Abstract

INTRODUCTION

Dengue fever has become a pandemic threat¹ 1. World Health Organization. World malaria report: 2011. Geneva: WHO; 2011.and a major public health challenge to the health officials and policy makers in nearly all the tropical areas of the world²2. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496:504-7.. Frequent outbreaks³3. Enserink M. Massive outbreak draws fresh attention to little-known virus. Science. 2006;311:1085., hospitalisations⁴4. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, et al. Dengue: a continuing global threat. Nat Rev Microbiol. 2010;8(12 Suppl):S7-16. and the increasing number of people at risk, over 40% of the world's population¹1. World Health Organization. World malaria report: 2011. Geneva: WHO; 2011., are reminders of the constant menace of this disease. Efforts to reverse the trend to a higher incidence of mosquito-borne outbreaks have mainly being based on the use of synthetic insecticides, particularly organophosphates and pyrethroids⁵5. World Health Organization. Guidelines for laboratory in field testing of mosquito larvicides. Geneva: WHO; 2005.. Dinetro-o-cresol was first used as a synthetic insecticide in 1892, and since then more potential insecticides have been discovered⁶6. Bara JJ, Montgomery A, Muturi EJ. Sublethal effects of atrazine and glyphosate on life history traits of Aedes aegypti and Aedes albopictus (Diptera: Culicidae). Parasitol Res. 2014;113:2879-86.. Although insecticide-based strategies have sometimes been successful⁷7. Da-Cunha MP, Lima JB, Brogdon WG, Moya GE, Valle D. Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003. Mem Inst Oswaldo Cruz. 2005;100:441-4.^,88. Montella IR, Martins AJ, Viana-Medeiros PF, Lima JB, Braga IA, Valle D. Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. Am J Trop Med Hyg. 2007;77:467-77., the monolithic reliance on these two classes of products has led to adverse effects. Their widespread misuse have caused the development of mosquitoes⁹9. Das N, Goswami D, Rabha B. Preliminary evaluation of mosquito larvicidal efficacy of plant extracts. J Vector Borne Dis. 2007;44:145-8. resistance, with the main vector, Aedes aegypti, ranking eighth in the list of species with the highest reported number of resistant cases worldwide¹⁰10. Whalon ME, Mota-Sanchez D, Hollingworth RM, editors. Global pesticide resistance in arthropods. Wallingford: Cabi; 2008.. The fact that these two classes of insecticides have identical modes of action has generated a cross-resistance problem¹¹11. Bisset JA, Marín R, Rodríguez MM, Severson DW, Ricardo Y, French L, et al. Insecticide resistance in two Aedes aegypti (Diptera: Culicidae) strains from Costa Rica. J Med Entomol. 2013;50:352-61.^,1212. Rodríguez MM, Bisset J, Ruiz M, Soca A. Cross-resistance to pyrethroid and organophosphorus insecticides induced by selection with temephos in Aedes aegypti (Diptera: Culicidae) from Cuba. J Med Entomol. 2002;39:882-8.. These problems have hampered the effectiveness of current control measures. Despite the efforts to test other substances, only a few insecticides have become available¹³13. Nkya TE, Akhouayri I, Kisinza W, David JP. Impact of environment on mosquito response to pyrethroid insecticides: facts, evidences and prospects. Insect Biochem Mol Biol. 2013;43:407-16.. These circumstances have stimulated the search for new sustainable control strategies, such as the use of botanical insecticides¹⁴14. Regnault-Roger C. Essential oils in insect control. In: Ramawat KG, Mérillon JM, editors. Natural products. New York: Springer; 2013. p. 4088-102.^,1515. Ghosh A, Chowdhury N, Chandra G. Plant extracts as potential mosquito larvicides. Indian J Med Res. 2012;135:581-98..

