve our understanding of black fly bioecology.



To the master's students of Laboratório de Ficologia, UFPR and Dr PS Mera for helping to identify the microalgae; Dr Mera also helped with the photographs presented in this paper. To MSc. Marcelo Garcia who made helpful suggestions in many steps of this project and Dr PM Fearnside, who made helpful suggestions and revised the manuscript.



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+Fellowship from CNPq (139761/96-9) and corresponding author. Fax: +55-92-643.3195
Received 16 June 2000
Accepted 10 October 2000
This study received partial financial support from PPG-BTRN/INPA, PPI-2-3230 (INPA/MCT) and CNPq.rn

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