Research ArticleLarvicidal Effectiveness of Azolla pinnata againstAedes aegypti (Diptera Culicidae) with Its Effects on LarvalMorphology and Visualization of Behavioural Response

Nor Shaida Husna Zulkrnin1 Nurul Nadiah Rozhan1

Nur Amanina Zulkfili1 Nik Raihan Nik Yusoff1 Mohd Sukhairi Mat Rasat2

Nor Hakimin Abdullah2 Muhammad Iqbal Ahmad2 Rajiv Ravi 1

Intan H Ishak 34 andMohamad Faiz Mohd Amin 1

1Faculty of Earth Science Universiti Malaysia Kelantan Jeli Campus Jeli Kelantan Malaysia2Faculty of Bioengineering and Technology Universiti Malaysia Kelantan Jeli Campus Jeli Kelantan Malaysia3School of Biological Sciences Universiti Sains Malaysia Minden Penang Malaysia4Vector Control Research Unit School of Biological Sciences Universiti Sains Malaysia Minden Penang Malaysia

Correspondence should be addressed to Rajiv Ravi rajiv ravi86yahoocom Intan H Ishak intanishakusmmyand Mohamad Faiz Mohd Amin mohamadfaizumkedumy

Received 7 March 2018 Accepted 29 May 2018 Published 27 June 2018

Academic Editor Bernard Marchand

Copyright copy 2018 Nor Shaida Husna Zulkrnin et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Dengue is vector-borne diseases with 390 million infections per year extending over 120 countries of the world Aedes aegypti (L)(Diptera Culicidae) is a primary vector for dengue viral infections for humans Current focus on application of natural productagainst mosquito vectors has been the main priority for research due to its eco-safety The extensive use of chemical insecticideshas led to severe health problems environmental pollution toxic hazards to human and nontarget species and development ofinsecticide resistance on mosquitoes Azolla pinnata is an aquatic fern and predominantly used as feed in poultry industry andas fertilizer in agricultural field for enhancing the fertility of rice paddy soil The present study was conducted to explore thelarvicidal efficacy of A pinnata using fresh and powdered form against late third-stage larvae (6 days 5 mm in larvae body length)of Ae aegypti (L) (Diptera Culicidae) The larvicidal bioassays were performed using World Health Organization standard larvalsusceptibility test method for different concentration for powdered and fresh A pinnata Powdered A pinnata concentration usedduring larvicidal bioassay ranges from 500ppm to 2000ppm meanwhile fresh A pinnata ranges from 500ppm to 9000000 ppmThe highest mortality was at 1853 ppm for powderedA pinnata compared with freshA pinnata at 2521535 ppm while the LC

50for

both powdered and fresh A pinnata recorded at 1262 ppm and 1853 ppm respectively Finally the analysis of variance (ANOVA)showed significant difference onAe aegypti larval mortality (F=30439 df=1 ple0001) and concentration (F=20002 df=1 ple0001)compared to powdered and fresh A pinnata at 24-hour bioassay test In conclusion the powdered A pinnata serves as a goodlarvicidal agent against Ae aegypti (L) (Diptera Culicidae) and this study provided information on the lethal concentration thatmay have potential for a more eco-friendly Aedesmosquito control program

1 Introduction

The intense increase on arthropod-borne viruses (arbovi-ruses) diseases such as dengue Zika and Chikugunya ismainly contributed by primary vectorAe aegypti Recently inMalaysia from January till July 2017 therewere 55744 dengue

cases with 131 deaths [1] Previously Ishak [2] reported that50 million vector-borne disease cases worldwide were dueto dengue and Malaysia has reported 46171 cases in 2010with 134 deaths In addition to that the planning of infras-tructure development and management has also contributedin arthropod-borne viruses (arboviruses) diseases World

HindawiJournal of Parasitology ResearchVolume 2018 Article ID 1383186 5 pageshttpsdoiorg10115520181383186

2 Journal of Parasitology Research

Health Organization [3] and Mohd Amin [4 5] have statedthat unplanned urbanization with the defect in water supplyand solid waste managementrsquos have mainly contributed inarboviral diseases spread by the mosquitoes Currently thecontrol measures of dengue vector in Malaysia are adulticid-ing with permethrin deltamethrin and malathion and lar-viciding using temephos and Bacillus thuringiensis israelensis(Bti) [2] Additionally Malaysian Ministry of Health (MoH)operators private companies and household communitiesare using insecticides to control dengue vectors Howeverthe intense exposures from all this usage have contributedto insecticide resistance in Malaysian Aedes populations InMalaysia evidence of resistance towards permethrin andtemephos has been recorded from both Ae aegypti and Aealbopictus in Kuala Lumpur and Penang regions [6 7] Ishak[2] has reported that insecticide resistance was caused by twomain factors due to increase in rate of insecticide metabolismand alterations in its target sites Additionally alterations intarget sites are caused by mutations from target genes suchas knockdown (kdr) resistance acetylcholinesterase (Ace-1)gene and GABA receptors [2]

Hence due to all these problems in controlling denguevector alternative method is required In such situationalternative usage of biological control can provide a moresuitable and sustainable solution against Ae aegypti Fol-lowing this safer and greener alternative conception Azollapinnata plant has the potential as bioinsecticidemay solve theproblems of resistance and chemical pollution In additionto that this conception was also supported by Ghosh [8]who mentioned that bioinsecticide from botanical origin issimple and sustainable method compared to conventionalinsecticides Unlike conventional insecticides advantages ofplant-derived insecticides which is composed by botanicalblends of chemical compounds will act concertedly on bothphysiological and behavioural processes [8] Hence therewould be only very minute possibilities of vectors in devel-oping resistance to such substances

