Present investigations evaluated the impact of Argemone mexicana stem extracts on the reproductive fitness of dengue vector, Aedes aegypti, assessed in terms of oviposition deterrent and ovicidal potential. The oviposition deterrent studies of the extracts, prepared using petroleum ether, hexane, benzene, acetone, and ethanol as the solvents, revealed the maximum deterrence potency of the petroleum ether extracts with a significant 15.6% ED at 60 ppm rising by 83.8% at 1000 ppm to 99.4% effective deterrence (ED). Other stem extracts were found to be ineffective at 60 ppm, though resulted in 85.3–96.2% ED and diminished fecundity in A. aegypti at 1000 ppm. Further, A. mexicana stem extracts exhibited moderate ovicidal potential against A. aegypti eggs causing only 42.65–67.85% egg mortality at 1000 ppm, the lowest hatch of 32.15% caused by the benzene extract. Other stem extracts also failed to express effective ovicidal potency with the percent egg hatch ranging between 96.6 and 99.0 at 400 ppm, and 78.8 and 99.0 at 600 ppm. Our results suggest the significant but variable efficacy of A. mexicana stem extracts causing reproductive disadvantage in A. aegypti.
Introduction
Vector-borne diseases are a major source of infirmity worldwide. Mosquitoes constitute a major public health problem as vectors of severe human diseases, such as malaria, filariasis, yellow fever, dengue, and Japanese encephalitis, contributing significantly to an increasing number of fatalities in tropical and subtropical countries of the world.1,2 In India, with an annual incidence of 700 million clinically manifested cases and a death toll of 1.1–2.7 million, dengue is considered as one of the most important diseases.3 As till date there is neither any specific medication nor an effective vaccine available against dengue virus, the only way to control the disease is to control the mosquito vector, Aedes aegypti, agent of disease transmission.
During the past few decades, worldwide efforts have been made to control the mosquito population. The synthetic chemical insecticides have been used since long as the quickest and most sought-after approach to deal with the increasing and widely spreading mosquito population. However, the indiscriminate and persistent use of insecticides has produced serious repercussions, such as insect resistance to insecticides, mammalian toxicity, harm to non-target organisms, bioaccumulation, and environment damage.4,5 These problems have directed the attention of researchers toward the immediate need for novel insecticides.6
Over the past few years, the botanical insecticides have caught researchers' attention as alternate mosquito control agents, being environmentally safe and non-hazardous to non-target organisms.
Several phytochemicals isolated from various botanical sources have been reported to have detrimental effects on different species of mosquitoes, inducing multiple effects such as larvicidal, fecundity suppression, ovicidal activity, oviposition deterrence, growth inhibition, etc.7891011–12
Weeds, the so-called nuisance plants, belong to one such group, which has attracted researchers' attention as eco-friendly substitutes to chemical insecticides for the mosquito management.9,10 The larvicidal potential of the extracts prepared from the different parts of Mexican prickly poppy, Argemone mexicana, has been reported against the early fourth instars of A. aegypti.10 These extracts have also been shown to induce behavioral and morphological modifications in the larvae of A. aegypti. Researchers have also reported the larvicidal and chemosterilant activity of phytochemicals derived from A. mexicana seeds against A. aegypti.13
The literature available, however, reveals that the impact of this plant on the reproductive fitness and behavior of mosquito vectors, in terms of ovicidal and oviposition deterrent potential, has not been explored extensively. Hence, the present study was undertaken to assess the ovicidal efficacy and oviposition deterrence potential of the extracts prepared from the stems of A. mexicana against A. aegypti. These investigations may provide significant information in formulating a new agent modifying the reproductive fitness of the mosquito adversely and formulating an appropriate mosquito management strategy against the dengue vector.