Plants produce a broad range of bioactive chemical compounds¹⁶16. Bernhoft A. A brief review on bioactive compounds in plants: proceedings from a symposium held at The Norwegian Academy of Science and Letters, Oslo, 13 - 14 November 2008. Oslo: The Norwegian Academy of Science and Letters; 2010.. Several plants have been screened due to their insecticidal properties and crude extracts of many plant species have shown promising mosquitocidal activities¹⁷17. Sukumar K, Perich MJ, Boobar LR. Botanical derivatives in mosquito control: a review. J Am Mosq Control Assoc. 1991;7:210-37.^,1818. Tennyson S, Ravindran KJ, Arivoli S. Screening of twenty five plant extracts for larvicidal activity against Culex quinquefasciatus Say (Diptera: Culicidae). Asian Pacific J Trop Biomed. 2012;2 Suppl:S1130-4.. Some phytochemical insecticides are even more effective than synthetic ones¹⁹19. Olaitan A, Abiodun A. Comparative toxicity of botanical and synthetic insecticides against major field insect pests of Cowpea (Vigna unquiculata (L) Walp). J Nat Prod Plant Resourc. 2011;1:86-95.. Aedes aegypti in Thailand was found to be susceptible to the essential oil of the plants Carum carvi, Curcuma zedoria, Piper longum, Apium graveolens and Illicium verum even though these mosquitos had already shown resistance to the permethrin insecticide²⁰20. Chaiyasit D, Choochote W, Rattanachanpichai E, Chaithong U, Chaiwong P, Jitpakdi A, et al. Essential oils as potential adulticides against two populations of Aedes aegypti, the laboratory and natural field strains, in Chiang Mai province, northern Thailand. Parasitol Res. 2006;99:715-21.. Besides being generally target-specific²¹21. Cetin H, Erler F, Yanikoglu A. Larvicidal activity of a botanical natural product, AkseBio2, against Culex pipiens. Fitoterapia. 2004;75:724-8.^,2222. Mann RS, Kaufman PE. Natural product pesticides: their development, delivery and use against insect vectors. Mini Rev Org Chem. 2012;9:185-202., plant-derived insecticides decompose quickly²³23. Khater HF. Prospects of botanical biopesticides in insect pest management. Pharmacologia. 2012;3:641-56., and they are not stored in organisms²⁴24. Isman MB. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol. 2006;51:45-66., and in ecosystems²⁵25. Stark JD, Walter JF. Persistence of azadirachtin A and B in soil: effects of temperature and microbial activity. J Env Sci Health. 1995;30:685-98.. Due to a poor penetrance in ecosystems, large animals are not affected by plant-derived insecticides²⁶26. Evergetis E, Michaelakis A, Haroutounian SA. Essential oils of Umbelliferae (Apiaceae) family taxa as emerging potent agents for mosquito control. In: Soloneski S, editor. Integrated pest management and pest control: current and future tactics. Rijeca: InTech; 2012. [cited 2015 Dec 14]. Available from: http://www.intechopen.com/books/integrated-pest-management-and-pestcontrol-current-and-future-tactics/essential-oils-of-umbelliferae-apiaceae-family-taxa-as-emerging-potentagents-for-mosquito-control
http://www.intechopen.com/books/integrat... . They contain no halogen molecules and are bioactive at lower concentrations²⁷27. Vyvyan JR. Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron. 2002;58:1631-46.. Reductions in progeny size and longevity, delayed development and repellency to adults parasitoids²⁸28. Tunca H, Kilinçer N, Ozkan C. Side-effects of some botanical insecticides and extracts on the parasitoid, Venturia canescens (Grav.) (Hymenoptera: Ichneumonidae). Türk Entomol Derg. 2012;36:205-14. as well as the development of resistance due to overuse²⁹29. Zhao JZ, Li YX, Collins HL, Gusukuma-Minuto L, Mau RF, Thompson GD, et al. Monitoring and characterization of diamondback moth (Lepidoptera: Plutellidae) resistance to spinosad. J Econ Entomol. 2002;95:430-6., are characteristics that have been attributed to botanical insecticides. These characteristics have raised some arguments on the need to reduce the amount of botanical insecticides applied³⁰30. Scott TW. Containment of arthropod disease vectors. ILAR J. 2005;46:53-61.. Phototoxic activities, the geographical source of plant, the use of solvents during extraction are factors that could also affect the effectiveness of plant-derived insecticides on mosquitoes¹⁷17. Sukumar K, Perich MJ, Boobar LR. Botanical derivatives in mosquito control: a review. J Am Mosq Control Assoc. 1991;7:210-37.. The formulation of spot treatment applications are recommended rather than broadcasting over large areas, as this represents a serious menace for non-target invertebrates and biological control agents. This strategy is also important for dengue control perspectives due to an increased interest of integrating botanical and microbial pesticides to control dengue mosquitoes.

One way to avoid pesticide residue hazards and the associated disruption of natural enemies would be to combat the evolution of resistance by reducing the amount of insecticide applied. The use of sublethal doses can help minimizing the effects on the physiology and behaviour of individuals that have been exposed to a pesticide³¹31. Desneux N, Decourtye A, Delpuech JM. The sublethal effects of pesticides on beneficial arthropods. Annu Rev Entomol. 2007;52:81-106.. Exposure to sublethal doses of botanical insecticides has been proven to disrupt the insect nervous system and cause hormonal changes, which alter both the behaviour and the physiological processes³²32. Sanil D, Shetty NJ. The effect of sublethal exposure to temephos and propoxur on reproductive fitness and its influence on circadian rhythms of pupation and adult emergence in Anopheles stephensi Liston-a malaria vector. Parasitol Res. 2012;111:423-32.. In Ae. aegypti, exposure to sublethal doses of botanical insecticides disrupts the development, the larval eclosion and the reproduction outcome³³33. Ma Z, Gulia-Nuss M, Zhang X, Brown MR. Effects of the botanical insecticide, Toosendanin, on blood digestion and egg production by female Aedes aegypti (Diptera: Culicidae): topical application and ingestion. J Med Entomol. 2013;50:112-21.. Two key fitness parameters for all the insects³⁴34. Ellers J, Jervis M. Body size and the timing of egg production in parasitoid wasps. Oikos. 2003;102:164-72.^,3535. Thornhill R, Alcock J. The evolution of insect mating systems. Cambridge: Harvard University; 1983., the body size and reproduction have always been ignored by researchers as main sublethal effects of botanicals extracts, including dengue mosquitoes³⁶36. Ponlawat A, Harrington LC. Age and body size influence male sperm capacity of the dengue vector Aedes aegypti (Diptera: Culicidae). J Med Entomol. 2007;44:422-6.^,3737. McMeniman CJ, O'Neill SL. A virulent Wolbachia infection decreases the viability of the dengue vector Aedes aegypti during periods of embryonic quiescence. PLoS Negl Trop Dis. 2010;4:e748.. Applied in the field, this efficiency may be offset by many environmental factors such as temperature and rain³⁸38. Ghidiu GM, Hitchner EM, Henninger MR. Effect of simulated rainfall on the control of Colorado Potato Beetle (Coleoptera: Chrysomelidae) and Potato Leafhopper (Homoptera: Cicadellidae) with at-plant applications of Imidacloprid, Thiamethoxam or Dinotefuran on potatoes in laboratory and field. In: Stoytcheva M, Zlatev R, editors. Agricultural chemistry. Rijeca: InTech; 2013. [cited 2015 Dec 14]. Available from: http://www.intechopen.com/books/howtoreference/agricultural-chemistry/effect-of-simulated-rainfall-on-the-control-of-colorado-potato-beetle-coleoptera-chrysomelidae-and-p
http://www.intechopen.com/books/howtoref... . It seems likely, therefore, that many insect pests targeted by the application of lethal insecticide doses in nature, end up with sublethal doses. This situation is likely to be more prevalent in countries like Malaysia where the rain falls all year round.

