Larvicidal activity of Copaifera reticulata ducke oil-resin against Culex quinquefasciatus Say (Diptera: Culicidae)

Atividade larvicida do óleo-resina de Copaifera reticulata sobre Culex quinquefasciatus Say (Diptera: Culicidae)

SCIENTIFIC NOTE

Larvicidal activity of Copaifera reticulata ducke oil-resin against Culex quinquefasciatus Say (Diptera: Culicidae)

Atividade larvicida do óleo-resina de Copaifera reticulata sobre Culex quinquefasciatus Say (Diptera: Culicidae)

Ionizete G. Silva; Valéria O.M. Zanon; Heloisa H.G. Silva

Inst. Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, C. postal 131, 74001-970, Goiânia, GO

ABSTRACT

The appearance of resistance of Culex quinquefasciatus Say to chemical insecticides and the modification of the susceptibility of this mosquito to pyrethroid has stimulated studies on the activity of plants as alternative for its control. In this study, bioassays were carried out to verify the larvicidal activity of the oil-resin of Copaifera reticulata on C. quinquefasciatus. The bioassays were carried out with the oil dissolved in dimethylsulfoxide (DMSO), at the proportion of 0.4 ml of DMSO to 24.6 ml of distilled water. For each instar, the bioassays were carried out in plastic glasses with 100 ml solution using 20 larvae, wit four replications to adjust the lethal concentrations. The mortality observations were made after 48h exposure of the larvae to the solution. The control was carried out in distilled water and DMSO, with the same number of replications as the bioassays. The experiments were carried out in a biological chamber, controlled at 28 ± 1°C, 80 ± 5% relative humidity and 12h light. The larvae without movement were considered dead. The C. reticulata oil-resin demonstrated larvicide activity for all the C. quinquefasciatus instars. The LC₅₀ found for the 1^st, 2^nd, 3^rd and 4^th larval instars were: 0.4, 0.9, 39 and 80 ppm, and the LC₉₉ were 15, 15, 50 and 180 ppm, respectively.

Key words: Botanical insecticide, elephantiasis, control, Culicinae

RESUMO

A evolução de resistência de Culex quinquefasciatus Say aos inseticidas sintéticos e a modificação da suscetibilidade desse mosquito aos piretróides, estimularam estudos sobre a atividade de plantas inseticidas, como alternativa para o seu controle. Neste trabalho foram realizados ensaios biológicos para verificar a atividade larvicida do óleo-resina de Copaifera reticulata sobre C. quinquefasciatus. Para a realização dos bioensaios o óleo-resina foi solubilizado com dimetilsulfóxido (DMSO), na proporção de 0,4 ml do DMSO para 24,6 ml de água destilada. Para cada estádio, os bioensaios foram realizados em copos descartáveis, contendo 100 ml de solução na qual colocavam-se 20 larvas, com quatro réplicas para ajuste das concentrações letais. As leituras de mortalidade foram feitas após 48h de exposição das larvas à solução. O controle foi feito em água destilada e DMSO, com o mesmo número de réplicas dos bioensaios. Os experimentos foram realizados numa câmara biológica, climatizada a 28 ± 1°C, umidade relativa de 80 ± 5 % e fotofase de 12h e posteriormente, no campo. Foram consideradas mortas as larvas que não possuíam movimentos. O óleo-resina de C. reticulata demonstrou atividade larvicida para todos os estádios de C. quinquefasciatus. As LC₅₀ encontradas para larvas de 1º, 2º, 3º e 4º estádios foram de 0,4; 0,9; 39 e 80 ppm, e as LC₉₉ foram de 15; 15; 50 e 180 ppm, respectivamente.

Palavras-chave: Inseticida botânico, elefantíase, controle, Culicinae

Culex quinquefasciatus Say is the main transmitter of bancroftian filariasis in the world and is the only vector in the Americas in very humid areas. In Brazil, transmission is restricted to the states of Alagoas, Pará and Pernambuco (Rocha & Fontes 1998). Filariasis is caused by Wucheria bancrofti (Cobbald), a helminth that lives in the lymph glands and vessels that provokes edemas by lymph obstruction. It is responsible for the more severe clinical manifestations in the lower limbs (elephantiasis) and the scrotum (World Health Organization 1992,1994).