Materials and Methods
Mosquito Rearing
Different stages of the dengue fever mosquito, A. aegypti, were collected from seven different locations across Delhi, India, and the surrounding areas (Fig. 1). The colony was maintained in a rearing laboratory at 28 ± 1°C, 80 ± 5% Relative Humidity (RH), and 14:10 Light/Dark (L/D) photoperiod.14 The adults were reared in screened cloth cages (45 × 40 × 40 cm) with a moist cotton pad placed on the top of cage to provide water for them. Water-soaked split raisins were placed in the cage as a source of food primarily for the male mosquitoes. Intermittent blood meals were provided to the females, for the maturation of egg follicles, by keeping restrained albino rats in the cages. The eggs were collected in an enamel bowl half-filled with dechlorinated tap water and lined with Whatman filter paper on all the sides. The eggs were transferred to the trays (12” × 16”) filled with dechlorinated water and allowed to hatch. Hatched larvae were fed upon a mixture of finely ground dog biscuits (procured from M/s Lal Pet Products, India) and yeast (procured from M/s Solar Sales, India) in the ratio of 3:1 by weight with utmost care in order to prevent formation of any scum on the surface of water. The pupae formed were collected in an enamel bowl and transferred to the cages for adult emergence.
Figure 1.
Areas selected in Delhi and NCR for the collection of mosquito larvae. http://d-maps.com/carte.php?num_car=16531&lang=en.
Plant Collection
The A. mexicana plant was collected from New Delhi, India and surrounding regions, and brought to the laboratory. The stems were carefully cut from the plant and thoroughly washed with dechlorinated tap water to remove traces of dust. The stems were critically scrutinized under microscope, and disease-free, healthy stems were separated. The selected stems were kept under shade at room temperature of 27 ± 2°C for about 20 days till they dried completely. The dried stems were crushed into powdered form and sieved through a nylon strainer to obtain fine powder.
Preparation of the Extract
The powdered stem material was weighed and 200 g of the material was extracted separately with 1000 mL of five different solvents of varying polarities, viz, petroleum ether, hexane, benzene, acetone, and ethanol, using a Soxhlet extraction apparatus. The extraction continued for 3 days, 8 hours per day, at a temperature not beyond the boiling point of the respective solvent. The five crude extracts, thus formed, were concentrated using a rotary vacuum evaporator at 45°C under low pressure and stored in a refrigerator at 4°C as the stock solution of 1000 ppm for investigating their oviposition deterrent and ovicidal potential against A. aegypti. The dilutions of each extract were prepared using ethanol as the solvent.
Oviposition Deterrence Studies
The oviposition deterrence studies were performed against A. aegypti under controlled temperature, humidity, and photoperiod conditions using multiple concentration tests of A. mexicana stem extract prepared in a particular solvent. The experiment was conducted on 25 blood-fed females of A. aegypti released in the cage along with an equal number of males. For each extract, a total of 10 small bowls lined with Whatman filter paper were prepared for oviposition. Nine of these bowls were filled with 99 mL of dechlorinated distilled water and 1 mL of a particular concentration of the selected stem extract. The tenth oviposition bowl served as the control and contained 99 mL of dechlorinated distilled water and 1 mL of ethanol. These oviposition bowls were placed in the cage at random places for egg laying. The position of each cup was changed each day to negate the position effect, if any. The bowls were kept till no further egg laying was noticed, for at least 48 hours. The eggs laid into each cup were collected every day separately and scored using a dissecting microscope at the magnification of x40. Identical protocol was followed for investigation on the oviposition deterrent efficacy of other stem extracts against A. aegypti. Three replicates of each concentration of each extract were carried out for evaluating the deterrent efficacy of extracts.
The impact of the stem extracts of A. mexicana on the egg-laying capacity, and thus, the reproductive fitness of female A. aegypti was assessed by calculating the fecundity rate of females at each concentration of every extract using the following formula:
The percent effective deterrence of each extract was evaluated by the following formula in order to evaluate their potential to reduce the reproductive fitness of females and deter them for egg laying.
where ED = effective deterrence, NC = total number of eggs laid in control bowl, and NT = total number of eggs laid in an experimental bowl. Based on the above observations, the Oviposition Activity Index (OAI) of female A. aegypti was also calculated in each case using the following formula:
The results obtained were analyzed by one-way analysis of variance (ANOVA), and means were separated using Tukey's test for statistical significance considered for P ≤ 0.05.
