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In vitro larvicidal evaluation of Annona muricata L., A. diversifolia Saff. and A. lutescens Saff. Extracts against Anastrepha ludens larvae (Diptera, Tephritidae)/Evaluacion in vitro de extractos de Annona muricata L., A. diversifolia Saff. Y A. lutescens Saff. Sobre larvas de Anastrepha ludens (Diptera, Tephritidae)/Avaliacao in vitro de extratos de Annona muricata L., A. diversifolia...

SUMMARY

Annonaceae (custard apple family) extracts have demonstrated their insecticidal potential against a variety of insect pests. This study is aimed to determine the activity of ethanolic and aqueous extracts of the stems and leaves of Annona muricata L., A. diversifolia Saff. and A. lutescens Saff. against larvae of Anastrepha ludens (Mexican fruit fly). Ethanolie extracts were obtained by Soxhlet extraction, and aqueous extracts were obtained by boiling. Larvicidal activity against third in star A. ludens larvae was determined after 24 and 72h of larval exposure to the extracts at concentrations of 100, 1000 and 2000[micro]g x [ml.sup.-1]. Additionally, toxicity was quantified by the Artemia salina (brine shrimp) assay. The results indicate that extracts of the three aforementioned Annona species show time-dependent larvicidal activity against A. ludens, with variable mortality rates at 72h of exposure, as follows: A. lutescens 8794%, A. diversifolia 70-90% and A. muricata 63-74%.

RESUMEN

Los extractos de Annonaceae han demostrado tener potencial insecticida contra diferentes insectos plaga. Este trabajo tuvo como objetivo determinar la actividad de extractos etanolieos y aeuosos de tallos y hojas de Annona muricata, A. diversifolia y A. lutescens sobre larvas de Anastrepha ludens. Se obtuvieron extractos etanolicos por el metodo de Soxhlet y acuosos por ebullicion. La actividad larvicida se determino a las 24 y 72h de exposicion sobre larvas de tercer estadio con concentraciones de 100, 1000 y 2000[micro]g x [m1.sup.-1]. Adicionalmente se cuantifico la toxicidad con el ensayo de Artemia salina. Los resultados sehalan que los extractos de las tres especies estudiadas presentaron actividad larvicida tiempo dependiente. Las actividades (% de mortalidad) fueron significativas a las 72h de exposicion con diferencias entre las especies estudiadas: A. lutescens 87-94%, A. diversifolia 70-90% y A. muricata 63-74%.

RESUMO

Os extratos de Annonaceae tem demostrado potencial inseticida contra diferentes pragas. Dentre as especies ainda nao avaliadas no controle de pragas como a Anastrepha ludens se encontram a Annona muricata, A. diversifolia e A. lutescens. Este trabalho teve como objetivo determinar a atividade de extratos etanolieos e aquosos de caules e folhas destas especies sobre larvas de A. ludens. Os extratos etanolicos foram obtidos pelo metodo de Soxhlet e o aquoso por ebulicao; a atividade larvicida foi determinada durante 24 e 72h de exposicao sobre larvas de terceiro estadio com eoncentracoes de 100, 1000 e 2000[micro]g x [ml.sup.-1]. Adicionalmente foi quantificada a toxicidade com ensaio de Artemia salina. Os resultados demonstram que os extratos das tres especies estudadas apresentaram atividade larvicida dependente do tempo de exposicao. Com 72h de exposicao observou-se mortalidade de 87-94% com os extratos de A. lutescens, 70-90% com A. diversifolia e 63-74% com A. muricata.

KEY WORDS / Anastrepha ludens / Annona / Larvicidal Effect / Plant Extracts /

Received: 11/20/2010. Modified: 03/05/2012. Accepted: 03/08/2012.

Introduction

Annonaceae (custard apple family) plants have been intensively studied since they were discovered to contain compounds with important biological properties. These properties include cytotoxic, antitumor, antiparasitic, antifungal, antispasmodic, repellent and insecticidal activities (Cave et al., 1997).

