Bioactivity of the latex from Parahancornia amapa (Apocynaceae) on the development of Rhodnius nasutus (Hemiptera, Reduviidae, Triatominae) under laboratory conditions

^ILaboratório de Transmissores de Leishmanioses - Setor de Entomologia Médica e Forense/IOC, FIOCRUZ, Av. Brasil, 4365, 21045 - 900, Rio de Janeiro-RJ, Brasil. [e-mail]

^IIDepartamento de Química Orgânica - Universidade Federal Rural do Rio de Janeiro/UFRRJ

^IIIPrograma de Pós-Graduação em Ciências Veterinárias - Universidade Federal Rural do Rio de Janeiro/UFRRJ

^IVPrograma de Pós-Graduação em Biologia Animal - Universidade Federal Rural do Rio de Janeiro/UFRRJ, Br 465, Km 7 Seropédica, 23890 - 000, Rio de Janeiro, RJ, Brasil

ABSTRACT

Bioactivity of the latex from Parahancornia amapa (Apocynaceae) on the development of Rhodnius nasutus (Hemiptera, Reduviidae, Triatominae) under laboratory conditions. The aim of this study was to verify the effects of the methanolic fraction of the latex from Parahancornia amapa (Apocynaceae) (PALAM) on individuals of the species Rhodnius nasutus Stål (Hemiptera, Triatominae). Many of the insects treated with the substance presented deformities and these may interfere in the feeding and possibly hinder the reproductive capacity. They also presented significant mortality during the molt when compared to the control group, noting a gradual increase in mortality. The treated insects also presented delayed nymphal development (5^th instar) and higher adult longevity.

key words: Laboratory conditions; natural products; PALAM; triatomine; vector control.

Rhodnius nasutus Stål, 1859 (Reduviidae, Triatominae) is geographically restricted to the northeast region of Brazil (Galvão et al. 2003). This species could be considered of secondary importance in the transmission of Trypanosoma cruzi Chagas, 1909 (Alencar 1987). In studies conducted in Piauí, several authors have found that R. nasutus is a typical wild triatomine that colonizes natural ecotypes such as birds' nests and palm trees, mainly the babaçu (Orbignya marti) and carnaúba (Copernicia cerifera) and is less abundant in artificial ecotypes (Soares et al. 1995). According to Lima & Sarquis (2007), the deforestation of carnaúba for raising cattle and agricultural development is diminishing the wild areas for this species, and thus this insect is being found in peri-urban environments. Therefore, changes in relation to the environment of this species have increased its vector potential as a possible transmitter of Chagas disease. Many cases of Chagas disease caused by R. nasutus have been described and this triatomine species has been found to be highly infected by T. cruzi (Lima et al. 2008). Thus to combat Chagas disease effectively it is essential to control the vectors (Rey 2008).

Biopesticides provide an alternative to synthetic pesticides because they have low impact on the environment, low toxicity to humans, and low costs as well as other advantages (Liu et al. 2000). Traditionally plants from the family Apocynaceae are widely used as medicinal plants and as insecticides. Parahancornia amapa (Huber) Ducke (Apocynaceae) is a native tree from the State of Amapá in the Amazon region. Like other species of the family Apocynaceae, this plant produces a white latex, commonly known as "leite do Amapá", which is mixed with honey or drinks for human consumption (Van den Berg 1982). The phytochemical study of P. amapa latex revealed the presence of acyl-lupeol esters besides a large amount of carbohydrate and phenylethanoids detected as principal compounds in the methanol fraction such as the methylmyoinositol and the glycoside known as cornoside (Carvalho et al. 2008).

Based on these data, we decided to evaluate the bioactivity of a methanolic fraction of P. amapa latex on the 5^th instar nymphs of R. nasutus. The third generation of a stock colony of this insect established at the Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ was used for this study. The insects were maintained under laboratory conditions (24 - 30 °C; 60 - 85% RH). The photoperiod was not controlled.

The plant material used was collected from one specimen identified by the botanist Benedito Vitor Rabello in the State of Amapá, Brazil. The voucher specimen (nº 07231) is deposited at the Herbário Amapaense (HAMAB) of the Divisão de Botânica do Museu Ângelo Moreira da Costa Lima, Instituto de Estudos e Pesquisas do Amapá (IEPA), Macapá-AP, Brazil.