Ovicidal Studies
The potential of all the five extracts prepared from the stems of A. mexicana was also estimated for their efficacy as ovicidal agents against A. aegypti. The investigations were performed at 28 ± 1°C on the freshly laid eggs of A. aegypti collected in oviposition bowls filled with dechlorinated distilled water and lined with Whatman filter paper. The eggs were counted using a dissecting microscope at the magnification of 40x.
A total of 250 freshly laid eggs of A. aegypti were counted and transferred to a 250 mL glass jar containing homogenous test solution of 1 mL of a particular concentration of the extract and 99 mL of distilled water. Likewise, nine glass jars were prepared, each containing 250 eggs and 1 mL of a particular concentration of the extract and 99 mL of distilled water. Control treatments of the eggs were carried out by replacing the extract with only ethanol. After the exposure period of 24 hours, the treated eggs from each jar were carefully transferred to another glass jar containing distilled water and were washed thoroughly to ensure the removal of any traces of the extract. Thereafter, the eggs were submerged in trays containing only dechlorinated water and were allowed to hatch. The hatched larvae were counted, and the percent hatch was calculated in each instance.
The percent hatch in control treatment was compared with the percent hatch observed in each concentration of the stem extract to assess the ovicidal potential of the extract. Identical protocol was followed for all the stem extracts, and three replicates were carried out simultaneously for each concentration of the extract making a total of 750 eggs for each test. The results obtained were analyzed by one-way ANOVA, and means were separated using Tukey's test for statistical significance considered for P ≤ 0.05. After treatment, the eggs unable to hatch were scrutinized under light microscopy for any morphological alterations.
Results
The present investigation evidently indicates the potential of stem extracts of A. mexicana to deter the oviposition by A. aegypti and diminish their reproductive fitness (Tables 1–3 and Fig. 2). Conversely, these extracts were found only moderately effective as ovicidal agents as they could not establish significantly high ovicidal efficacy even at very high concentrations (Tables 4 and 5).
Table 1.
Oviposition deterrent activity of the stem extracts of Argemone mexicana against female Aedes aegypti.
Table 2.
Statistical analysis (ANOVA) for Oviposition deterrence activity of the stem extracts of Argemone mexicana against female Aedes aegypti.
Table 3.
Oviposition activity index of the stem extracts of Argemone mexicana against females Aedes aegypti.
Figure 2.
Percent effective deterrence of various stem extracts of Argemone mexicana against female Aedes aegypti.
Table 4.
Ovicidal effects of the stem extracts of Argemone mexicana on freshly laid eggs of Aedes aegypti.
Table 5.
Statistical analysis (ANOVA) for ovicidal activity of the stem extracts of Argemone mexicana against female Aedes aegypti.
Our studies clearly concluded the increasing oviposition deterrence of the stem extracts of A. mexicana with their increasing concentration. The petroleum ether extract Petroleum ether Stem Extract (PSE) was found to be the most effective extract to deter the egg laying and cause reproductive disadvantage in female A. aegypti. The extract established a significant %ED of 15.6% (P < 0.001; F = 121.28) at as low as 60 ppm (Fig. 2) causing a total of 202 eggs laid in the PSE bowls as against 239 eggs laid in control bowl (Table 1). The percent effective deterrence of the extract rose by 83.8% at 1000 ppm causing significant reproductive disadvantage resulting in only two eggs being laid by female A. aegypti (Table 1). The efficacy of other extracts was found in the decreasing order of the moderately significant oviposition deterrence exhibited by the hexane extract followed by that shown by benzene and ethanol stem extracts. The hexane stem extract caused an appreciable increase in the %ED against A. aegypti ranging between 18.98 and 96.1% (P < 0.001; F = 435.42) with the concentration of extract increasing from 80 to 1000 ppm. Likewise, Benzene Stem Extract (BSE) resulted in a significant 83% increase in reproductive disadvantage of A. aegypti females with 13.2% (P < 0.01) ED at 80 ppm rising up to 96.2% (P < 0.01; F = 397.93) at 1000 ppm. A more drastic decrease in reproductive fitness was seen in Ethanol Stem Extract (ESE)-containing oviposition medium, which revealed an 88% decreased egg laying with increase in concentration from 80 to 1000 ppm (P < 0.01, F = 291.03). On the other hand, the Acetone Stem Extract (ASE) was found to be the least effective affecting reproductive fitness of A. aegypti being the only extract with a positive OAI of 0.01 at 80 ppm (Table 3).