Among the reports of pest control attributed to this plant family are the assessment of the insecticidal activity of acetonic and ethanolic extracts of Annona squamosa L. seeds against the cowpea weevil Callosobruchus maculates (Dharmasena et al., 2001), and the demonstration of acaricidal activity of Uvaria versicolor and U. klaineana against the dust mite Dermatophagoides pteronyssinus (Akendengue et al., 2003). In addition, ingestion of and contact with methanolic extracts of Annona muricata seeds are lethal to first and second instar larvae of fall armyworm Spodoptera frugiperda (Gomez, 2005). Furthermore, A. squamosa extracts impart larvicidal activity, and the susceptibility of different larval instars of diamondback moth Plutella xylostella L. and cabbage looper Trichoplusia ni Hubner to these extracts have been documented by Leatemia and Isman (2004). Other studies regarding compounds isolated from Annonaceae have indicated both larvicidal and antifeedant activities (Guadano et al., 2000, Gonzalez-Coloma et al., 2002, Alvarez et al., 2007).

The only documented Anastrepha study to date was carried out by Perales and Martinez (1999). This study investigated the effect of aqueous extracts or A. squamosa leaves against Anastrepha obliqua during oviposition, hatching and adult emergence. The results indicated that the infusion of A. squamosa leaf extract at a concentration of 1% caused a decrease in hatching of up to 60%, whereas an extract concentration of 5% reduced adult emergence by 20%. However, the potential of extracts derived from Annonaceae species for the control of Anastrepha ludens (Mexican fruit fly) has not yet been demonstrated.

Annona muricata, A. diversifolia and A. lutescens are perennial tropical trees with edible fruits. These species are used almost exclusively for consumption or, in some regions, for the formation of living hedges. Coupled with this, these three species produce large amounts of cytotoxic compounds (Zafra-Polo et al., 1998; Abrajan, 2002; Schlie et al., 2009; de la Cruz et al., 2011), making them promising candidates in the ongoing search for natural resources with insecticidal /pesticidal properties. Therefore, the goal of this research was to evaluate the effectiveness of ethanolic and aqueous extracts of the stems and leaves of A. muricata, A. diversifolia and A. lutescens against A. ludens larvae.

Materials and Methods

Plant material

The plant material (stems and leaves) was collected in 2005 in Tuxtla Gutierrez, Chiapas, Mexico. The reference specimens, Annona diversifolia (351), A. lutescens (352) and A. murieata (4152), were deposited at the Herbario Eizi Matuda (HEM) herbarium of the Universidad de Ciencias y Artes de Chiapas.

Extracts

The stems and leaves were dried in the shade at room temperature. The ethanolic extracts were obtained using 100g of dry powdered plant material extracted three times with absolute ethanol (400ml) in a Soxhlet extraction apparatus under reflux for 8h. Next, the ethanolic extracts were concentrated under low pressure at 40[degrees]C. The aqueous extracts were prepared by heating 200g of dry powdered plant material in 500ml of boiling distilled water for 20min. The infusion was decanted and filtered through a Buchner funnel, and the aqueous extract was concentrated until completely dry in Pyrex glass containers at room temperature. These extracts were used to prepare various concentrations of ethanolic and aqueous stem and leaf extracts, diluted in 5% ethanol or distilled water, respectively, for the evaluation of larvicidal activity.

Bioassay with Anastrepha ludens larvae

Evaluation of the stem and leaf extracts was conducted in the laboratories of the Subdireccion de Desarrollo de Metodos of Moscafrut, SAGARPA-IICA (Secretaria de Agricultura, Ganaderia, Desarrollo Rural, Pesca, y Alimentacion--Inter-American Institute for Cooperation on Agriculture) program, located in Metapa de Dominguez, Chiapas, Mexico. Biological material was obtained from Moscafrut, located in Metapa de Dominguez, Chiapas, Mexico. The larvae were obtained from the same facility and were randomly chosen for testing.

To determine larvicidal activity, the ethanolic or aqueous extracts were incorporated into larvae feed at final concentrations of 100, 1000 and 2000[micro]g x [ml.sup.-1]. Fifty A. ludens larvae in the third instar were introduced to each concentration of stem and leaf extract. Two negative controls were used, one consisting of distilled water and the other consisting of 5% ethanol. GF-120 fruit fly bait was employed as the positive control. The data corresponding to the mortality of A. ludens larvae were recorded after 24 and 72h of exposure to the stem and leaf extracts. Five replicates of the experiment were performed, and the results were adjusted according to Abbott's formula (Bobadilla et al., 2002).