One of the first field studies from genus of Azolla plantfor its effects on mosquito breeding was by Bao-Lin L [9]from Hunan China on Azolla filiculoides which has reducedthe larval density of Culex tritaeniorhynchus by an averageof 69 for 3 months at 75 coverage of water surface at800 hectare of paddy land Moreover reduction of Anophelessinensis larval densities was not clear in his observationsbut the pupal densities were reduced for both larval species[9] On the other hand another field study report with Apinnata as mosquito breeding control was by Pandey [10]from Gujerat India It was mentioned that A pinnata hasan effect on the oviposition of Anopheles culicifacies and Ansubpictusmosquitoes in rice fields [10]

Similarly Pandey [10] have reported a lab based studywith A pinnata which affects the oviposition of Culex quin-quefasciatus and Cx culicifacies mosquitoes Lab containerscovered with 50 of A pinnata result in a suppression ofegg laying properties with its significant behavioural changeson Cx quinquefasciatus and Cx culicifacies mosquitoes [10]Additionally in paddy fields of Tanzania Africa MwingiraV S [11] found Anabaena azollae have reduced the lar-vae productivity and larvae densities of An gambiae An

funestus and Cx quinquefasciatus Mwingira V S [11] find-ings suggest that the mosquito productivity is low when theAzolla coverage is high (gt80) in paddy fields

Despite the fact of many studies on Azolla plant withmosquitoes none of them have mentioned the use of freshand powdered form of the plant and its direct applications onAe aegypti However understanding these future potentialsbased on the papers reviewed it seems that A pinnatahas bioinsecticides potentials with its alterations againstbehavioural changes in mosquito vectors In addition tothat none of the papers have investigated the larvicidalmorphological and behavioural efficacies on Ae aegypti

Hence this study will be focused on A pinnata freshand powdered form as a biological control agent againstAe aegypti larvae To date no other studies have beenconducted with A pinnata plant for its larvicidal efficaciesand morphological and behavioural responses on Ae aegyptilarvae

2 Material and Methods

21 Research Area and Design This research follows WorldHealth OrganizationWHO [3] guidelines formosquito larvi-cidal bioassay A total of 50 kg fresh A pinnata was sampledfrom Kuala Krai Kelantan (5∘311015840N 102∘121015840E) and its specieswas identified based on leaves phyllotaxis morphologicalpattern [12]

22 Sample Preparation

221 Powdered Samples Total of 30 kgA pinnata fresh sam-ple was prepared using sun-dried technique for 2 days Thenthe dried samples were powdered electrically with grindingmachine Faber FBG-460K and sieved as fine powder NexttheA pinnata powder was been stored in zipper bag and keptat room temperature (Figure 1)

222 Fresh Samples Fresh A pinnata plant larvicidal testswere conducted with 20 kg of fresh plant The fresh sampleswere then washed with chlorine free water to remove anyimpurities from other sources before using it for larvicidalbioassay (Figure 1)

23 Aedes Larvae Rearing Susceptible lab strain eggs ofAe aegypti were obtained from Vector Control ResearchUnit (VCRU) at University Sains Malaysia (USM) PenangMalaysia Then the eggs were hatched in seasoned waterfor 24 hours The hatching process was triggered with 02g of larval food (food ratio 211 of cat biscuit beef liveryeast and milk powder) The eggs were maintained at 25∘Cto 30∘C (room temperature) a pH of 695 to 703 and relativehumidity of 80 plusmn 10 and dissolved oxygen from 55 to 61mgL in the laboratory After 6 days the larvae turned intoinstars third stages

24 Larvicidal Bioassay Larvicidal bioassays were per-formed in accordance with the standard World HealthOrganization [1 3] larval susceptibility testmethods (distilled

Journal of Parasitology Research 3

(a) (b)

Figure 1 Azolla pinnata powder (a) and fresh Azolla pinnata (b)

water and plant solution) The bioassay tests were donewith four replicates of Ae aegypti late third-stage larvae (6days 5 mm in larvae body length) Plastic containers (eachcontains 500 mL of distilled water A Pinnata powder and20 mosquito larvae) were used in this test [1] After 24 hoursthe mortalities of Ae aegypti larvae were determined [1]Larvae with total absence of movement even after touchwere considered as dead

25 Morphological View and Visualization Ae aegypti latethird-stage larvae was observed under the opticalmicroscope(Leica USA) magnification 40-400x Visualization was con-ducted by Samsung ES80 camera

26 Data Analysis The percentages of mortality were sub-jected to log-probit analysis for computing LC

50and LC

90

with 95 confidence limit using the SPSS 200 (StatisticalPackage of Social Sciences) software Analysis of variance(ANOVA) was performed using the concentration and mor-tality between powdered and fresh in 24-hour bioassaytest Homogeneity of variance was tested using (Shapiro-Wilk) prior to analysis according to guidelines provided byAndy [13] 24 hours of experimental time was served asindependent variable and concentration and mortality weretreated as dependent factors