The high C. quinquefasciatus population density in the cosmotropical area has triggered several interventions by the public health authorities using wide synthetic insecticide application as the main means of combat and control. However, the inefficiency of the organophosphate and carbamate insecticides (Bracco et al. 1999, Hemingway et al. 1985, Failloux et al. 1994), along with the need for safer methods regarding toxicity to man and the environment has stimulated the search for new means of control. Oil-resin or plant extracts are an alternative with potential for use.

Studies have shown the potential of plants for use in C. quinquefasciatus control, such as Piper nigrum (Chahad & Boof 1994), Azadirachta indica, Rhazya sticta and Syzygium aromaticum (Mishra et al. 1995, Su & Mulla 1998, Das et al. 1999, El-Hag et al. 1999), Agave americana and Kaempferia galanga (Dharmshaktu et al. 1987, Choochote et al. 1999, Pizarro et al. 1999), Atriplex halimus (El-Gougary 1998, Massoud & Labib 2000) and Commiphora molmol (Pitasawat et al. 1998).

The objective of this study was to investigate insecticide substances in plants to find an alternative for C. quinquefasciatus control. After previous studies with dozens of plant species, Copaifera reticulata (Leguminosae) presented the greatest potential. The oil-resin of this plant has been used up to now only as a phytotherapeutic, anti-inflammatory and antimicrobial of the respiratory, urinary and dermatological systems and as a healer for ulcers and wounds (Corrêa1984, Veiga Jr & Pinto 2002).

The C. reticulata oil-resin used in the bioassays was obtained in August 2001 in Jacundá, PA, by exudation through a perforation of approximately 70 cm in the base of the trunk of the plant (about 50 cm above soil level). The oil-resin was placed in amber-colored glass flasks until the bioassays with C. quinquefasciatus.

The C. quinquefasciatus larvae used in the tests were reared in plastic basins with water from the public water supply, similar to the methodology by Silva et al. (1998) in an acclimatized chamber with temperature 28 ± 1^oC moisture 80 ± 5% and 12h photoperiod.

The stock solutions were prepared by weighing the C. reticulata oil-resin on analytical scales using dimethylsulfoxide as solubilizer (DMSO). The quantity of DMSO used to prepare the solution had been determined previously by tolerance experiments with C. quinquefasciatus larvae to find the non-lethal concentrations. These were observed at a proportion of 0.4 ml DMSO to 24.6 ml distilled water.The stock solution was prepared at the 500 ppm concentration, using 500 mg C. reticulata oil-resin dissolved in 16.2 ml DMSO and the volume completed to 1000 ml with distilled water. The other solutions were obtained from this by dilution with distilled water to the required concentrations. Later 100 ml of the solutions with appropriate concentrations were transferred to disposable plastic cups to carry out the tests, in which the larvae were placed with the help of a disposable plastic pipette. In the laboratory 20 larvae were used at each instar for each experiment, with four replications, and the same number for their respective controls, using only DMSO at the proportion of 0.4 ml to 24.6 ml distilled water. The laboratory experiments were carried out in an acclimatized chamber under the same conditions as the mosquito rearing. In the field, the experiments were carried out in six different types of recipients, considered as potential breeding-grounds (ceramic pot, cement and asbestos vase, plastic bottle, tire and glass aquarium) with two replications, placing 1.5 liters of the solution in each one. One hundred 4^th-instar C. quinquefasciatus larvae were placed in each one of the recipients. The same quantity of larvae was used for the control, placed in the DMSO and distilled water solution keeping the proportion of the laboratory experiments.

In the laboratory, the mortality was read 48h after the start of the tests and after 24, 48 and 72h in the field. The larvae were considered dead when there was total absence of movement, with darkening on the body and head capsule. The lethal concentrations were interpoled by the Probit analysis by the Statistical Product and Service Solution program (SPSS) only for the laboratory tests.