All experimental data for larvicidal activity were expressed as means [+ or -] SD (standard deviation) for the five experimental replicates. To ascertain the differences in larvicidal activity between the various extracts, an analysis of variance (ANOVA) was performed. Significant differences between means were determined by Bonferroni's multiple comparison test. P values <0.05 were regarded as significant. Finally, a paired-sample analysis was performed to establish whether the larvicidal activity was more closely related to the type of extract (ethanolic vs aqueous) or to a particular plant organ (stem vs leaf).

Anastrepha ludens larvae diet

In all assays, the larvae diet consisted of a mixture of 18.0% corncob powder, 9.1% suga1, 7.0% yeast, 5.3% cornmeal, 0.2% nipagin sodium, 0.4% sodium benzoate, 0.4% citric acid and 0.1% Guar gum.

Artemia salina bioassay

The toxicity level of all extracts was evaluated by using the brine shrimp (Artemia salina) bioassay, as described by Meyer et al. (1982) and McLaughlin et al. (1998). To obtain A. salina larvae, eggs (20mg) were incubated in saline solution (250ml; 25% artificial sea salt CORALIFE[R] dissolved in distilled water) for 48h under artificial light and at room temperature in a small pond divided into two compartments, one dark and one illuminated. Eggs were deposited in the dark side of the pond. At hatching, the larvae migrated to the illuminated side of the pond and from there were collected for testing.

Four concentrations (1, 10, 100 and 1000[micro]g x [ml.sup.-1]) of each extract were prepared in 5ml of artificial saline medium, and 10 crustacean larvae were transferred into vials containing each of the plant samples to be evaluated. The negative control was a sample of artificial saline medium containing no plant extract. Incubation of the larvae with the extracts or the negative control was conducted at room temperature. The number of dead larvae was counted at 6 and 24h, adjusting for dead larvae in the negative control according to Abbott's formula (Bobadilla et al., 2002). Five experimental replications were performed for each concentration of each plant extract. L[C.sub.50] (half-maximal lethal concentration) values for each extract were determined using a Probit regression analysis to determine the 95% confidence interval. L[C.sub.50] values >1000[micro]g x [ml.sup.-1] for all plant extracts were considered inactive. All statistical analyses for toxicity were performed using Stata 8.0 statistical software.

[FIGURE 1 OMITTED]

Results

To study the larvicidal potential of ethanolic and aqueous extracts of the stems and leaves of Annona muricata, A. diversifolia and A. lutescens, all extracts were evaluated against Anastrepha ludens larvae at 72h. Among the ethanolic leaf extracts, the most active extracts, in terms of the percentage of larvae killed at the concentration of 2000[micro]g x [ml.sup.-1], were those derived from A. diversifolia (75.3%) and A. muricata (72.8%); P<0.0l), while the greatest mortality rates stemming from the A. lutescens extracts occurred at 100[micro]g x [ml.sup.-1] (Figure la). The aqueous leaf extracts of all three species were more effective than the corresponding ethanolic extracts and showed similar activity at 72h: A. lutescens 92.8%; A. muricata 84.3%, and A. diversifolia 75.8% at 2000[micro]g x [ml.sup.-1]; P<0.01 (Figure lb).

The results with the ethanolic stem extracts indicated that the activities of the A. diversifolia and A. muricata extracts were somewhat concentration-dependent (Figure 1c), in that the 2000[micro]g x [ml.sup.-1] concentration was more efficacious (90.0% and 82.8% larvicidal activity at 2000[micro]g x [ml.sup.-1], respectively; P<0.01) than either the 100 or the 1000[micro]g x [ml.sup.-1] concentration. The same concentration of ethanolic A. lutescens stem extract showed lower larvicidal activity (30.9%) compared with the other ethanolic stem extracts. With respect to the aqueous stem extracts, A. luteseens and A. muricata extracts at the 100[micro]g x [ml.sup.-1] concentration had the highest larvicidal activity (95.9% and 86.0%, respectively; P<0.01). However, the activity of the aqueous A. lutescens extract decreased with increasing extract concentration (Figure 1d).