3 Results

The bioassay testing was conducted on critical range of 5 to95mortality for powderedA pinnata in 500 ppm 750 ppm1000 ppm 1250 ppm 1500 ppm 1750 ppm and 2000 ppm andfreshA pinnata consisting of 500 ppm 2500 ppm 5000 ppm10000 ppm 20000 ppm 30000 ppm 40000 ppm 50000 ppm60000 ppm 500000 ppm 4000000 ppm and 9000000ppm concentrations (Figures 2 and 3) The results showed asignificant increase in mortality percentage with the increaseof concentration in both cases The highest mortality wasdemonstrated at 1853 ppm for powderedA pinnata comparedwith fresh A pinnata at 2521535 ppm (Table 1) Meanwhilethe LC

50of powdered and fresh A pinnata was recorded at

1262 ppm and 1853 ppm respectively (Table 1) Table 2 showsthe annova results of Ae aegypti larval mortality betweenpowdered and fresh A pinnata exposure for 24 hours

Figure 2 Percentagemeanmortality of late third-stage larvae ofAeaegypti after 24 hours in response to powdered A pinnata

Figure 3 Percentagemeanmortality of late third-stage larvae ofAeaegypti after 24 hours in response to fresh A pinnata

Figures 4 and 5 showmorphological deformities melani-sation and behavioural effects ofAe aegypti larval during thebioassay test meanwhile the attached supplementary video(available here) file could intelligible to their behaviouralresponses Hence it can be assertive that fresh and powderedA pinnata acts as a biological control against Ae aegypti

4 Journal of Parasitology Research

(a) (b) (c)

Figure 4 Morphological deformities and melanisation effects of Ae aegypti late third-stage larvae after 24 hours in response to powderedand fresh Azolla pinnata

Table 1 Larvicidal activity of Aedes aegypti using Azolla pinnata powdered form and fresh plant

Time TreatmentLC50 (ppm)

with 95 confidenceinterval

LC90 (ppm)with 95 confidence

intervalRegression equation

24 hoursPowdered

Azolla pinnata1262794

(1030588-1928775)1853238

(1030588-1928775) Y= 7692X ndash 23857

FreshAzolla pinnata

192517205(111397462-478710318)

2521535166(855514813-19888028270) Y= 1147X ndash 6062

LC50 lethal concentration required to kill 50 of the population exposed LC90 lethal concentration required to kill 95 of the population exposed and ppmparts per million

Table 2 Analysis of variance on Aedes aegypti larval mortality between powdered and fresh Azolla pinnata exposure for 24 hours

Source of variation df MS F-value P-valueConcentration 1 7622200694444 30439 ple0001lowastMortality 1 532900 20002 ple0001lowastdf degree of freedom MS mean-squared valueSignificant values are given in bold

Figure 5 Visualization video captured on behavioural responseeffects of Ae aegypti late third-stage larvae after 24-hour bioassaytest in powdered and fresh Azolla pinnata

4 Discussion

This study has demonstrated the biocontrol efficacies of freshand powdered A pinnata against Ae aegypti late third-stage larvae Additionally the Ae aegypti late third-stagelarvae show some morphological deformities and abnormalbehavioural responses during these bioassays Interestingly

the physical morphological observation of the larvae during24 hours of exposure showed a brownish colour abdominalsegment which changes into whitish colour (Figure 4) Asshown in Figure 5 and visualization videos it could be furtherdiscussed as toxic accumulation in body effects upon Apinnata applications These are due to the adaptation on newenvironment whereby the larva possesses passive movementand swims at the bottom showing sluggish and wigglingbehavioural responses In addition to that the larvae wereflexing to clean their siphon with mouthparts which led tohigh possibilities of toxic accumulation in their body

Lakshmi Naidu [14] reported that plant produces a broadrange of bioactive chemical compounds consisting secondarymetabolites such as flavonoids tannins terpenoids and alka-loids which would significantly produce biological activitiesand chemical defences against insects Hence A pinnatamight have contributed in body effects of larvae due toits natural bioactive chemical compounds and secondarymetabolites Results from this current study indicate thepotential application fromA pinnata plant asmosquito larvi-cidal agentThis is because even-through low concentrationswe could observe larvae mortalities Mogi [15] and Baolin[9] showed the effects of Azolla by covering the total watersurface which affects the mosquitoes breeding barrier on

Journal of Parasitology Research 5

oviposition and emergence of pupae further reducing thelarvae densities in rice fields

Comparison of efficacies between powdered and freshA pinnata plant from this present study has shown thatpowdered application is the best in lab scale experimentSimilarly Rao [16] have applied the neem cake powderwhich resulted in drastic reduction of late-instar larvae andpupae of Culicine mosquitoes from paddy fields comparedto fresh plant application Thus this current study wouldbe an important framework in the future investigation ofbioinsecticidal from A pinnata plant used for larvicidal test

In conclusion discovering an environmentally friendlyinsecticide to control mosquito vectors is considered to bea vital role which reduces the negative impacts caused bychemical insecticides in our environment Thus this currentresearch provides evidential framework for A pinnata aspotential larvicidal agents Furthermore its application mayalter the morphology and behavioural process of Ae aegyptilarvae Therefore it can be concluded that A pinnata hasdesired compounds in larvicidal bioassays and future initia-tives are necessary to investigate its bioactive compounds

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

There are no conflicts of interest by all authors

Acknowledgments

This study is supported by Ministry of Higher EducationMalaysia through the Fundamental Research Grant Scheme(RFRGSA080000425A0022017000440)

Supplementary Materials

Visualization video on behavioural response effects of Aeaegypti late third-stage larvae after 24-hour bioassay testin powdered and fresh Azolla pinnata The movement oflarvae is inactive from both observations with the presenceof whitish pigmented cuticles (Supplementary Materials)