The dried latex (100.0 g) was extracted by maceration three times with hexane, dichloromethane and with methanol at room temperature and lyophilized to afford the residues PALAH (methanolic fraction of hexane partition) (37.5 g), PALAD (methanolic fraction of dichloromethane partition) (30.0 g) and PALAM (methanolic fraction of methanol partition) (25.0 g). The residue PALAM (15.0 g) was dissolved in methanol and submitted to Sephadex LH-20 column chromatography, eluted with methanol. The ¹H and ¹³C NMR spectra showed signals of glycopyranoside (1) together with signals compatible with other phenylethanoid derivatives such as 1b, 2, 2a, and 3. The GC-MS analysis afforded a chromatogram whose analysis of each corresponding mass spectrum considering the main peaks led us to identify 1b as [4-hydroxy-4-(2-hydroxyethyl)-2,5-cyclohexadien-1-one, certainly yielded from 1]; 2 as [ethanol-2-(ciclohexyl-1-hydroxy-5-methoxy-2-en-4-one); and 3 as [4-hydroxyphenylethanol (tyrosol). The absence of a corresponding peak for 2a suggested it was a derivative from 2. Quinol glucoside 1, known as cornoside (Jiménez & Riguera 1994), was identified by IR, NMR spectra, including 2D-NMR experiments, besides mass spectrum and comparison with literature data (Khan et al. 1992). 1 (50.0 mg), which was treated with acetic anhydride and pyridine (1:1) at room temperature overnight, following usual work-up and filtration through a silica gel column, yielded the tetra-acetyl derivative 1a (m.p. 219 - 220 ºC, 40.0 mg). The analysis of 1H and 13C NMR spectra of a solid material (300.00 mg), revealed the presence of the carbohydrate (4). 30.0 mg of 4 was acetylated in the same way as 1 to obtain the derivative 4a (Fig. 1) (Carvalho et al. 2008).

The soluble methanol fraction, named PALAM, was applied topically on the abdomen in the concentrations of 2, 3 and 4% (based on Mendonça et al. 2011), diluted in distilled water. A total of 360 individuals were separated in test and control groups (divided in three replicates). The in vivo feeding was done weekly on mice of the species Mus musculus L. During the experiment, the observations were made every other day.

The parameters analyzed were: number of ecdyses, duration and viability of the 5^th instar, as well as viability and longevity of the adults. Morphological alterations of the adults from the test groups were analyzed using scanning electron microscopy (SEM), as described by Santos-Mallet et al. (2005). The statistical program used to analyze the data was GraphPad InStat. The results were analyzed by ANOVA and the means were compared with the Tukey-Kramer test (Sokal & Rohlf 1979).

The longevity of adults of R. nasutus from the treated groups did not differ significantly from those of the control group (29.61 ± 17.71days). However, there was significant difference among the insects of the treated groups (Table I). Rocha et al. (1997) observed a prolongation of the longevity of Rhodnius pictipes Stål, 1872 fed on mice when compared to the use of silica membranes. The treatment caused a significant elongation of the 5^th instar nymph period when compared to the control group (22.4 ± 10.1 days) (Table I). On the other hand, no difference was observed among the treatments (Table I). Arévalo et al. (2007) reported duration of 29.7 days for the 5^th instar of R. prolixus reared on chicken. According to Carcavallo et al. (2000), R. nasutus belongs to the same complex as R. prolixus and the biological behavior of these species is very similar.

Morphological alterations were observed only for the treated groups (Figs. 2 and 3). There was a significant difference among the three tested concentrations, but only for legs deformities (Table II). Concurrent deformity of the wings and legs was not significantly different and represented 14.6 and 13.9% for the 3 and 4% treated groups, respectively (Table II). There is no report of any morphological alterations of R. nasutus in the literature. However, some authors have reported that natural extracts can cause morphological alterations in insect species (Fernandes et al. 2012; Cabral et al. 2000). These morphological alterations found using the scanning electron microscopy can hinder the locomotion of the insect and decrease the dispersion activity of the triatomine. The dispersion of the insect is an important factor for epidemiology (Brenner et al. 2000).

No significant difference was observed for the mortality of the nymphs. The mortality of the nymphs was higher at 2 and 3%. However, the mortality during the ecdysis was significantly different when comparing the treated groups with the control, demonstrating a summation total of nymph mortality/mortality during the ecdysis of 42.1, 41 and 67.7%, respectively 2, 3 and 4% (Table III).

In conclusion, the results showed that the triatomine R. nasutus suffered morphological alterations and the development of the insect from 5^th instar nymphs to adult was affected, as well as the longevity of adults and the mortality. We can suggest that these results are due to the activity of a methylmyoinositol and the glycoside known as cornoside, which were detected as the principal compounds in the methanolic fraction obtained from the latex extraction "PALAM" (Carvalho et al. 2008).

ACKNOWLEDGMENTS

The authors are grateful for the financial support received from the Instituto Oswaldo Cruz (IOC/FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

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Received 24 March 2014

Accepted 24 September 2014

Associate Editor: Maria Anice M. Sallum

Bioactivity of the latex from Parahancornia amapa (Apocynaceae) on the development of Rhodnius nasutus (Hemiptera, Reduviidae, Triatominae) under laboratory conditions

Marcio B. P. Lopes^{I, IV}; Paloma M. Mendonça^{I, III}; Jacenir R. S. Mallet^I; Mário G. Carvalho^II; Margareth M. C. Queiroz^I

Publication Dates

• Publication in this collection
  21 Nov 2014
• Date of issue
  Dec 2014

History

• Received
  24 Mar 2014
• Accepted
  24 Sept 2014