Two extract concentrations, 100 and 1000[micro]g x [ml.sup.-1], were selected to determine the effect of exposure time to all plant extracts (ethanolic versus aqueous, stem versus leaf) on larvicidal activity. The activity of all plant extracts at 24h was relatively minor, yielding only ~5.4% mortality. However, the larvicidal activity increased significantly at 72h (Table I). To determine whether the larvicidal activity was more closely related to the type of plant extract (ethanolic vs aqueous) or the organ (stem vs leaf), appropriate statistical analyses were performed (Table II). Significant differences were observed in both cases for A. lutescens. For A. diversifolia, a significant difference was found between the stem and leaf extract for the ethanolic but not the aqueous extract. By contrast, no statistically significant differences were found between any of the extract activities for A. muricata, although, as noted above, the aqueous leaf extract tended to be more effective than the ethanolic leaf extract only at 100[micro]g x [ml.sup.-1] (Table I).

The results from the A. salina bioassay revealed that for all three Annona species, the toxicity of each ethanolic extract was higher than that of the corresponding aqueous extract. For example, the L[C.sub.50] values for the ethanolic A. diversifolia leaf and stem extracts were 52.0 and 409.1[micro]g x [ml.sup.-1], respectively, after 24h of larval exposure. Aqueous extracts, with the exception of the stem extract derived from A. lutescens, were comparatively less toxic (Table III).

Discussion

Ecosystem deterioration is caused by the inappropriate use of insecticides and pesticides and has stimulated the search for new biodegradable alternatives for pest management. This study therefore evaluated the activity of three Annona species against third instar Anastrepha ludens larvae. Ethanolic and aqueous extraets derived from Annona muricata, A. diversifolia and A. lutescens showed time-dependent larvicidal potential, with significantly higher mortality of larvae at 72 vs 24h. The aqueous extracts from A. lutescens stand out in this regard, with activities reaching 95.9% (stem extract at 100[micro]g x [ml.sup.-1]) and 94.4% (leaf extract at 1000[micro]g x [ml.sup.-1]). By comparison, the larvicidal activities of the aqueous extracts derived from A. muricata were 86% (stem extract at 100[micro]g x [ml.sup.-1]) and 84.8% (leaf extract at 1000[micro]g x [ml.sup.-1]), whereas the larvicidal activity of the ethanolic stem extract derived from A. diversifolia was 89% at 1000[micro]g x [ml.sup.-1].

Similar larvicidal activity levels were previously found with aqueous leaf and stem extracts of A. muricata against fourth instar Aedes aegypti larvae after 40h of exposure, but at 10-fold higher concentrations (Bobadilla et al., 2005). Likewise, the larvicidal activity described in this study is superior to that demonstrated with ethanolic extracts of Annona triloba branches against the Mexican bean beetle Epilachna varivestis (60% mortality, 100ppm) after 72h of exposure (Johnson et al., 1996). However, with the exception of A. lutescens and A. diversifolia (limited to ethanolic stem vs leaf extract for the latter), the analysis of variance (95% confidence) employed in the current study did not distinguish between the larvicidal activity of aqueous stem, aqueous leaf, ethanolic stem or ethanolic leaf extracts.

Possession of similar larvicidal activity by most of the extracts employed herein could be related to both the common polarity of the ethanol and water solvents (both polar), as well as to the broad distribution of cytotoxic compounds in numerous plant organs. For example, various alkaloids and acetogenins have been reported in the stems and leaves of Annona species (A. muricata in particular), as well as in their roots and seeds (Leboeuf et al., 1982; Cave et al., 1997; Fang-Rong et al., 2000). Larvicidal potential has been attributed to acetogenins due to their potent inhibitory activity against mitochondrial respiratory complex I. This inhibition was first described in the mitochondria of the intestinal cells of fifth instar European corn borer Ostrinia nubilalis larvae using asimicine (a polyketide, Lewis et al., 1993), and in the mitochondria of tobaeeo hookworm Manduca sexta larvae using bullatacine (an antitumor acetogenin; Ahammadsahib et al., 1993). Furthermore, benzylisoquinoline alkaloids derived from Annona species are characterized by their inhibitory activity against topoisomerase II (Woo et al., 1997), which is another potential larvicidal mechanism of the plant extracts described in this study.