References

[1] World Health Organization (2017) Update on the denguesituation in the Western Pacific Region Update (522) WHO(2005) Guidelines for laboratory and field testing of mosquitolarvicides World Health Organization 1ndash41

[2] I H Ishak Z Jaal H Ranson and C S Wondji ldquoContrastingpatterns of insecticide resistance and knockdown resistance(kdr) in the dengue vectors Aedes aegypti and Aedes albopictusfrom Malaysiardquo Parasites amp Vectors vol 8 no 1 article no 1812015

[3] WHO Dengue and Dengue Haemorrhagic Fever Investing toOvercome the Global Impact of Neglected Tropical DiseasesWorld Health Organization Geneva Switzerland 2015

[4] M F Mohd Amin S G J Heijman S I C Lopes andL C Rietveld ldquoPolyelectrolytes ability in reducing atrazineconcentration in water surface effectsrdquo The Scientific WorldJournal vol 2014 Article ID 162157 6 pages 2014

[5] M F Mohd Amin S G Heijman and L C Rietveld ldquoClay-starch combination for micropollutants removal from wastew-ater treatment plant effluentrdquoWST vol 71 no 7 pp 1719ndash17272014

[6] W A Nazni S Selvi H L Lee et al ldquoSusceptibility statusof transgenic Aedes aegypti (L) against insecticidesrdquo DengueBulletin vol 33 no 1 pp 124ndash129 2009

[7] H Ranson J Burhani N Lumjuan and W C Black ldquoInsecti-cide resistance in dengue vectorsrdquo TropIKAnet Journal vol 1no 1 2010

[8] A Ghosh N Chowdhury and G Chandra ldquoPlant extractsas potential mosquito larvicidesrdquo Indian Journal of MedicalResearch vol 135 no 5 pp 581ndash598 2012

[9] L Bao-lin ldquoThe effect of Azolla on mosquito breedingrdquo Para-sitology Today vol 4 no 11 pp 328-329 1988

[10] S D Pandey S K Sharma and R Kant ldquoRole of biologicalagents for the control ofmosquito breeding in rice fieldsrdquo IndianJournal of Malariology vol 33 no 4 pp 209ndash215 1996

[11] V S Mwingira B KMayala K P Senkoro et al ldquoMosquito lar-val productivity in rice-fields infested with Azolla in MvomeroDistrict Tanzaniardquo Tanzania Journal of Health Research vol 11no 1 pp 17ndash22 2009

[12] R Rajendran and R Reuben ldquoLaboratory Evaluation of theWater Fern Azolla pinnata for Mosquito Controlrdquo BiologicalControl vol 2 no 2 pp 114ndash116 1988

[13] Andy Field 2005 Discovering statistics using SPSS 2nd edition[14] P V Lakshmi Naidu K Kishore Kumar C Mohan Kumar

G Gunesh and M Narasimha Rao ldquoAntimicrobial activity ofAchyranthes asperardquo Biosciences Biotechnology Research Asiavol 3 no 1 A pp 171ndash174 2006

[15] M Mogi T Okazawa I Miyagi et al ldquoDevelopment andsurvival of anopheline immatures (Diptera Culicidae) in ricefields in northernThailandrdquo Journal ofMedical Entomology vol23 no 3 pp 244ndash250 1986

[16] D R Rao R Reuben M S Venugopal B A Nagasampagiand H Schmutterer ldquoEvaluation of neem Azadirachta indicawith and without water management for the control of culicinemosquito larvae in rice-fieldsrdquoMedical andVeterinary Entomol-ogy vol 6 no 4 pp 318ndash324 1992

Journal of Parasitology Research 3

(a) (b)

Figure 1 Azolla pinnata powder (a) and fresh Azolla pinnata (b)

water and plant solution) The bioassay tests were donewith four replicates of Ae aegypti late third-stage larvae (6days 5 mm in larvae body length) Plastic containers (eachcontains 500 mL of distilled water A Pinnata powder and20 mosquito larvae) were used in this test [1] After 24 hoursthe mortalities of Ae aegypti larvae were determined [1]Larvae with total absence of movement even after touchwere considered as dead

25 Morphological View and Visualization Ae aegypti latethird-stage larvae was observed under the opticalmicroscope(Leica USA) magnification 40-400x Visualization was con-ducted by Samsung ES80 camera

26 Data Analysis The percentages of mortality were sub-jected to log-probit analysis for computing LC

50and LC

90

with 95 confidence limit using the SPSS 200 (StatisticalPackage of Social Sciences) software Analysis of variance(ANOVA) was performed using the concentration and mor-tality between powdered and fresh in 24-hour bioassaytest Homogeneity of variance was tested using (Shapiro-Wilk) prior to analysis according to guidelines provided byAndy [13] 24 hours of experimental time was served asindependent variable and concentration and mortality weretreated as dependent factors

3 Results

The bioassay testing was conducted on critical range of 5 to95mortality for powderedA pinnata in 500 ppm 750 ppm1000 ppm 1250 ppm 1500 ppm 1750 ppm and 2000 ppm andfreshA pinnata consisting of 500 ppm 2500 ppm 5000 ppm10000 ppm 20000 ppm 30000 ppm 40000 ppm 50000 ppm60000 ppm 500000 ppm 4000000 ppm and 9000000ppm concentrations (Figures 2 and 3) The results showed asignificant increase in mortality percentage with the increaseof concentration in both cases The highest mortality wasdemonstrated at 1853 ppm for powderedA pinnata comparedwith fresh A pinnata at 2521535 ppm (Table 1) Meanwhilethe LC

