Insecticidal activity of 2-tridecanone against the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

The effect of 2-tridecanone vapor on the cowpea weevil (Callosobruchus maculatus) development was determined. Seeds of cowpea were infested with adults and exposed to different doses of 2-tridecanone isolated from Pilocarpus microphyllus Stapf ex Holm, a plant species native from northeastern Brazil. The pure monoterpene was evaluated both undiluted as well as in the dilutions 1:10, 1:100 and 1:1,000 (v/v). The following parameters of the cowpea weevil life cycle were analyzed in response to decreasing doses of 2-tridecanone: number of eggs laid, percentage of egg hatching on seeds, percentage of adult emergence, adult weight at emergence, mean developmental time and number of adults emerged. Vapor of 2-tridecanone caused a significant (P < 0.05) reduction in the number of eggs laid, in the percentage of eggs hatched and in the number of emerged adults in infested seeds. The fumigant insecticidal effect of 2-tridecanone was mainly due to its ovicidal activity.


INTRODUCTION
Cowpea [Vigna unguiculata (L.) Walp.] is one of the most widely adapted, versatile, and nutritious grain legumes (Ehlers and Halla 1997).It has been consumed by humans since the earliest practice of agriculture in developing countries of Africa, Asia and Latin America, where it is especially valuable as a source of dietary proteins as well as vitamins and minerals (Singh et al. 2003) Substantial quantities of cowpea are produced in semiarid northeastern Brazil, however before harvest or during storage the seeds are vulnerable to infestation by many insect pests, constituting the major constraint on the food availability.In this region, over 90% of the insect damage to cowpea seeds is caused by the " cowpea weevil" Callosobruchus maculatus F. (Coeloptera: Bruchidae), a pest of several pulses including chickpeas (Cicer arietinum L.), lentils (Lens culinars Medik.), soybeans (Glycine max Mer.) and common beans (Phaseolus vulgaris L.).Indeed C. maculatus infestation on stored legumes may reach 50% within 3-4 months of storage (Pascual-Villalobos and Ballesta-Acosta 2003).
It has been well reported that terpenes from a variety of essential oils have potent insect pest-control properties, and they have being found to affect the biology of target insects in different modes such as ovicides, repellents, antifeedants, fumigants and contact toxicants, and insecticides (Watanabe et al. 1993, Hough-Goldstein 1990, Karr and Coats 1988, Rice and Coats 1994, Tsao An Acad Bras Cienc (2007)  79 (1) et al. 1995).The effectiveness of many essential oils as fumigants and contact insecticides in protecting cowpea seeds against C. maculatus infestation has been studied, and this beetle has shown susceptibility to some plantderived chemicals (Shaaya et al. 1997, Keita et al. 2000, 2001, Raja et al. 2001).
With the goal to identify plant-produced substances active against economically important insect pests, we report in the present work the fumigant activity of the 2tridecanone, the major constituent of the seed essential oil from Pilocarpus microphyllus, against the cowpea weevil C. maculatus.53.0 g of the seeds from Pilocarpus microphyllus were submitted to hydrodistillation for 3 hours in a Clevengertype apparatus.The essential oil was dried over anhydrous sodium sulfate to yield a clear yellow oil in 0.15% yield.

ANALYSIS OF THE VOLATILE CONSTITUENTS
Analysis of the volatile constituents of the essential oil was performed by CG/MS on a Hewlett-Packard 5971 GC/MS instrument equipped with a dimethylpolysiloxane DB-5 fused silica capillary column (30 m × 0.25 mm; 0.25µm fi lm thickness) and Helium as carrier gas at a flow-rate of 1 mL/min and the temperature programmed from 35 • C-180 • C at a 4 • C/min rate and 180 • C-280 • C at a 20 • C/min rate.The injector and detector were maintained at 250 • C and 200 • C, respectively.The mass spectra were taken over the m/z 28-400 range with an ionizing voltage of 70 eV.Identifi cation of individual components of the essential oil was performed by computer library MS search based on their Kovat's retention indices as a preselection routine (Craveiro et al. 1984, Alencar et al. 1990), and comparison of the fragmentation pattern with published spectral data (Adams 2001, Stenhagen et al. 1974).Analysis by GC/MS revealed the 2-tridecanone as the only constituent of the essential oil (100%) (Figure 1).Its structural characterization was accomplished by mass spectral analysis and NMR experiments, uni and bidimensional, run on a Bruker DRX 500 spectrometer, and posterior comparison with published data.