The extracts derived from A. lutescens showed a marked difference in larvicidal activity relative to the extracts derived from A. muricata and A. diversifolia. Notably, lower A. lutescens extract doses were more active against A. ludens larvae compared with higher doses. This is suggestive of ah antifeedant effect, already noted in the activity of Asimina triloba ethanolic extracts and an annonaceous acetogenin against O. nubilalis fourth instar larvae (Lewis et al., 1993). Liriodenine alkaloid and cherimoline-2 and rolliniastatine-2 acetogenins have all been isolated from A. lutescens (Abrajan, 2002), which may account for the antifeedant and larvicidal activities observed for this plant species. In the present study, A. lutescens extracts were also highly toxic against A. salina. If a possible antifeedant of a deterrent-type activity is considered, it might be hypothesized that ah aliment with a low concentration extract rather than one with a high concentration would be preferable so as to avoid potential cytotoxicity.

As noted above, the larvicidal/insecticidal activity of Annonaceae extracts might be related to their content of acetogenins, as evidenced by reports of the antifeedant, larvicidal, pupicidal and pesticidal effects of these compounds. Moreover, the toxicity of certain acetogenins can be selective for a particular insect; for example, annonatine exhibits antifeedant activity against the Colorado potato beetle Leptinotarsa decemlineata, while squamocine exhibits insecticidal activity against L. deeemlineata and green peach aphid Myzus persicae (Guadafio et al., 2000). On the other hand, an acetogenin can have various targets; for example, in addition to their above-noted targets, squamocine and asimicine are also toxic to the melon aphid Aphis gossypii and the bean beetle Epilachna varivestis (Cave et al., 1997). In either case, it can be inferred that larvicidal properties of A. muricata, A. diversifolia and A. lutescens against A. ludens are due, in large part, to their content of aectogenins, but the contribution of Annona species-derived bioactive alkaloids cannot be overlooked.

When analyzing the toxicity of the plant extracts against A. salina, a close relationship was found between toxicity and extract concentration after 24h of exposure for all three plant species. Ethanolic extracts of the leaves and stems were the most toxic, highlighting ethanolic A. diversifolia leaf and stem extracts ([LC.sub.50]= 52.0 and 409.1[micro]g x [ml.sup.-1], respectively) and ethanolic A. lutescens leaf extract ([LC.sub.50]= 319.0[micro]g x [ml.sup.-1]). However, the extracts were not toxic to A. ludens larvae at this exposure time, which indicates a type of species-specific activity and throws into question the value of this assay as a toxicity indicator for A. ludens.

Conclusions

Extracts derived from the Annona species A. muricata, A. diversifolia and A. lutescens all showed time-dependent larvieidal activity against third instar A. ludens larvae. However, with the exception of A. luteseens extracts, the results of this study showed that neither aqueous vs ethanolic extracts, nor stem vs leaf extracts will figure predominantly in the development of environmentally friendly, Annona based insecticidal strategies to control A. ludens. The effects of these extracts must also be demonstrated in A. ludens adults and pupae, which represent the exposed stages of this pest. In conclusion, the three Annona species studied in this work constitute an extremely valuable, commercially feasible natural resource, due to their larvicidal activity and high content of readily attainable acetogenins and alkaloids.

ACKNOWLEDGMENTS

The authors thank FOMIX-Chiapas for support of the project 'Actividad Potential Insecticida en Tres Especies de Annona' (CHIS-2005-C03-003); the Moscafrut Program, SAGARPA-IICA, for its support of this work and the facilities provided; and the Laboratorio de Alimento Vivo y Monitoreo de Marea Roja del Acuario de Veracruz, Mexico.

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Bobadilla M, Zavala F, Sisniegas M, Zavaleta G, Mostacero J, Taramona L (2005) Evaluacion larvicida de suspensiones acuosas de Annona muricata Linnaeus "guanabana" sobre Aedes aegypti Linnaeus (Diptera, Culicidae). Rev. Per. Biol. 12: 145-152.

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Guadano A, Gutierrez C, De la Pena E, Cortes D, Gonzalez-Coloma A (2000) Insecticidal and mutagenic evaluation of two annonaceous acetogenins. J. Nat. Prod. 63: 773-776.

Johnson HA, Gordon J, McLaughlin JL (1996) Monthly variations in biological activity of Asimina triloba. In Janick J, Simon JE (Eds.) Progress in New Crops. ASHS Press. Arlington, VA, USA. pp. 609-614.