Ipomoea cairica Linn. (Family Convolvulaceae) is an herbaceous perennial plant distributed worldwide³⁹39. Liu RS, Qiu YL, Wei DM, Liu HH, Zhu XY, Tian HQ, et al. Distribution of starch and neutral lipids in the developing anthers of Ipomoea cairica. Ann Bot Fennici. 2011;48:256-62.. This plant was chosen in this study due to the fact that it is considered as unwanted invasive weeds in nature, and it is widely used in fencing of domestic and peri-domestic areas. The study on sublethal effects of this particular plant against Aedes mosquitos has been little or has not been investigated. Therefore, this study was conducted to verify whether sublethal doses of I. cairica acethonilic extracts on parent generation would modify the fecundity, fertility, and the development in the life history traits of progenies of Ae. albopictus and Ae. aegypti. Both of the Aedes species tested are main vectors of dengue and dengue haemorrhagic fever in Malaysia.

MATERIALS AND METHODS

Collection of plant samples

Samples of Ipomoea cairica (Linn.) (Convolvulaceae) were collected from Bayan Baru, Penang, Malaysia (5°25'N, 100°19'E) by using the random sampling method and the taxonomic authentication was made by the Botany Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia with the identification number HERB No 9251.

Mosquitoes colony

Eggs of Ae. aegypti were obtained by placing ovitraps in Bagan Dalam, Butterworth, Penang (5º24'N, 100º23'E), while Ae. albopictus was obtained from Durian Valley, University of Sains Malaysia, Penang, (5º26'N, 100º49'E) two weeks prior to the experiment. To start the colony, eggs were hatched in enamel trays containing dechlorinated tap water until reached early 3^rd instar larvae to be used in sublethal studies. Larvae were fed daily with 0.5 gm of fine powder made from dog biscuit, beef liver, yeast, and milk powder at the ratio of 2:1:1:1. Mosquito cultures were maintained at (28 ± 2 ºC), with 70%-85% relative humidity (RH), and a period of 14 h of light and 10 h of darkness.

Preparation of plant extracts

Ipomoea cairica leaves were air-dried for 7-10 days in the shadow. Dried materials were powdered mechanically by using a commercial electrical stainless steel blender (Panasonic: MX-899TM). The finely ground leaves were extracted using the Soxhlet apparatus (Favorit, Malaysia) with acetone solvent (2000 mL, Qualigens). A total of 40 g of ground leaves were placed in paper thimble (Favorit cellulose extraction thimbles: size 43 X 123 mm), together with some clean pebbles to ensure optimum solvent flow through the plant powder. The apparatus was set to the boiling point of 56 ºC for acetone⁴⁰40. Amer HH, Paxton RR, van Winkle M. Methanol-ethanol-acetone: vapour liquid equilibria. Ind Eng Chem. 1956;48:142-6.. The apparatus ran for three hours, until the solvent colour in the siphon side arm became almost clear. The procedure was repeated twice by replacing the 40 g of leaves powder.

The excess of solvents from collected crude extracts was removed by using a rotary evaporator machine. The speed was set up to 100 rpm and the temperature of the water bath was fixed to the boiling point of acetone at 56 ºC. The solvent from the concentrated crude extract was further removed by placing it in an electrical oven at 37 ºC for a week. The yield of the crude residue from plants varied according to the solvents used. The collected crude extract was weighed and stored in petri dishes at 4 ºC until use.

Dose preparation

A stock solution at 10,000 ppm was prepared by dissolving one gram of crude extracts in 100 ml of acetone solvent. From the stock solution, 1,000 ppm was prepared by dissolving 50 mL of the stock solution in 450 mL of distilled water and subsequent dilutions were made as needed. The crude extract obtained and the stock solutions were stored in a refrigerator until use⁴¹41. Thomas TG, Rao S, Lal S. Mosquito larvicidal properties of essential oil of an indigenous plant, Ipomoea cairica Linn. Jpn J Infect Dis. 2004;57:176-7..

The preliminary bioassay tests revealed that crude acetone extracts of I. cairica leaves were more toxic than the methanol extract from other parts of the plant⁴²42. Ahbirami R, Zuharah WF, Yahaya ZS, Dieng H, Thiagaletchumi M, Fadzly N, et al. Oviposition deterring and oviciding potentials of Ipomoea cairica L. leaf extract against dengue vectors. Trop Biomed. 2014;31:456-65.. Hence this fraction of acethonilic leaves extract was selected to study the sublethal effects on two species of Aedes mosquitoes: Ae. albopictus and Ae. aegypti. The sublethal dose (LC₅₀) which caused 50% of larval mortality was used in this study. In this case, we assumed that 50% of the parent mosquitoes would stay alive and continue to have the first generation progeny. The LC₅₀ value for Ae. albopictus was 105.59 ppm, while for Ae. aegypti it was 101.94 ppm. All of the solutions mentioned above were prepared by using serial dilutions from the stock solution. Mosquito larvae that were treated with LC₅₀ concentrations were considered as treated groups. The control group was exposed only to the distilled water with addition of 1 mL of acetone and used for comparisons. Acetone was added to the control experiment to ensure that the control group would be identical in every aspect of the test solution since we have predicted that a little amount of solvent would still be present in the crude extract.

