Occurrence of filamentous fungi in Simulium goeldii Cerqueira & Nunes de Mello (diptera: simuliidae) larvae in central Amazonia, Brazil.

The family Simuliidae is the host of simbiontes fungi that inhabit the digestive tracts of arthropods. This paper reports the presence of fungi in Simulium goeldii Cerqueira & Nunes de Mello larvae in Amazonia. We observed that the larvae are a good component of aquatic systems to isolate filamentous fungi.

The family Simuliidae (Diptera: Nematocera) is known worldwide for its medical importance. Several species have been listed as hosts of Trichomycetes fungi that are obligate inhabitants of the digestive tracts of several Arthropoda; this class of fungi is included in the group of organisms with biotechnological potential, being of interest for pharmacological use and for use in the insecticide industry. Several species of Trichomycetes have been described in the Amazon region (1,19,23). However, there is a lack of studies in this region on the relationship of these fungi to other microorganisms associated with aquatic insects.

Simulium goeldii Cerqueira & Nunes de Mello has a wide distribution in the Amazon region. Its immatures inhabit small acidic streams in the forest (6,12,13). Larvae are filter feeders and can be found on different substrates, occurring in higher density on the leaves of aquatic macrophytes, and on deciduous leaves and roots from trees that grow on the stream banks (11). Although the female of this species is not anthropophilic, studies of the population of this species can contribute to knowledge of the biology and ecology of black flies in Amazonia. The objective of the present study is to isolate and identify the fungi associated with S. goeldii larvae in Central Amazonia, Brazil. This study is the first step towards selecting fungal lineages of biotechnological interest with a view to their use in the biological control of insect vectors of tropical diseases.

This study was conducted from April to October 2004 at different localities in the municipalities of Manaus, Presidente Figueiredo and Rio Preto da Eva (Amazonas) (Table 1).

Table 1

Simulium goeldii (Diptera: Simuliidae) collection sites in Amazonia.

Site	Habitat	Date	Collector	Longitud/ Latitud
1	HW AM010 km 24, Reserva Florestal Adolpho Ducke, Igarapé Barro Branco, Manaus municipality	28/4/2004 02/5/2004	Fonseca, Q.R.	02°34’S; 60°07’W
2	HW AM010, Ramal Acará, Balneário da SEDUC, Manaus municipality	16/7/2004	Fonseca, Q.R.	02°56’S; 59°59’W
3	HW BR174 km 107, Igarapé da Santa Cláudia,Presidente Figueiredo municipality	18/5/2004	Fonseca, Q.R.	02°02’S; 60°00’W
4	HW AM010 km 02, Ramal Baixo Rio, Igarapé Família Dó-re-mi, Rio Preto da Eva municipality	28/9/2004	Alencar, Y.B.	02°42’S; 59°42’W

Larvae of S. goeldii were collected using forceps, placed in containers and stored under refrigeration before being dissected in the laboratory. The larvae were dissected in distilled water with forceps and needles. Morphological characters used for species characterization were the same as those used in conventional studies of black-fly systematics (5,12). Voucher specimens are deposited in the Invertebrate Collection of the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Amazonas, Brazil.

Larvae were separated and stored in test tubes in groups of ten specimens and sterilized by consecutive baths in sterile distilled water and 70% alcohol for 2 seconds each; Each Simuliidae species pool was composed of 10 insects that were macerated in 0.2 ml of saline solution (0.9%). The macerated samples were processed using the technique of Alves (2), 0.1 ml of the macerated sample was seeded onto Petri dishes containing PDA (potato dextrose agar) culture media, to which 0.05 g per l of chloramphenicol was added. The plates were incubated at 28°C and examined every three days for 20 days. To preserve the genus of each isolate the colonies were transferred to test tubes (16 x 100 mm) containing 5 ml of PDA (2). These tubes were kept in a chamber under the same conditions of temperature (28°C) and relative humidity (80%).

To observe the macroscopic characteristics for identification of the various genera in each isolate, fragments of the colony grown in the test tubes were transplanted using a platinum loop to Petri dishes containing the media potato dextrose agar (PDA), Czapek-Dox-agar (CZ) and malt extract agar (MEA) (Difco), and were incubated at 28°C.

The cultures were identified by microscopic characteristics (sexual and asexual) using the slide culture technique and specific literature (3,8,14,20,21). Species were mounted in Amann lactophenol plus cotton blue and observed under a compound microscope.

Voucher cultures were preserved in hemolysis tubes (15 x 100 mm) with PDA under a 1-cm layer of mineral oil and incorporated in the fungus culture collection of the Instituto Nacional de Pesquisas da Amazônia (INPA) and Coleção de Culturas de Fungos do Departamento de Micologia, Instituto Oswaldo Cruz- FIOCRUZ/IOC.