The stem extracts of A. mexicana, however, could establish just moderately effective ovicidal potential against A. aegypti eggs failing to cause high levels of egg mortality even at 1000 ppm. The results of the ovicidal potential of different stem extracts of A. mexicana against A. aegypti eggs are presented in Tables 4 and 5. Among the five different stem extracts tested, the BSE caused the lowest percent egg hatch of 32.15% at 1000 ppm reducing by 21% at 800 ppm though still causing toxicity to 46.85% eggs (P < 0.05; F = 51.76). Other stem extracts also failed to express effective ovicidal potency resulting in 98–99% egg hatch till 400 ppm and only 51–58% at 1000 ppm (Table 4).
Discussion
The rapid increase in global population of mosquitoes has caused alarming rise in the mosquito-borne diseases, which have become a source of major annoyance and fatalities. The synthetic chemical insecticides have been used since long as the quickest and most sought-after approach to deal with the increasing and widely spreading mosquito population. However, the indiscriminate and persistent use of insecticides has elicited detrimental effects, such as development of resistance amongst the target mosquito population, and everlasting toxicity to non-target organisms, environment, and humans. To address and overcome these issues, botanical insecticides are now being used widely as mosquito control agents, being environmentally safe and non-hazardous to non-target organisms. Researchers have reported use of botanicals as larvicides, ovicides, growth regulators, and behavioral modifiers against mosquitoes.4,9,10,12 Despite various efforts, the mosquito population is on continual rise leading to increasing incidences of mosquito-borne diseases. A. aegypti, a dengue vector, has become the major nuisance in tropical and subtropical countries.
Scientists have reported that oviposition is one of the most critical events in the life cycle of mosquitoes that chiefly determines several life parameters, such as population densities, species propagation, and dispersal of species in different geographical zones.15 In some mosquito species, female choice in oviposition site is adaptive.16 Thus, selection of the appropriate egg-laying site can dramatically increase the survival, causing increased egg hatch and efficient development of their offspring. Keeping in view that assessment of the oviposition decisions of mosquitoes may provide additional information on the factors influencing reproductive fitness and regulation of the Aedes population, further assisting in predicting the response of mosquito population to various control measures, the impact of the extracts prepared from the weed A. mexicana stems was evaluated against the reproductive fitness of the females of A. aegypti.
Our study demonstrated the significant oviposition deterrent potential of the five different stem extracts of A. mexicana against gravid females of A. aegypti leading to appreciable reproductive disadvantage. With the percent effective oviposition deterrence ranging between 85 and 99% at 1000 ppm, the PSE of A. mexicana was found to be the most efficient deterrent agent against A. aegypti while the ASE showed the least effective oviposition deterrent potential. These results are in conformity with those of Warikoo and Kumar4 who reported the 100% oviposition deterrence potential of 1000 ppm petroleum ether root extract of A. mexicana against an Indian strain of A. aegypti. Similarly, Kumar et al12 reported the appreciable efficacy of 1000 ppm diethyl ether leaf extract of Parthenium hysterophorus causing the significantly diminished fecundity in A. aegypti with 99.7% effective repellency and considerably high deterrence potential of petroleum ether leaf extracts resulting in 94% effective repellency at 1000 ppm. The oviposition deterrence effects of ethanolic leaf extract of Cassia obtusifolia was studied by Rajkumar and Jebanesan17 who revealed the 75.5% ED at 100 mg/L rising to 92.5% at 400 mg/L. Elango et al18 had reported that 500 ppm of acetone, ethyl acetate, and methanol leaf extracts of some indigenous plants exhibited strong deterrent effect against Anopheles subpictus.