Method of exposure of third instar larvae to sublethal doses of the crude extract

Six batches of 25 third instar larvae of Ae. albopictus and Ae. aegypti exposed to sublethal concentrations (LC₅₀) of the leaves crude extract were tested separately following the WHO procedures for standard bioassay tests³⁵35. Thornhill R, Alcock J. The evolution of insect mating systems. Cambridge: Harvard University; 1983.. Experiments were carried out at 28 ± 2 ºC and 70%-85% of relative humidity (RH). Twenty-four hours post-treatment, larvae that survived were counted, isolated and rinsed with seasoned water and reared in enamel trays until pupal emergence³²32. Sanil D, Shetty NJ. The effect of sublethal exposure to temephos and propoxur on reproductive fitness and its influence on circadian rhythms of pupation and adult emergence in Anopheles stephensi Liston-a malaria vector. Parasitol Res. 2012;111:423-32.. Larvae in both treated and control groups were fed with a mixture of dog biscuit, beef liver, yeast, and milk powder in ratio of 2:1:1:1. Adults were allowed to emerge in emergence cages (45 × 45 × 40 cm) and were provided with cotton wick soaked with 10% sucrose. Subsequent studies on life traits were conducted on the next generation.

Egg production (fecundity)

After five days of the adults emergence, both treated and control mosquitoes, 30 males and 30 females from each species, Ae. albopictus and Ae. aegypti were transferred separately into oviposition cages using a manual aspirator. Females and males mosquitoes were allowed to mate. Females were then provided with blood meal from restrained rats placed in resting cages during the day (for 12 hours) and they were subsequently supplied with 10% sucrose solutions in a tube with cotton wick for two days. Two days after the blood meal, 30 engorged females were transferred into individual paper cups covered with mesh net. A moist of no.1 Whatman filter paper cut in cone shape was used as the oviposition site. The eggs laid by the mosquitoes were counted daily for a maximum period of seven days under a dissecting microscope (×20).

Egg hatchability (fertility)

To investigate the hatchability of both Aedes mosquito species, 10 paper cones with eggs were randomly selected. Paper cones were allowed to dry for 24 hours before hatching in enamel culture trays containing seasoned water. Eggs that hatched and turned into first instar larvae were counted.

Egg length

For the measurement of the eggs length, paper cones containing eggs from the fecundity study were used. Three paper cones from both control and treated groups of each Aedes sp were selected. The length of 10 randomly selected eggs from each paper cone was measured using a stereomicroscope (Olympus EX41), at the Electron Microscopy Unit, School of Biological Sciences, University of Sains Malaysia (100×) (Fig. 1) and the data were recorded.

Width of larval head capsule

The eggs obtained from the fecundity study were hatched and reared until they turned into third instar larvae. Thirty larvae from the treated and the control groups of each mosquito species were selected for the measurement of the width of the larval head capsule. Larvae were immobilised using 60% alcohol. The width of the head capsule across the dorsal surface at the base of the antennae was measured using a stereomicroscope (100×) (Fig. 2).

Adult wing length

The measurement of the adult mosquito wing length which estimates the body size of aedes mosquitoes was used in this study⁶¹61. Robert LL, Olson JK. Effects of sublethal dosages of insecticides on Culex quinquefasciatus. J Am Mosq Control Assoc. 1989;5:239-46.. After the adult emergence (from the fertility studies), 30 females and 30 males of both species (Ae. albopictus and Ae. aegypti) and tested groups (treated and control) were randomly aspirated and maintained in separate cages. Then, all the individuals were anesthetized with 60% alcohol and the right wing of each individual was detached from the thorax and the length from the axial vein to the outmost extreme of the R1 vein⁵⁰50. Pelizza SA, Scorsetti AC, Tranchida MC. The sublethal effects of the entomopathic fungus Leptolegnia chapmanii on some biological parameters of the dengue vector Aedes aegypti. J Insect Sci. 2013;13:22. was measured using a stereomicroscope (100×) (Fig. 3).

Data analysis

The effective reduction percentage (ER%) for the fecundity was calculated as follows²⁹29. Zhao JZ, Li YX, Collins HL, Gusukuma-Minuto L, Mau RF, Thompson GD, et al. Monitoring and characterization of diamondback moth (Lepidoptera: Plutellidae) resistance to spinosad. J Econ Entomol. 2002;95:430-6.:

ER% = [NC-NT/NC] × 100

NC- number of eggs in the control group

NT- number of eggs in the treated group

The percentage of hatchability (fertility) was calculated as follows³²32. Sanil D, Shetty NJ. The effect of sublethal exposure to temephos and propoxur on reproductive fitness and its influence on circadian rhythms of pupation and adult emergence in Anopheles stephensi Liston-a malaria vector. Parasitol Res. 2012;111:423-32.:

% hatchability = (number of larvae/ number of eggs) × 100

Data from all the experiments of the sublethal effect studies were tested for normality (Shapiro-Wilk) prior to analysis. All the data were natural-log transformed (ln[y+1]) due to violations of the homogeneity of variance¹¹11. Bisset JA, Marín R, Rodríguez MM, Severson DW, Ricardo Y, French L, et al. Insecticide resistance in two Aedes aegypti (Diptera: Culicidae) strains from Costa Rica. J Med Entomol. 2013;50:352-61.. The transformed data were analysed using independent T-test by means of the SPSS software (version 20) to determine the significant differences at p < 0.05 between treated and untreated groups (control). An Arc sine transformation was performed before the analysis of the percentage of data from the fertility study.