50of powdered and fresh A pinnata was recorded at

1262 ppm and 1853 ppm respectively (Table 1) Table 2 showsthe annova results of Ae aegypti larval mortality betweenpowdered and fresh A pinnata exposure for 24 hours

Figure 2 Percentagemeanmortality of late third-stage larvae ofAeaegypti after 24 hours in response to powdered A pinnata

Figure 3 Percentagemeanmortality of late third-stage larvae ofAeaegypti after 24 hours in response to fresh A pinnata

Figures 4 and 5 showmorphological deformities melani-sation and behavioural effects ofAe aegypti larval during thebioassay test meanwhile the attached supplementary video(available here) file could intelligible to their behaviouralresponses Hence it can be assertive that fresh and powderedA pinnata acts as a biological control against Ae aegypti

4 Journal of Parasitology Research

(a) (b) (c)

Figure 4 Morphological deformities and melanisation effects of Ae aegypti late third-stage larvae after 24 hours in response to powderedand fresh Azolla pinnata

Table 1 Larvicidal activity of Aedes aegypti using Azolla pinnata powdered form and fresh plant

Time TreatmentLC50 (ppm)

with 95 confidenceinterval

LC90 (ppm)with 95 confidence

intervalRegression equation

24 hoursPowdered

Azolla pinnata1262794

(1030588-1928775)1853238

(1030588-1928775) Y= 7692X ndash 23857

FreshAzolla pinnata

192517205(111397462-478710318)

2521535166(855514813-19888028270) Y= 1147X ndash 6062

LC50 lethal concentration required to kill 50 of the population exposed LC90 lethal concentration required to kill 95 of the population exposed and ppmparts per million

Table 2 Analysis of variance on Aedes aegypti larval mortality between powdered and fresh Azolla pinnata exposure for 24 hours

Source of variation df MS F-value P-valueConcentration 1 7622200694444 30439 ple0001lowastMortality 1 532900 20002 ple0001lowastdf degree of freedom MS mean-squared valueSignificant values are given in bold

Figure 5 Visualization video captured on behavioural responseeffects of Ae aegypti late third-stage larvae after 24-hour bioassaytest in powdered and fresh Azolla pinnata

4 Discussion

This study has demonstrated the biocontrol efficacies of freshand powdered A pinnata against Ae aegypti late third-stage larvae Additionally the Ae aegypti late third-stagelarvae show some morphological deformities and abnormalbehavioural responses during these bioassays Interestingly

the physical morphological observation of the larvae during24 hours of exposure showed a brownish colour abdominalsegment which changes into whitish colour (Figure 4) Asshown in Figure 5 and visualization videos it could be furtherdiscussed as toxic accumulation in body effects upon Apinnata applications These are due to the adaptation on newenvironment whereby the larva possesses passive movementand swims at the bottom showing sluggish and wigglingbehavioural responses In addition to that the larvae wereflexing to clean their siphon with mouthparts which led tohigh possibilities of toxic accumulation in their body

Lakshmi Naidu [14] reported that plant produces a broadrange of bioactive chemical compounds consisting secondarymetabolites such as flavonoids tannins terpenoids and alka-loids which would significantly produce biological activitiesand chemical defences against insects Hence A pinnatamight have contributed in body effects of larvae due toits natural bioactive chemical compounds and secondarymetabolites Results from this current study indicate thepotential application fromA pinnata plant asmosquito larvi-cidal agentThis is because even-through low concentrationswe could observe larvae mortalities Mogi [15] and Baolin[9] showed the effects of Azolla by covering the total watersurface which affects the mosquitoes breeding barrier on

Journal of Parasitology Research 5

oviposition and emergence of pupae further reducing thelarvae densities in rice fields

Comparison of efficacies between powdered and freshA pinnata plant from this present study has shown thatpowdered application is the best in lab scale experimentSimilarly Rao [16] have applied the neem cake powderwhich resulted in drastic reduction of late-instar larvae andpupae of Culicine mosquitoes from paddy fields comparedto fresh plant application Thus this current study wouldbe an important framework in the future investigation ofbioinsecticidal from A pinnata plant used for larvicidal test

In conclusion discovering an environmentally friendlyinsecticide to control mosquito vectors is considered to bea vital role which reduces the negative impacts caused bychemical insecticides in our environment Thus this currentresearch provides evidential framework for A pinnata aspotential larvicidal agents Furthermore its application mayalter the morphology and behavioural process of Ae aegyptilarvae Therefore it can be concluded that A pinnata hasdesired compounds in larvicidal bioassays and future initia-tives are necessary to investigate its bioactive compounds

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

There are no conflicts of interest by all authors

Acknowledgments

This study is supported by Ministry of Higher EducationMalaysia through the Fundamental Research Grant Scheme(RFRGSA080000425A0022017000440)

Supplementary Materials

Visualization video on behavioural response effects of Aeaegypti late third-stage larvae after 24-hour bioassay testin powdered and fresh Azolla pinnata The movement oflarvae is inactive from both observations with the presenceof whitish pigmented cuticles (Supplementary Materials)

References

[1] World Health Organization (2017) Update on the denguesituation in the Western Pacific Region Update (522) WHO(2005) Guidelines for laboratory and field testing of mosquitolarvicides World Health Organization 1ndash41