The investigation of the oviposition deterrent potential of A. mexicana stem extracts against A. aegypti expresses the OAI of extract, which signifies a global perspective of the relative preference of a substrate by gravid females.19 Positive index values indicate more oviposition in the experimental bowls than in the control bowls proving the inefficacy of extracts affecting reproductive fitness of A. aegypti. On the contrary, more egg laying in the control medium as compared to the experimental media results in negative index values indicating the preference of distilled water for egg deposition over the water-extract mixture. Present investigation resulted in negative OAI values when mixture of petroleum ether stem extracts of A. mexicana and water was used as oviposition medium. It caused much reduced fecundity of A. aegypti affecting its reproductive fitness adversely. Chenniappan and Kadarkarai20 evaluated the oviposition deterrent effects of ethanolic extract of Andrographis paniculata against females of Anopheles stephensi, and reported OAI values of (-)0.28, (-)0.45, (-)0.49, and (-)0.59 for extract concentrations of 29, 35, 41, and 46 ppm, respectively. Kweka et al16 reported the significantly increased oviposition by gravid Anopheles gambiae in the control cups as compared to that in water containing essential oils from Ocimum kilimandscharicum and Ocimum suave. The OAI in water–oil mixture was found to be negative ranging from (-)0.19 to (-)1.0 indicating the potential of Ocimum sp. to deter oviposition in A. gambiae leading to reproductive disadvantage.
Present investigations have also tried to assess the ovicidal potential of A. mexicana stem extracts, which could then allow formulation of effective ovicides for reduction of A. aegypti population in future. Our results revealed that the stem extracts of A. mexicana could result in only moderate ovicidal effect against the freshly laid eggs of A. aegypti. Among the five extracts tested, the BSE resulted in the lowest percent egg hatch of 32.15% at 1000 ppm, whereas the percent hatch caused by other stem extracts ranged between 51 and 58% only. Similar studies by Kumar et al (2011) revealed the moderate ovicidal effect of 41% shown by petroleum ether leaf extract of P. hysterophorus against eggs of A. aegypti resulting in 59% hatch at 1000 ppm. On the contrary, Govindarajan21 reported that at 200 ppm, the methanol leaf extract of Coccinia indica exerted zero egg hatchability in A. aegypti eggs. Krishnappa and Elumalai6 also reported 100% mortality of A. aegypti eggs at 300 ppm extracts of Basella rubra and at 420 ppm extracts of Cleome viscosa.
Our results clearly indicate the significant and variable efficacy of various extracts prepared from the stems of A. mexicana leading to reproductive disadvantage in A. aegypti. The extracts have established their potential as oviposition deterrents though they resulted in only moderate ovicidal efficacy. However, the mechanism causing these variable impacts of stem extracts of A. mexicana is still unknown and needs to be investigated further. However, it seems probable that variety of types and levels of bioactive constituents in each extract may be responsible for their variable potential against reproductive behavior of A. aegypti. Further investigations are thus recommended to establish A. mexicana as eco-friendly botanical to be used in mosquito control program replacing the harmful conventional insecticides.
Author Contributions
Conceived and designed the experiments: SK, RW. Analyzed the data: RW. Wrote the first draft of the manuscript: RW. Contributed to the writing of the manuscript: SK. Agree with manuscript results and conclusions: SK. Jointly developed the structure and arguments for the paper: SK, RW. Made critical revisions and approved final version: SK. All authors reviewed and approved of the final manuscript.
Acknowledgments
The authors are highly grateful to University Grants Commission, New Delhi, for providing a Senior Research Fellowship to carry out the present investigations. Thanks are extended to Dr Savithri Singh, Principal, Acharya Narendra Dev College, for providing infrastructure and research facilities.