RESULTS

Our data suggested that acethonilic leaves extract of I. cairica had a significantly higher impact on the reduction of fecundity and fertility of Ae. Albopictus, while in Ae. aegypti a lower impact was observed in the first generation. Our results indicated that I. cairica acethonilic extract can reduce the populations of Ae. albopictus in parent generation and subsequently reduce the next generation population. Exposure to a sublethal dose (LC₅₀) of I. cairica crude extract of third instar larvae of Ae. albopictus and Ae. aegypti decreased their fecundity by 34.68% and 16.61% compared to the respective controls. Significant reduction on the number of eggs laid by female mosquitoes was only observed in Ae. albopictus mosquitoes (t = 3.12, df = 58, p = 0.030; Table 1).

Fertility of Ae. albopictus was measured by the percentage of hatched eggs, and it was significantly lower in treated groups compared to controls, a 49.06% of reduction (t =-4.857, df = 3.833, p = 0.009; Table 2), whereas in Ae. aegypti, the eggs hatchability was reduced only by 18.55% a not significant reduction in comparison to the control group (t = -2.592, df = 2.969, p = 0.082; Table 2). The statistical significance obtained has clearly depicted that eggs become less fertile or less productive after the parental mosquitoes exposure to the sublethal dose of I. cairica.

However, we found that the length of treated eggs for both species was significantly different from control groups (p < 0.05), as well as the decreased hatchability. The sublethal dose of crude extracts reduced the egg length in Ae. aegypti by 0.02 mm followed by Ae. albopictus (0.01 mm) in comparison to controls.

Even though the size of eggs was not affected by the sublethal dose, the size of the head capsule in third instar larvae was significantly reduced in both Ae. albopictus (t = -3.560, df = 56.645, p = 0.001) and Ae. aegypti (t = -2.505, df = 57.972, p = 0.015). Significantly narrower head capsules were found in both species with reductions of 0.03 mm for Ae. albopictus and 0.02 mm for Ae. aegypti, resulting in smaller larvae in comparison to the control group.

In relation to the smaller size of head capsule, the emerged female and male adult mosquitoes had shorter wing length which indicates smaller body sizes of the adult mosquitoes. As shown in Table 3, the mean wing length of both treated female and male mosquitoes was significantly reduced compared to controls for both mosquito species, Ae. albopictus (Female: t = -5.109, df = 46.39, p = 0.000; Male: t = -2.113, df = 57.70, p = 0.039) and Ae. aegypti (Female: t = -6.526, df = 58, p = 0.000; Male: t = -2.519, df = 57.50, p = 0.015).

DISCUSSION

The present study has shown that exposure of Aedes mosquitoes to sublethal doses of Ipomoea cairica acethonilic extracts has affected the life history traits of these mosquitoes under laboratory conditions. Ae. albopictus was found to be the most affected species regarding their progeny after exposure to lethal concentrations (LC₅₀) of acethonilic I. cairica extracts. The acethonilic extract of I. cairica has been found to be more effective to cause mortality in both Aedes species compared to the methanolic extract⁴²42. Ahbirami R, Zuharah WF, Yahaya ZS, Dieng H, Thiagaletchumi M, Fadzly N, et al. Oviposition deterring and oviciding potentials of Ipomoea cairica L. leaf extract against dengue vectors. Trop Biomed. 2014;31:456-65.. However, Ae. aegypti fecundity and fertility were less influenced by the I. cairica acethonilic extract.

The decrement in fecundity was observed in Ae. albopictus females, indicating that the sublethal dose of I. cairica leaf extract has induced reproductive disadvantages in the exposed group. A similar result has been previously reported following exposure of Culex quinquefasciatus, Anopheles stephensi, and Ae. aegypti larvae to other plant extracts such as Calophyllum inophyllum, Solanum suratense, Samadera indica and Rhinocanthus nasutus extracts that caused a significant reduction in fecundity⁴³43. Muthukrishnan J, Pushpalatha E. Effects of plant extracts on fecundity and fertility of mosquitoes. J App Entomol. 2001;125:31-5.. The methanolic extract of seed and leaf of chinaberry tree, Melia azedarach⁴⁴44. Nathan SS, Kalaivani K, Murugan K. Effects of neem limonoids on the malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Acta Trop. 2005;96:47-55., methanolic extract of Indian white cedar, Dysoxyllum malabaricum⁴⁵45. Nathan SS, Kalaivani K, Sehoon K. Effects of Dysoxylum malabaricum Bedd.(Meliaceae) extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Bioresour Technol. 2006;97:2077-83., limonoids of neem, Azadirachta indica⁴⁴44. Nathan SS, Kalaivani K, Murugan K. Effects of neem limonoids on the malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Acta Trop. 2005;96:47-55. at various concentrations have remarkably decreased the fecundity of An. stephensi. Pyrethrin derived from Tanacetum cinerariaefolium which is known to be a highly effective insecticide against both Aedes species can cause a reduction of eggs production in emerging adults after exposure⁴⁶46. Sulaiman S, Fadhlina K, Hidayatulfathi O. Evaluation of pyrethrin formulations on dengue/dengue haemorrhagic fever vectors in the laboratory and sublethal effects. Iranian J Arthropod-Borne Dis. 2007;1:1-6.. In addition, the naturally-derived insecticide spinosad has significantly decreased the fecundity in exposed females compared to unexposed females of Ae. aegypti⁴⁷47. Antonio GE, Sanchez D, Williams T, Marina CF. Paradoxical effects of sublethal exposure to the naturally derived insecticide spinosad in the dengue vector mosquito, Aedes aegypti. Pest Manag Sci. 2009;65:323-6..