[2] I H Ishak Z Jaal H Ranson and C S Wondji ldquoContrastingpatterns of insecticide resistance and knockdown resistance(kdr) in the dengue vectors Aedes aegypti and Aedes albopictusfrom Malaysiardquo Parasites amp Vectors vol 8 no 1 article no 1812015

[3] WHO Dengue and Dengue Haemorrhagic Fever Investing toOvercome the Global Impact of Neglected Tropical DiseasesWorld Health Organization Geneva Switzerland 2015

[4] M F Mohd Amin S G J Heijman S I C Lopes andL C Rietveld ldquoPolyelectrolytes ability in reducing atrazineconcentration in water surface effectsrdquo The Scientific WorldJournal vol 2014 Article ID 162157 6 pages 2014

[5] M F Mohd Amin S G Heijman and L C Rietveld ldquoClay-starch combination for micropollutants removal from wastew-ater treatment plant effluentrdquoWST vol 71 no 7 pp 1719ndash17272014

[6] W A Nazni S Selvi H L Lee et al ldquoSusceptibility statusof transgenic Aedes aegypti (L) against insecticidesrdquo DengueBulletin vol 33 no 1 pp 124ndash129 2009

[7] H Ranson J Burhani N Lumjuan and W C Black ldquoInsecti-cide resistance in dengue vectorsrdquo TropIKAnet Journal vol 1no 1 2010

[8] A Ghosh N Chowdhury and G Chandra ldquoPlant extractsas potential mosquito larvicidesrdquo Indian Journal of MedicalResearch vol 135 no 5 pp 581ndash598 2012

[9] L Bao-lin ldquoThe effect of Azolla on mosquito breedingrdquo Para-sitology Today vol 4 no 11 pp 328-329 1988

[10] S D Pandey S K Sharma and R Kant ldquoRole of biologicalagents for the control ofmosquito breeding in rice fieldsrdquo IndianJournal of Malariology vol 33 no 4 pp 209ndash215 1996

[11] V S Mwingira B KMayala K P Senkoro et al ldquoMosquito lar-val productivity in rice-fields infested with Azolla in MvomeroDistrict Tanzaniardquo Tanzania Journal of Health Research vol 11no 1 pp 17ndash22 2009

[12] R Rajendran and R Reuben ldquoLaboratory Evaluation of theWater Fern Azolla pinnata for Mosquito Controlrdquo BiologicalControl vol 2 no 2 pp 114ndash116 1988

[13] Andy Field 2005 Discovering statistics using SPSS 2nd edition[14] P V Lakshmi Naidu K Kishore Kumar C Mohan Kumar

G Gunesh and M Narasimha Rao ldquoAntimicrobial activity ofAchyranthes asperardquo Biosciences Biotechnology Research Asiavol 3 no 1 A pp 171ndash174 2006

[15] M Mogi T Okazawa I Miyagi et al ldquoDevelopment andsurvival of anopheline immatures (Diptera Culicidae) in ricefields in northernThailandrdquo Journal ofMedical Entomology vol23 no 3 pp 244ndash250 1986

[16] D R Rao R Reuben M S Venugopal B A Nagasampagiand H Schmutterer ldquoEvaluation of neem Azadirachta indicawith and without water management for the control of culicinemosquito larvae in rice-fieldsrdquoMedical andVeterinary Entomol-ogy vol 6 no 4 pp 318ndash324 1992

4 Journal of Parasitology Research

(a) (b) (c)

Figure 4 Morphological deformities and melanisation effects of Ae aegypti late third-stage larvae after 24 hours in response to powderedand fresh Azolla pinnata

Table 1 Larvicidal activity of Aedes aegypti using Azolla pinnata powdered form and fresh plant

Time TreatmentLC50 (ppm)

with 95 confidenceinterval

LC90 (ppm)with 95 confidence

intervalRegression equation

24 hoursPowdered

Azolla pinnata1262794

(1030588-1928775)1853238

(1030588-1928775) Y= 7692X ndash 23857

FreshAzolla pinnata

192517205(111397462-478710318)

2521535166(855514813-19888028270) Y= 1147X ndash 6062

LC50 lethal concentration required to kill 50 of the population exposed LC90 lethal concentration required to kill 95 of the population exposed and ppmparts per million

Table 2 Analysis of variance on Aedes aegypti larval mortality between powdered and fresh Azolla pinnata exposure for 24 hours

Source of variation df MS F-value P-valueConcentration 1 7622200694444 30439 ple0001lowastMortality 1 532900 20002 ple0001lowastdf degree of freedom MS mean-squared valueSignificant values are given in bold

Figure 5 Visualization video captured on behavioural responseeffects of Ae aegypti late third-stage larvae after 24-hour bioassaytest in powdered and fresh Azolla pinnata

4 Discussion

This study has demonstrated the biocontrol efficacies of freshand powdered A pinnata against Ae aegypti late third-stage larvae Additionally the Ae aegypti late third-stagelarvae show some morphological deformities and abnormalbehavioural responses during these bioassays Interestingly

the physical morphological observation of the larvae during24 hours of exposure showed a brownish colour abdominalsegment which changes into whitish colour (Figure 4) Asshown in Figure 5 and visualization videos it could be furtherdiscussed as toxic accumulation in body effects upon Apinnata applications These are due to the adaptation on newenvironment whereby the larva possesses passive movementand swims at the bottom showing sluggish and wigglingbehavioural responses In addition to that the larvae wereflexing to clean their siphon with mouthparts which led tohigh possibilities of toxic accumulation in their body