INSECTICIDAL BIOASSAYS
To investigate the fumigant effects of 2-tridecanone on C. maculatus development, the pure compound was evaluated both without any dilution as well as diluted (1:10, 1:100, and 1:1,000, v/v) in 100% ethanol.All bioassays were carried out in small plastic containers (3 cm diameter × 4.7 cm height) and the sample to be tested (100µL) was applied on a small fi lter paper disk (2 cm diameter) which was then laid on the inner downside of the container.Each fi lter paper disk was then covered with a disk of cheese cloth with the same diameter so as to avoid direct contact of the sample with the insects.Thereafter, for each treatment as well for the control (seeds exposed to 100% ethanol), 200 seeds (three replicates) were introduced in each treated container and provided as oviposition sites for fi ve males and fi ve females of C. maculatus (aged 0-24 h).
An Acad Bras Cienc (2007) 79 (1) After the oviposition period had fi nished (about one week) the total number of eggs laid (hatched and unhatched) was recorded, and seeds were observed for 50 days for adult emergence.Moreover, the disks of fi lter paper where the tested samples were applied were maintained inside the containers until the last day of the experiment.The date when each adult emerged and its weight were then recorded.The developmental period (time of egg laying to adult emergence) and the percentage of adult emergence (number of adults emerged × 100/number of hatched eggs) were calculated.All data obtained were subjected to analysis of variance (ANOVA) and means were compared using Tukey´s test.
2-tridecanone is the major constituent of the essential oils from Pilocarpus species (Rutaceae) native from northeastern Brazil: P. jaborandi, P. trachyllophus, P. grandiflorus and P. spicatus (Andrade-Neto et al. 1995, 2002, Craveiro et al. 1979).In this work, the vapor of the pure 2-tridecanone isolated from Pilocarpus microphyllus proved to be toxic against the cowpea weevil, Callosobruchus maculatus.Six biological parameters of C. maculatus development were analyzed and vapor of 2-tridecanone signifi cantly (P < 0.05) affected the total number of eggs laid on seeds, the percentage of egg hatching, the total number of emerged adults and the percentage of adult emergence (Table I).The fumigant toxic effects of 2-tridecanone resulted in lower oviposition, higher egg mortality and decreased numbers of emerged adults from treated seeds.For all these affected biological parameters of C. maculatus by 2-tridecanone vapor, the intensity of the effects was directly proportional to the concentration of the applied doses (Table I).
A signifi cant (P < 0.05) reduction (ca.48.2%) in the mean number of eggs laid was observed when adult females were exposed to undiluted 2-tridecanone.In contrast the highest dilution of 2-tridecanone (1:1,000) produced an increase in the mean number of eggs laid, although this was not signifi cantly higher (P > 0.05) than the average oviposition rate observed in the control.Furthermore, the percentage of hatched eggs was signifi cantly (P < 0.05) reduced in the presence of either undiluted 2-tridecanone (89%) or 2-tridecanone 1:10 (51%).In addition, the percentage of adult emergence in the treatment with undiluted 2-tridecanone was significantly (P < 0.05) lower (66%) in relation to the control (91%).On the other hand, the vapor of 2-tridecanone did not show any signifi cant (P > 0.05) effect on the mean developmental time and the average weight of freshly emerged adults of C. maculatus (Table I).
The exposure of the cowpea seeds to the vapor of 2-tridecanone was very effective to control their infestation by C. maculatus since the total number of adults emerged was signifi cantly (P < 0.05) reduced (96%) when compared to untreated seeds.The results show that 2-tridecanone exhibit fumigant toxicity and its effi cacy in protecting the cowpea seeds against C. maculatus is mainly due to its ovicidal activity.Since the percentage of adult emergence is based on the proportion of hatched eggs that develop into adults inside the seeds, the results also suggest that 2-tridecanone vapor can cross the seed coat and therefore interfere with the larvae development.
To our best knowledge this is the fi rst report on the insecticidal activity of 2-tridecanone against the cowpea weevil.
Cowpea is now one of the world's important legume food crops with an estimated cultivated area of about 14 million ha and an annual production of over 4.5 million metric tons (Mt) worldwide.A substantial part of cowpea production comes from Nigeria (about 5 million ha, with 2.1 Mt), Niger (about 3 million ha, with 0.6 Mt) and Brazil (about 1.9 million ha, with 0.7 Mt) (Singh et al. 2003).Production of cowpea in Brazil is concentrated in the northeast, an under-developed, semiarid region where the intensive use of industrialized pesticides by most farmers is impracticable.In this con- text, the identifi cation of naturally occurring substances with insecticidal activity to cowpea weevil is of special interest.Furthermore, the potential use of essential oils in integrated pest management strategy to control insect damage was confi rmed.However, further investigations are needed to understand their mechanism of action.

TABLE I Effects of 2-tridecanone on the cowpea weevil Callosobruchus maculatusa a
Means within a column followed by the same letter are not signifi cantly different at P = 0.05 (Tukey´s test). a