The mixture of oil-resin with DMSO and distilled water was milky-white in color, semi transparent with a balsamic smell. The results of the larval mortality assessed after 48h of exposure in the laboratory are shown in Table 1.

The C. reticulata oil-resin acted on all the C. quinquefasciatus larval stages. The 4^th instar larvae were the most tolerant to the lethal concentrations, indicating this concentration for use in the combat interventions of this mosquito. In the field, the larva mortality was total after 72h of contact with the solution in all the types of potential breeding grounds tested. However, in the glass, plastic and tire breeding grounds, 100% death occurred after 24h exposure. This is an interesting fact because these types of recipients are predominant in urban areas and thus there are perspectives and possibilities for successful use of the oil-resin. After 24h exposure in the recipients ceramic, cement and cement-asbestos the mortality was respectively, 90, 91 and 95%, and after 48h, it was 95, 95 and 98%.

Studies were not found in the literature on the larvicidal properties of C. reticulata oil-resin for culicids. However, studies with other plants taking as base their lethal concentrations and indication for use, served as reference for the potentiality of C. reticulata oil-resin for use in C. quinquefasciatus control. Pizzarro et al. (1999) studied the activity of the dehydrated gross extract and saponine fraction of Agave sisalana and estimated the lethal concentration LC₅₀, LC₉₀ and LC₉₅ for 3^rd instar C. quinquefasciatus, that were 183, 408 and 512 ppm, respectively. These concentrations were much higher than those reported in this study, but these authors suggested its use for control of this mosquito. Based on the lethal concentrations, C. reticulata presented the most promising form, and its potential for use increased when referring to its non toxic activity for hepatic cells demonstrated by Brito et al. (2000) in rats.

The larvicidal activity of plants in the Philippines was studied by Monzon et al. (1994) by the gross aqueous extract for 3^rd and 4^th C. quinquefasciatus instars and presented extremely high lethal concentrations and LC₉₀ of 37, 28, and 24g/100ml water, respectively, for Lansium domesticum, A. indica, Eucalyptus globosus and Codiaeum variegatum. This corresponded to a concentration one thousand times greater than reported in this study.

Chahad & Boof (1994) assessed the toxic effect of P. nigrum extracts obtained by the soxhlet methanolic and acetonic methods on 4^th-instar C. quinquefasciatus larvae after 36h exposure and reported LC₉₉ of 41.6 and 35.7 ppm, respectively. The results of the black pepper extracts were inferior but compatible with the results obtained with the in natura C. reticulata oil-resin.

The C. reticulata oil-resin LC₅₀ for the 4^thC. quinquefasciatus instar was 80 ppm. This larvicidal activity was similar to the isolated main ingredient, lemonine, from the Citrus reticulata seed in India by Jayaprakasha et al. (1997). However, the LC₅₀ of C. reticulata oil-resin for the same instar of this mosquito presented an inferior result to the isolated main ingredient, carvacrol, from the Egyptian plant Thymus capitatus whose lethal concentration was 100 ppm (Mansour et al. 2000).

In Thailand, the larvicidal activity of K. galanga extractions was studied with several solvents in 4^th instar C. quinquefasciatus larvae where the hexane fraction had a LC₅₀ of 42.3 ppm (Choochote et al. 1999). This result was slightly lower than that reported for the in natura C. reticulata oil-resin solution.

C. reticulata presented lower lethal concentration (LC₅₀ and LC₉₉) for C. quinquefasciatus than the corresponding concentration for the second most promising plant, Magonia pubescens, studied by Silva et al. (1996) and Guimaraes (2001) with 3^rd instar Aedes aegypti L.

Literature Cited

Corrêa, P. 1984. Dicionário das plantas úteis do Brasil e das cultivadas. Ed. Ministério da Agricultura - Instituto Brasileiro de Desenvolvimento Florestal. Rio de Janeiro, 707p.

Received 03/02/03

Accepted 06/06/03

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Publication Dates

• Publication in this collection
  09 Mar 2004
• Date of issue
  Dec 2003

History

• Received
  03 Feb 2003
• Accepted
  06 June 2003