Lakshmi Naidu [14] reported that plant produces a broadrange of bioactive chemical compounds consisting secondarymetabolites such as flavonoids tannins terpenoids and alka-loids which would significantly produce biological activitiesand chemical defences against insects Hence A pinnatamight have contributed in body effects of larvae due toits natural bioactive chemical compounds and secondarymetabolites Results from this current study indicate thepotential application fromA pinnata plant asmosquito larvi-cidal agentThis is because even-through low concentrationswe could observe larvae mortalities Mogi [15] and Baolin[9] showed the effects of Azolla by covering the total watersurface which affects the mosquitoes breeding barrier on

Journal of Parasitology Research 5

oviposition and emergence of pupae further reducing thelarvae densities in rice fields

Comparison of efficacies between powdered and freshA pinnata plant from this present study has shown thatpowdered application is the best in lab scale experimentSimilarly Rao [16] have applied the neem cake powderwhich resulted in drastic reduction of late-instar larvae andpupae of Culicine mosquitoes from paddy fields comparedto fresh plant application Thus this current study wouldbe an important framework in the future investigation ofbioinsecticidal from A pinnata plant used for larvicidal test

In conclusion discovering an environmentally friendlyinsecticide to control mosquito vectors is considered to bea vital role which reduces the negative impacts caused bychemical insecticides in our environment Thus this currentresearch provides evidential framework for A pinnata aspotential larvicidal agents Furthermore its application mayalter the morphology and behavioural process of Ae aegyptilarvae Therefore it can be concluded that A pinnata hasdesired compounds in larvicidal bioassays and future initia-tives are necessary to investigate its bioactive compounds

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

There are no conflicts of interest by all authors

Acknowledgments

This study is supported by Ministry of Higher EducationMalaysia through the Fundamental Research Grant Scheme(RFRGSA080000425A0022017000440)

Supplementary Materials

Visualization video on behavioural response effects of Aeaegypti late third-stage larvae after 24-hour bioassay testin powdered and fresh Azolla pinnata The movement oflarvae is inactive from both observations with the presenceof whitish pigmented cuticles (Supplementary Materials)

References

[1] World Health Organization (2017) Update on the denguesituation in the Western Pacific Region Update (522) WHO(2005) Guidelines for laboratory and field testing of mosquitolarvicides World Health Organization 1ndash41

[2] I H Ishak Z Jaal H Ranson and C S Wondji ldquoContrastingpatterns of insecticide resistance and knockdown resistance(kdr) in the dengue vectors Aedes aegypti and Aedes albopictusfrom Malaysiardquo Parasites amp Vectors vol 8 no 1 article no 1812015

[3] WHO Dengue and Dengue Haemorrhagic Fever Investing toOvercome the Global Impact of Neglected Tropical DiseasesWorld Health Organization Geneva Switzerland 2015

[4] M F Mohd Amin S G J Heijman S I C Lopes andL C Rietveld ldquoPolyelectrolytes ability in reducing atrazineconcentration in water surface effectsrdquo The Scientific WorldJournal vol 2014 Article ID 162157 6 pages 2014

[5] M F Mohd Amin S G Heijman and L C Rietveld ldquoClay-starch combination for micropollutants removal from wastew-ater treatment plant effluentrdquoWST vol 71 no 7 pp 1719ndash17272014

[6] W A Nazni S Selvi H L Lee et al ldquoSusceptibility statusof transgenic Aedes aegypti (L) against insecticidesrdquo DengueBulletin vol 33 no 1 pp 124ndash129 2009

[7] H Ranson J Burhani N Lumjuan and W C Black ldquoInsecti-cide resistance in dengue vectorsrdquo TropIKAnet Journal vol 1no 1 2010

[8] A Ghosh N Chowdhury and G Chandra ldquoPlant extractsas potential mosquito larvicidesrdquo Indian Journal of MedicalResearch vol 135 no 5 pp 581ndash598 2012

[9] L Bao-lin ldquoThe effect of Azolla on mosquito breedingrdquo Para-sitology Today vol 4 no 11 pp 328-329 1988

[10] S D Pandey S K Sharma and R Kant ldquoRole of biologicalagents for the control ofmosquito breeding in rice fieldsrdquo IndianJournal of Malariology vol 33 no 4 pp 209ndash215 1996

[11] V S Mwingira B KMayala K P Senkoro et al ldquoMosquito lar-val productivity in rice-fields infested with Azolla in MvomeroDistrict Tanzaniardquo Tanzania Journal of Health Research vol 11no 1 pp 17ndash22 2009

[12] R Rajendran and R Reuben ldquoLaboratory Evaluation of theWater Fern Azolla pinnata for Mosquito Controlrdquo BiologicalControl vol 2 no 2 pp 114ndash116 1988

[13] Andy Field 2005 Discovering statistics using SPSS 2nd edition[14] P V Lakshmi Naidu K Kishore Kumar C Mohan Kumar

G Gunesh and M Narasimha Rao ldquoAntimicrobial activity ofAchyranthes asperardquo Biosciences Biotechnology Research Asiavol 3 no 1 A pp 171ndash174 2006

[15] M Mogi T Okazawa I Miyagi et al ldquoDevelopment andsurvival of anopheline immatures (Diptera Culicidae) in ricefields in northernThailandrdquo Journal ofMedical Entomology vol23 no 3 pp 244ndash250 1986