A total of 50 larvae of S. goeldii were dissected, from which 36 fungal lineages were isolated. Of the isolated lineages, only 42% produced spores. The genera/species of fungi identified were: Aspergillus ochraceus Wilhelm, Penicillium bilaii Chalabuda, Penicillium chrysogenum Thom, Penicillium variabile Sopp, Paecilomyces aerugineus Samson, Beauveria bassiana (Bals.) Vuill., Metarhizium anisopliae (Metsch.) Sorokin, Trichoderma harzianum Rifai, Pestalotiopsis guepinii (Desm.) Stey, Scopulariopsis brevicaullis (Saccardo) Bainer, Acremonium sp. and Gliocadium sp. (Table 2).

Table 2

Frequency of fungal isolates of S. goeldii larvae (Diptera: Simuliidae) collected at different localities in Amazonia

Species	Isolated/ Frequency	Site
Acremonium sp.	1 (2.8)	1
Aspergillus ochraceus	1 (2.8)	1
Beauveria bassiana	1 (2.8)	1
Gliocadium sp.	2(5.5)	3
Metarhizium anisopliae	1 (2.8)	1
Paecilomyces aerugineus	2 (5.5)	1
Penicillium bilaii	1 (2.8)	2
Penicillium chrysogenum	1 (2.8)	4
Penicillium variabile	1 (2.8)	3
Pestalotiopsis guepinii	1 (2.8)	2
Scopulariopsis brevicaullis	1 (2.8)	1
Trichoderma harzianum	2 (5.5)	1
Mycelia sterilia	21 (58.3)	1,2,3,4

The lineages that did not sporulate were inoculated in a poor culture medium, such as agar-water and agar-oats to induce the production of spores; however, sporulation was not observed. The sterile lineages were preserved in sterilized distilled water and saved for later molecular identification.

The isolated fungi in this study are widely distributed in the environment and can be isolated from soil sample and plants (2). Some of the isolated lineages have associated entomopathogenic genera/species such as M. anisopliae, Acremonium, Paecilomyces, B. bassiana and T. harzianum (2,16,17,18).

B. bassiana is frequently obtained from soil samples and insects (3), and lineages of this species are used to control populations of pest insects in the orders Coleoptera, Hemiptera, Lepidoptera, and some species of Diptera such as Musca domestica Linnaeus and Anastrepha ludens Loew (3,16,17).

M. anisopliae was the first species of fungus to be used in the microbial control of insects and occurs naturally in more than 300 species of different orders, including important pest insects. It is frequently used in the control of Deois flavopicta Stal and Zulia entreriana Berg (2).

Wicklow et al. (27) reported that Acremonium produce peptides with antimicrobial activity, having toxic properties and hability to form pores in membranes. This same study demonstrated that the antibiotic produced by Acremonium zeae Gams & Sumner inhibits the growth of Aspegillus flavus Link and Fusarium verticillioides Sacc. (Nirenberg). Also, this antibiotic showed activity against Candida albicans Sullivan and several other Gram-negative bacteria.

The fungus A. ochraceus is pathogenic to humans and other animals, being known to produce ocratoxina A, a toxic substance that contaminates food, especially grains such as corn and peanuts (9,15).

In assays made with insects in the family Culicidae using lineages of the fungi Penicillium, Aspergillus and Trichoderma it was observed larval mortality of nearly 70% over a period that rarely reached 72 hours (18). Several papers about Trichoderma have reported its potential for producing antibiotic peptides and hydrolytic enzymes; however, there is no report of the use of these metabolites to control insects (4,10,24,25).

During this study some genera/species of fungi known worldwide as important in the biological control of crop pests such as M. anisopliae and B. bassiana, were isolated from black-fly larvae. Also, from this insect fungi were isolated that are known to produce compounds of biotechnological interest, such as T. harzianum and Acremonium, but there are few studies of its potential for biological control of insect vectors of tropical diseases. Future studies on the lineages obtained need to be done to increase our knowledge of microorganisms with biotechnological potential in the Amazon region, thereby contributing to strengthening applied research in this region.

RESUMO

Ocorrência de fungos filamentosos associados a larvas de Simulium goeldii Cerqueira & Nunes de Mello da Amazônia Central, Brasil

A família Simuliidae é hospedeira de fungos simbiontes que habitam o trato digestivo de artrópodos. Este estudo reporta a presença de fungos em larvas de Simulium goeldii Cerqueira & Nunes de Mello da Amazônia. Foi observado que as larvas são bons componentes do sistema aquático para isolar fungos filamentosos.

Palavras-chave: Borrachudo, Simulium, insetos aquáticos, fungos