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Schlie-Guzman MA, Garcia-Carranca A, Gonzalez-Esquinca (2009) In vitro and in viro antiproliferative activity of laherradurin and cherimolin-2 of Annona diversifolia Saff. Phytother. Res. 23: 1128-1133.

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Alma Rosa Gonzalez-Esquinca. Doctora en Ciencias Biologicas, Universidad Nacional Autonoma de Mexico (UNAM). Investigadora, Universidad de Cientias y Artes de Chiapas (UNICACH), Mexico. Direccion: Libramiento Norte Poniente # 1150, Col. Lajas Maciel, CP 29039, Tuxtla Gutierrez, Chiapaz, Mexico. e-mail: aesquinea@unicach.mx

Lorena Mercedes Luna-Cazares. Doctora en Ciencias Biologicas, UNAM, Mexico. Docente, UNICACH, Mexico. email: lucaz58@hotmail.com

Maria Adelina Schlie Guzman. Doctora en Ciencias Biologicas, UNAM, Mexico. Investigadora, UNICACH, Mexico. e-mail: adeymac@hotmail.com

Ivan De la Cruz Chacon C. Doctor en Ciencias Biologicas, UNAM, Mexico. Asistente de investigador. UNICACH, Mexico. e-mail: delacruz277@ hotmail.com

Guillermo Laguna Hernandez. Doctor en Ciencias Biologicas, UNAM, Mexico. Profesor, UNICACH, Mexico. e-mail: glh@hp.fciencias.unam.m x

Salvador Flores Breceda. Maestro en Ciencias en Sanidad Vegetal, Instituto Tecnologico y de Estudios Superiores de Monterrey, Mexico. Jefe, Departamento de Deteccion y Control, Programa Moscafrut SAGARPA-IICA, Mexico. email: sfbrceda@hotmail.com

Pablo Montoya Gerardo. Doctor en Ciencias Biologicas, UNAM, Mexico. Subdirector de Desarrollo de Metodos, Programa Moscafrut SAGARPA-IICA, Mexico. e-mail: pablojmgl7@ hotmail.com
TABLE I
LARVICIDAL ACTIVITY OF Annona SPP. EXTRACTS AGAINST THIRD INSTAR
LARVAE OF A. ludens AT DIFFERENT EXPOSURE TIMES (24 vs 72h)

                                           Mortality (%)
Specie         Extract   Tissue       100[micro]g x [ml.sup.-1]

                                             Exposures time
                                        24h                 72h

A. diver-     Ethanolic  Leaves  1.0 [+ or -] 1.0   22.0 [+ or -] 30
   sifolia    Ethanolic  Stems   1.2 [+ or -] 1.3    5.8 [+ or -] 0.7
              Aqueous    Leaves  0.0 [+ or -] 0     70.3 [+ or -] 18.4 *
              Aqueous    Stems   0.2 [+ or -] 0.4   44.3 [+ or -] 4.2 *

A. lutescens  Ethanolic  Leaves  2.4 [+ or -] 2.1   27.0 [+ or -] 7.7
              Ethanolic  Stems   0.2 [+ or -] 0.4   29.5 [+ or -] 1.5 *
              Aqueous    Leaves  1.0 [+ or -] 1.7   81.7 [+ or -] 4.9 *
              Aqueous    Stems   5.4 [+ or -] 4.4   95.9 [+ or -] 3.8 *

A. muricata   Ethanolic  Leaves  1.4 [+ or -] 0.9   63.3 [+ or -] 3.5 *
              Ethanolic  Stems   1.0 [+ or -] 1.2    2.0 [+ or -] 1.5
              Aqueous    Leaves  0.6 [+ or -] 0.9   78.3 [+ or -] 12 *
              Aqueous    Stems   1.0 [+ or -] 0.71  86.0 [+ or -] 2.8 *

                                     Mortality (%)
Specie         Extract        1000[micro]g x [ml.sup.-1]

                                     Exposures time
                               24h                  72h

A. diver-     Ethanolic  2.2 [+ or -] 1.8   68.3 [+ or -] 1.4 *
   sifolia    Ethanolic  1.5 [+ or -] 2.4   89.3 [+ or -] 14.1 *
              Aqueous    2.4 [+ or -] 2.6   70.3 [+ or -] 8.0 *
              Aqueous    0.8 [+ or -] 0.8   74.3 [+ or -] 14.0 *