Similarly, several available studies have reported a fecundity and reproductive potential reduction in mosquito species after exposure to a sublethal dose of chemical insecticides. Temephos and propoxur have induced a marginal fecundity reduction in An. stephensi³²32. Sanil D, Shetty NJ. The effect of sublethal exposure to temephos and propoxur on reproductive fitness and its influence on circadian rhythms of pupation and adult emergence in Anopheles stephensi Liston-a malaria vector. Parasitol Res. 2012;111:423-32. and the exposure of Ae. aegypti females to higher concentrations of temephos, malathion and alpamenthtin has shown decreasing numbers of eggs production⁴⁸48. Reyes-Villanueva F, Juarez-Eguia M, Flores-Leal A. Effects of sublethal dosages of Abate upon adult fecundity and longevity of Aedes aegypti. J Am Mosq Control Assoc. 1990;6:739-41.^,4949. Minn Z, Shetty N. Toxicological effect of malathion and alphamethrin on reproductive potential in Aedes aegypti, a yellow fever mosquito. Pestology. 2008;32:39-43.. Also, a decreased fecundity upon exposure to entomopathogenic fungi, Leptolegnia chapmanii⁵⁰50. Pelizza SA, Scorsetti AC, Tranchida MC. The sublethal effects of the entomopathic fungus Leptolegnia chapmanii on some biological parameters of the dengue vector Aedes aegypti. J Insect Sci. 2013;13:22. and bacterial agents, Bacillus thuringiensis H-14⁵¹51. Wang LY, Jaal Z. Sublethal effects of Bacillus thuringiensis H-14 on the survival rate, longevity, fecundity and F1 generation development period of Aedes aegypti. Dengue Bull. 2005;29:192-6. has been observed in Ae. aegypti.

The fecundity reduction in the present study led us to speculate that the plant extract used has a certain mode of action to induce reproductive disadvantages in the Aedes treated group. The insects that were fed on plant secondary metabolites have shown toxic effects that in turn have affected the insects' physiology in different ways and at various receptor sites¹⁵15. Ghosh A, Chowdhury N, Chandra G. Plant extracts as potential mosquito larvicides. Indian J Med Res. 2012;135:581-98.. It has been reported that I. cairica contains alkaloids⁵²52. Lin RJ, Chen CY, Lo WL. Cytotoxic activity of Ipomoea cairica. Nat Product Res. 2008;22:747-53. which act as plant phytochemicals⁵³53. Kennedy DO, Wightman EL. Herbal extracts and phytochemicals: plant secondary metabolites and the enhancement of human brain function. Adv Nutr. 2011;2:32-50. and natural mosquito larvicides⁵⁴54. Talontsi FM, Matasyoh JC, Ngoumfo RM, Chepkorir R. Mosquito larvicidal activity of alkaloids from Zanthoxylum lemairei against the malaria vector Anopheles gambiae. Pestic Biochem Physiol. 2011;99:82-5.. The mode of action of alkaloids on insect vectors varies according to the molecules structure, but they are mainly reported to act on acetylcholinestrase (AChE) and sodium channels, which are the most important physical disruption in insects¹⁵15. Ghosh A, Chowdhury N, Chandra G. Plant extracts as potential mosquito larvicides. Indian J Med Res. 2012;135:581-98.. The insects' neuro-hormone system plays a key role in regulating a complex behavioural and physiological mechanisms related to their reproduction. Exposure to insecticide toxins cause coordination disruption resulting in a diminished reproductive capability⁵⁵55. Haynes KF. Sublethal effects of neurotoxic insecticides on insect behavior. Annu Rev Entomol. 1988;33:149-68.^,5656. Lee CY. Sublethal effects of insecticides on longevity, fecundity and behaviour of insect pests: a review. J Biosci. 2000;11:107-12..