[16] D R Rao R Reuben M S Venugopal B A Nagasampagiand H Schmutterer ldquoEvaluation of neem Azadirachta indicawith and without water management for the control of culicinemosquito larvae in rice-fieldsrdquoMedical andVeterinary Entomol-ogy vol 6 no 4 pp 318ndash324 1992

Journal of Parasitology Research 5

oviposition and emergence of pupae further reducing thelarvae densities in rice fields

Comparison of efficacies between powdered and freshA pinnata plant from this present study has shown thatpowdered application is the best in lab scale experimentSimilarly Rao [16] have applied the neem cake powderwhich resulted in drastic reduction of late-instar larvae andpupae of Culicine mosquitoes from paddy fields comparedto fresh plant application Thus this current study wouldbe an important framework in the future investigation ofbioinsecticidal from A pinnata plant used for larvicidal test

In conclusion discovering an environmentally friendlyinsecticide to control mosquito vectors is considered to bea vital role which reduces the negative impacts caused bychemical insecticides in our environment Thus this currentresearch provides evidential framework for A pinnata aspotential larvicidal agents Furthermore its application mayalter the morphology and behavioural process of Ae aegyptilarvae Therefore it can be concluded that A pinnata hasdesired compounds in larvicidal bioassays and future initia-tives are necessary to investigate its bioactive compounds

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

There are no conflicts of interest by all authors

Acknowledgments

This study is supported by Ministry of Higher EducationMalaysia through the Fundamental Research Grant Scheme(RFRGSA080000425A0022017000440)

Supplementary Materials

Visualization video on behavioural response effects of Aeaegypti late third-stage larvae after 24-hour bioassay testin powdered and fresh Azolla pinnata The movement oflarvae is inactive from both observations with the presenceof whitish pigmented cuticles (Supplementary Materials)

References

[1] World Health Organization (2017) Update on the denguesituation in the Western Pacific Region Update (522) WHO(2005) Guidelines for laboratory and field testing of mosquitolarvicides World Health Organization 1ndash41

[2] I H Ishak Z Jaal H Ranson and C S Wondji ldquoContrastingpatterns of insecticide resistance and knockdown resistance(kdr) in the dengue vectors Aedes aegypti and Aedes albopictusfrom Malaysiardquo Parasites amp Vectors vol 8 no 1 article no 1812015

[3] WHO Dengue and Dengue Haemorrhagic Fever Investing toOvercome the Global Impact of Neglected Tropical DiseasesWorld Health Organization Geneva Switzerland 2015

[4] M F Mohd Amin S G J Heijman S I C Lopes andL C Rietveld ldquoPolyelectrolytes ability in reducing atrazineconcentration in water surface effectsrdquo The Scientific WorldJournal vol 2014 Article ID 162157 6 pages 2014

[5] M F Mohd Amin S G Heijman and L C Rietveld ldquoClay-starch combination for micropollutants removal from wastew-ater treatment plant effluentrdquoWST vol 71 no 7 pp 1719ndash17272014

[6] W A Nazni S Selvi H L Lee et al ldquoSusceptibility statusof transgenic Aedes aegypti (L) against insecticidesrdquo DengueBulletin vol 33 no 1 pp 124ndash129 2009

[7] H Ranson J Burhani N Lumjuan and W C Black ldquoInsecti-cide resistance in dengue vectorsrdquo TropIKAnet Journal vol 1no 1 2010

[8] A Ghosh N Chowdhury and G Chandra ldquoPlant extractsas potential mosquito larvicidesrdquo Indian Journal of MedicalResearch vol 135 no 5 pp 581ndash598 2012

[9] L Bao-lin ldquoThe effect of Azolla on mosquito breedingrdquo Para-sitology Today vol 4 no 11 pp 328-329 1988

[10] S D Pandey S K Sharma and R Kant ldquoRole of biologicalagents for the control ofmosquito breeding in rice fieldsrdquo IndianJournal of Malariology vol 33 no 4 pp 209ndash215 1996

[11] V S Mwingira B KMayala K P Senkoro et al ldquoMosquito lar-val productivity in rice-fields infested with Azolla in MvomeroDistrict Tanzaniardquo Tanzania Journal of Health Research vol 11no 1 pp 17ndash22 2009

[12] R Rajendran and R Reuben ldquoLaboratory Evaluation of theWater Fern Azolla pinnata for Mosquito Controlrdquo BiologicalControl vol 2 no 2 pp 114ndash116 1988

[13] Andy Field 2005 Discovering statistics using SPSS 2nd edition[14] P V Lakshmi Naidu K Kishore Kumar C Mohan Kumar

G Gunesh and M Narasimha Rao ldquoAntimicrobial activity ofAchyranthes asperardquo Biosciences Biotechnology Research Asiavol 3 no 1 A pp 171ndash174 2006

[15] M Mogi T Okazawa I Miyagi et al ldquoDevelopment andsurvival of anopheline immatures (Diptera Culicidae) in ricefields in northernThailandrdquo Journal ofMedical Entomology vol23 no 3 pp 244ndash250 1986

[16] D R Rao R Reuben M S Venugopal B A Nagasampagiand H Schmutterer ldquoEvaluation of neem Azadirachta indicawith and without water management for the control of culicinemosquito larvae in rice-fieldsrdquoMedical andVeterinary Entomol-ogy vol 6 no 4 pp 318ndash324 1992

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