A. lutescens  Ethanolic  1.6 [+ or -] 1.9   27.0 [+ or -] 4.9
              Ethanolic  0.4 [+ or -] 0.9   70.3 [+ or -] 14.9 *
              Aqueous    2.2 [+ or -] 1.8   94.4 [+ or -] 1 *
              Aqueous    1.8 [+ or -] 2.9   34.1 [+ or -] 13.4 *

A. muricata   Ethanolic  0.2 [+ or -] 0.4   48.3 [+ or -] 63 *
              Ethanolic  0.2 [+ or -] 0.5   61.3 [+ or -] 19.8 *
              Aqueous    1.4 [+ or -] 2.1   84.8 [+ or -] 2.1 *
              Aqueous    1.0 [+ or -] 1     75.5 [+ or -] 23.3 *

Mortality percentages are given as means [+ or -] SD. The values
followed by an asterisk are significantly different (P<0.05) by an F
test (Bonferroni 95% confidence interval).

TABLE II
COMPARISON OF EXTRACTS BY STATISTICAL ANALYSIS

                    Aqueous      Ethanolic       Leaf         Stem
                  leaf & stem   leaf & stem   aqueous &    aqueous &
                                              ethanolic    ethanolic

A. diversifolia   t= -0.6122    t= -3.4163    t=  0.4427   t= -1.68631
                   P= 0.5574     P= 0.0091    P=  0.6697   P=  0.1302
A. lutescens      t= 10.0752    t= -6.3222    t= 31.0563   t= -4.0928
                   P= 0.0000     P= 0.0002    P=  1.2 E-9  P=  0.0035
A. muricata        t= 0.9512    t= -0.5515    t=  1.6381   t=  1.08089
                   P= 0.3694     P= 0.5964    P=  0.1400   P=  0.3113

P<0.05 indicates significant differences

TABLE III
REGRESSION PARAMETERS OF PROBIT ANALYSIS FOR THE MORTALITY (EXPRESSED
IN TERMS OF [LC.sub.50] AND [LC.sub.90] ([micro]g x [ml.sup.-1])) OF
A. salina LARVAE FOLLOWING EXPOSURE TO Annona SPECIES EXTRACTS

                                     [CL.sub.50]     Inf.       Sup.
                                                     limit      limit

A. diversifolia   Aqueous     Stem     588.685       113.46     914.262
                  Aqueous     Leaf   >1000
                  Ethanolic   Stem     409.139       206.39     568.503
                  Ethanolic   Leaf      52.0284    -1089.4      478.278
A. lutescens      Aqueous     Stem    1016.61        893.93    1160.13
                  Aqueous     Leaf   >1000
                  Ethanolic   Stem    7115.48       1479.1
                  Ethanolic   Leaf     318.972       -26.239    546.138
A. muricata       Aqueous     Stem   >1000
                  Aqueous     Leaf   >1000
                  Ethanolic   Stem     865           113.92    1564.45
                  Ethanolic   Leaf     831.445       642.44    1024.48

                               [CL.sub.90]    Inf.         Sup.
                                              limit        limit

A. diversifolia   Aqueous       2898.4       2184.89      4806.1
                  Aqueous      11407
                  Ethanolic     1412.97      1174.94      1825.79
                  Ethanolic     2726.5       1971.26      5268.83
A. lutescens      Aqueous        341.84       138.682      488.207
                  Aqueous        707.00
                  Ethanolic       1<
                  Ethanolic       1<
A. muricata       Aqueous       3984.2                    2340.18
                  Aqueous       3852.6       1565.16
                  Ethanolic     4539         2966.69     13851.1
                  Ethanolic     2058.3       1735.15      2606.81

[LC.sub.50]: lethal concentration to cause 50% mortality in
population, LC: lethal concentration to cause 90% mortality in
population, CI: confidence interval.
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Title Annotation:Articulo en ingles
Author:Gonzalez-Esquinca, Alma Rosa; Luna-Cazares, Lorena Mercedes; Guzman, Maria Adelina Schlie; De la Cru
Publication:Interciencia
Date:Apr 1, 2012
Words:4745
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