Regarding the eggs hatchability, the present study has shown that the eggs produced after exposure of parent mosquitoes to a sublethal dose of I. cairica leaf extracts were less fertile. It has been reported that temephos and propoxur-treated females produced less viable eggs which may be due to the direct toxic effect of the insecticide that remained un-metabolized in the body of the parent and may have been transmitted to the eggs³²32. Sanil D, Shetty NJ. The effect of sublethal exposure to temephos and propoxur on reproductive fitness and its influence on circadian rhythms of pupation and adult emergence in Anopheles stephensi Liston-a malaria vector. Parasitol Res. 2012;111:423-32.. A decrease in the production of a viable offspring is a common end result of sublethal poisoning⁵⁵55. Haynes KF. Sublethal effects of neurotoxic insecticides on insect behavior. Annu Rev Entomol. 1988;33:149-68.. Besides lignans, I. cairica was reported to contain coumarins as one of its major constituents⁵⁷57. Lima OO, Braz-Filho R. Dibenzylbutyrolactone lignans and coumarins from Ipomoea cairica. J Braz Chem Soc. 1997;8:235-8., so it might be possible that the cytotoxicity of coumarines caused some unfavourable alterations on the embryonic development of Ae. albopictus eggs and thus decreased their hatchability. Similarly, coumarines from Mammea siamensis were believed to cause abnormalities in the development of reproductive organs, have direct effect on the eggs and affect some fundamental aspects related to the embryonic development⁵⁸58. Promsiri S, Naksathit A, Kruatrachue M, Thavara U. Evaluations of larvicidal activity of medicinal plant extracts to Aedes aegypti (Diptera: Culicidae) and other effects on a non target fish. Insect Sci. 2006;13:179-88.. Similar findings were reported in fertility studies of Stegomyia albopicta, in which the percentage of egg hatching was reduced after sublethal exposure of females to boric acid sugar bait⁵⁹59. Ali A, Xue RD, Barnard DR. Effects of sublethal exposure to boric acid sugar bait on adult survival, host-seeking, bloodfeeding behavior, and reproduction of Stegomyia albopicta. J Am Mosq Control Assoc. 2006;22:464-8.. The treatment of Cx. quinquefasciatus and Ae aegypti, with 50% EC₅₀ of Calophyllum inophyllum and Rhinocanthus nasutus leaf extract has reduced the fertility from 94.9 to 30.2 % and 82.2 to 37.7% respectively, when compared to controls⁴³43. Muthukrishnan J, Pushpalatha E. Effects of plant extracts on fecundity and fertility of mosquitoes. J App Entomol. 2001;125:31-5.. Also, adults of An. stephensi exposed as larvae to sublethal concentrations of alkaloids isolated from Annona squamosa seeds were found to produce eggs with a 27.7% reduced hatchability in comparison to controls and the plant extract has disrupted some growth stimulating activities in the tested mosquitoes⁶⁰60. Saxena RC, Harshan V, Saxena A, Sukumaran P, Sharma MC, Kumar ML. Larvicidal and chemosterilant activity of Annona squamosa alkaloids against Anopheles stephensi. J Am Mosq Control Assoc. 1993;9:84-7..

The present study showed a slight but not significant reduction of the egg length when the parent larval stage was exposed to a sublethal concentration of I. cairica plant extract in both mosquito species. However, few studies have reported the positive sublethal effects of phytochemicals on mosquito egg length. Females of Cx. quinquefasciatus laid shorter eggs than the control group following exposure to a sublethal dose of malathion and methoprene⁶¹61. Robert LL, Olson JK. Effects of sublethal dosages of insecticides on Culex quinquefasciatus. J Am Mosq Control Assoc. 1989;5:239-46.. In contrast, our study suggested that the eggs length were insignificantly reduced in size when a sublethal effect of the extract was used. Regarding the reproductive disadvantages caused by the plant extract, the sublethal dose did not affect the size or the length of the produced eggs. It is possible that the sublethal dose effect was more pronounced on the quantity and quality of the produced eggs. A similar finding was reported with respect to the turnip moth (Agrotis segetum). Females treated with the dithiocarbamate fungicide mancozeb showed a reduced fecundity but there were no significant differences in egg length⁶²62. Adamski Z, Krawiec J, Markiewicz E, Bankiet M, Rybska E, Ratajczak M, et al. Effect of dithiocarbamate fungicide mancozeb on development, reproduction and ultrastructure of fat body of Agrotis segetum moths. Karaelmas Sci Eng J. 2011;1:7-16..

The results from our study have also revealed that third instar larvae of both Aedes species which emerged from eggs of treated parents had significantly narrower width of head capsules in comparison to the control group. The measurement of the larval head capsule is an important indicator of insect larvae developmental stages⁶³63. Daly HV. Insect morphometrics. Annu Rev Entomol. 1985;30:415-38.. So far, there are no published reports regarding sublethal effects of plant extracts on the width of mosquito larval head capsules. As mentioned previously, the egg length was not significantly reduced, but we have observed a significant decrease in the larval head capsule. Thus, it might be possible that properties of phytochemicals from plant extracts have produced morphological abnormalities in various developmental stages of Aedes mosquitoes. In this case, narrower head capsules indicated abnormal growth and development among the treated Aedes larvae compared to the control group. Herbicides such as atrizin and glyphosate were reported to decrease Ae. albopictus vectorial capacity in term of sex ratio distortion, adults emergence rate and body size⁶6. Bara JJ, Montgomery A, Muturi EJ. Sublethal effects of atrazine and glyphosate on life history traits of Aedes aegypti and Aedes albopictus (Diptera: Culicidae). Parasitol Res. 2014;113:2879-86.. Abnormalities were also observed at pupal stages of Ae. albopictus exposured to LC50 dose of Citrus contains R-(+)-limonene with larvae that failed to turn into adults and died in the pupa form which became elongated, showing the emergence only of the head and thorax from the puparium, with legs still attached to the pupal case or crumpled wing in adults⁶⁴64. Giatropoulos A, Papachristos DP, Kimbaris A, Koliopoulos G, Polissiou MG, Emmanouel N, et al. Evaluation of bioefficacy of three Citrus essential oils against the dengue vector Aedes albopictus (Diptera: Culicidae) in correlation to their components enantiomeric distribution. Parasitol Res. 2012;111:2253-63.. A sublethal dose did not only alter the morphology, but also the genetic structure of Ae. albopictus. Exposure to cadmium, inorganic mercury and methylmercury metals can change the morphology of Ae. albopictus' cell line, affecting the development of filopodia⁶⁵65. Braeckman B, Simoens C, Rzeznik U, Raes H. Effect of sublethal doses of cadmium, inorganic mercury and methylmercury on the cell morphology of an insect cell line (Aedes albopictus, C6/36). Cell Biol Int. 1997;21:823-32..

Following the reduction in width of larval head capsules, the wing lengths of both males and females from the first generation of Ae. albopictus and Ae. aegypti were found to be significantly reduced after treatment with I. cairica leaf extract. A similar finding was reported regarding the wing length of males and females of Ae. aegypti which emerged from larvae treated with microalgal chloorophytes, Scenedesmus quadricaula and Chlorococcum sp.⁶⁶66. Rohani A, Wan-Loy C, Han-Lim L, Siew-Moi P. Effect of four chlorophytes on larval survival, development and adult body size of the mosquito Aedes aegypti. J App Phycol. 2001;13:369-74., and entomopathogenic fungus, Leptolegnia chapmanii⁵⁰50. Pelizza SA, Scorsetti AC, Tranchida MC. The sublethal effects of the entomopathic fungus Leptolegnia chapmanii on some biological parameters of the dengue vector Aedes aegypti. J Insect Sci. 2013;13:22.. However, a contrasting finding has also been reported in females of Ae. aegypti that survived the treatment with spinosad at LC_50, resulting in progenies with significantly longer wings compared to the control group⁴⁷47. Antonio GE, Sanchez D, Williams T, Marina CF. Paradoxical effects of sublethal exposure to the naturally derived insecticide spinosad in the dengue vector mosquito, Aedes aegypti. Pest Manag Sci. 2009;65:323-6..

Reduction in wing length is considered as one of the morphological abnormalities caused by plant phytochemicals in insects. Wing length is a reliable parameter used as an indicator of body size in mosquitoes compared to their weight³⁰30. Scott TW. Containment of arthropod disease vectors. ILAR J. 2005;46:53-61.^,6767. Dieng H, Boots M, Eshita Y. Some insights into the concept of body size in mosquitoes. House Household Insect Pests. 2006;28:47-62.. It has been reported that the body size positively correlates with the fecundity of mosquitoes, which means larger females tend to lay more eggs compared to smaller ones⁶⁸68. Armbruster P, Hutchinson RA. Pupal mass and wing length as indicators of fecundity in Aedes albopictus and Aedes geniculatus (Diptera: Culicidae). J Med Entomol. 2002;39:699-704.^,6969. Blackmore MS, Lord CC. The relationship between size and fecundity in Aedes albopictus. J Vector Ecol. 2000;25:212-7.. In addition, shorter wing length may lead to a reduction of flight capabilities. Here, we postulate that the survival of Aedes mosquito treated with phytochemicals tend to be lower in time. Mosquitoes with shorter wings are unable to a long-distance migration and the host-seeking ability of blood feeding will be reduced as well. Smaller females tend to die before reaching hosts due to a lack of substantial energy⁶⁷67. Dieng H, Boots M, Eshita Y. Some insights into the concept of body size in mosquitoes. House Household Insect Pests. 2006;28:47-62.. Blood meals are essential for ovarian maturation and eggs production and therefore, we can predict a decrease in mosquito size leading to a reduced egg production. An observation of reciprocity in body size and ovarioles production in Ae. aegypti⁷⁰ 70. van den Heuvel M. The effect of rearing temperature on the wing length, thorax length, leg length and ovariole number of the adult mosquito, Aedes aegypti (L.). Trans Royal Entomol Soc London. 1963;115:197-216.seems to support this statement. In addition, a decreased blood feeding activity reduces the chances of female mosquitoes in acquiring and transmitting the dengue virus⁷¹71. Aziz AT, Dieng H, Ahmad AH, Mahyoub JA, Messed H, Koshike S, et al. Temporal variations on abundance, blood feeding activity of Aedes aegypti (Diptera: Culicidae) and dengue incidence in Makkah City, Saudi Arabia. Asian Pacific J Trop Biomed. 2012;1-7.. Hence, the reduction in wing length of male and female mosquitoes of Ae. albopictus and Ae. aegypti after exposure of parents to a sublethal dose of I. cairica leaf extract is a collateral advantage for the control of these mosquitoes species and the associated disease.

While most of the literature information resides on the larvicidal, ovicidal, adulticidal and repellency effects of phytochemicals on mosquitoes species, the present study is the first attempt to use a sublethal dose of I. cairica plant crude extract on the reproductive responses (fecundity), eggs hatchability (fertility), egg length, width of larval head capsules and wing length of Aedes mosquitoes.

CONCLUSIONS

The crude extract of Ipomoea cairica leaf has not only caused a high mortality rate in larvae of dengue vectors but has also caused sublethal effects on the offsprings of treated mosquitoes in terms of biological and morphological aspects. The reproductive potential (fecundity and fertility), width of larval head capsule and wing length were significantly reduced, especially in Ae. albopictus, indicating that this plant extract could be incorporated to integrated pest management aedes control programs.

Acknowledgements

We would like to thank the staffs of the School of Biological Sciences, Universiti Sains Malaysia and the Vector Control Research Unit, Universiti Sains Malaysia for field assistance and mosquito culture. This study received financial support from the Short Term Grant USM (304/BIOLOGI/6312026) and the Fundamental Research Grant Scheme, Ministry of Education, Malaysia (203/PBIOLOGI/6711359).

REFERENCES

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