apium paper_khondkar ehteshamul kabir_ report_b6

8/14/2019 Apium Paper_Khondkar Ehteshamul Kabir_ Report_B6

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A potent larvicidal and growth disruption activities ofApium graveolens (Apiaceae) seed extract on the denguefever mosquito,Aedes aegypti(Diptera: Culicidae)

Khondkar Ehteshamul Kabira, Rajput MuhammedTariqb,ShakilAhmedc, Muhammad Iqbal Choudharya*

aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,

University of Karachi, Karachi-75270, PakistanbM. A. H. Qadri Biological Research Center, University of Karachi, Karachi-75270, Pakistan

cIndustrial Analytical Center, H.E.J. Research Institute of Chemistry, International Center for Chemical

and Biological Sciences, University of Karachi, Karachi-75270, Pakistan

ABSTRACT

The ethanol extract of the seed of celery,Apium graveolenswas assayed for their toxicological

and biological activities against fourth instar larvae of the mosquito,Aedes aegypti, the vector of

dengue fever were originally collected from Karachi city, Pakistan. The ethanol-extracted

product produced a high level larval knockdown with a KD50of 238.15 ppm and subsequently

showed a potent toxicity to the larvae at 24 and 48 hrs after treatment with a LD50 of 126.13 ppm

and LD50 of 112.53 ppm respectively. The plant extract when applied to the larvae at lower

concentration (100 ppm) results the detrimental effects on the pupation and adult emergence

rate and further produced clear morphological malformations. Phototoxic compounds entiched

ethanol extract derived from the seed of celery are recommended as a promising agent for the

development of photo activated bioinsecticides to control the vector mosquitoes. The larval

knockdown and high mortality with a identifiable structural disruptions on larvae, pupae andadults upon exposure to the extract formulation indicated that it might affects through a process

of cytolysis on the neuro-muscular, endocrine and digestive system of the test subjects. The

physiological and molecular mode of action of the compounds in the bioactive extract fraction is

yet to be confirmed.

Keywords:Celery; Apium graveolens; phototoxin; Aedes aegypti; larvicidal; growth disruptor;peritrophic membrane; photo activated bioinsecticides

*Tel.: +92-21-4824924-5 ; fax: +92-21-4819018-9 .E-mail address: [email protected]


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2

1. Introduction

Mosquitoes play a predominant role as a vector for the transmission of malaria, yellowfever, dengue fever, filariasis and several other infectious diseases which are greatly affectedhumans and their domesticated animals worldwide. The control of mosquito larvae worldwidedepends primarily on continued applications of synthetic chemicals includes organophosphates

such as temephos, fenthion and insect growth regulators such as diflubenzuron andmethoprene (Dame et al.,1998).Although effective, repeated use of these products for mosquitocontrol has been gradually contaminated the bio-ecosystems, increaed the development ofresistance (Rivero et al., 2010), undesirable effects on non-target organisms (Devine andFurlong, 2007). It has been reported that in developing countries like Pakistan, the incidence ofpesticide poisoning may even be greater than reported due to under-reporting, lack of data andmisdiagnosis (Tariq et al., 2007).These problems have highlighted the need for the bioassayguided development of bioinsecticides for mosquito control with the use of plant secondarymetabolites (PSMs) enriched botanical extracts which would be more cheaper and potent,benign to non target organisms, degrading after sometime, and as they have different actionmechanisms, development of resistance in insects is limited. In addition to application asgeneral toxicants against mosquitoes these products may also have potentials as adulticides,

repellents, oviposition deterrents, growth and morphological disruptors. Plants are the promisingalternative source for the control of insect pests to replace conventional synthetic insecticidesbecause they bio-synthesize a diverse array of low molecular weight PSMs or specializedmetabolites such as alkaloids, polyphenols, terpenoids, steroids, lignans, phototoxicfuranocoumarin etc. to reduce insect attacks, both constitutive and inducible (Isman, 2006;

Arnason and Bernards, 2010). Much effort has, therefore, been focused on the plant extractsenriched with PSMs as potential sources for the developmet of novel bioinsecticides for insectpest control.

Celery, Apium graveolens L. is a biennial plant from the family of Apiaceae(Umbelliferae), commonly known in Pakistan as ajowan-khurasani, can be found throughoutEurope, the Mediterranean, and part of Asia. Although it extensively cultivated, consumed and

used as a popular vegetable, the dried ripe fruit are produced as a spice in China, France,India, Italy, Pakistan, the United States, and United Kingdom due to its pleasant aroma andmedicinal values for humans and animals (Mimica-Dukiand Popovi, 2007). This plant is alsoan effective remedy for various ailments such as bronchitis, liver and spleen disease, arthriticpain and this natural holistic approach to health is becoming more and more popular now a days(Kolarovic et al., 2010). Major bioactive secondary compounds has in celery include a class ofphenolic compounds called furanocoumarns.The main linear furanocoumarins in celery includethe three phototoxic furanocoumarins, psoralen (P), xanthotoxin (8-methoxypsoralen) (X) andbergapten (5 methoxypsoralen) (B) (Berenbaum, 1991) as well as isopimpinellin (5, 8-dimethoxypsoralen), which is not photo-biologically active (Ashwood Smith et al., 1992). Inaddition some other bioactive properties from Apiaceae family has been also reported recentlyfrom Mimica-Duki and Popovi (2007). Lombaert et al. (2001) reported that among the

biologically active furanocoumarins in celery, xanthotoxin and bergapten were the mostcommonly detected furanocoumarins (68 and 63%). Considering these facts in the presentstudy an attempt has been made to find out the larvicidal, growth disruption and behaviouraleffects of the ethanol extract of the seed of celery, A. graveolens against the dengue fevermosquito,Aedes qegypti (L.) (Diptera: Culicidae) which is a sole vector of the dengue viruses tohumans. Dengue is the most important human viral disease transmitted by arthropod vectors.

Annually, there are an estimated 50-100 million cases of dengue fever, and 250,000 to 500,000cases of dengue haemorrhagic fever (DHF) has been reported in tropical and subtropical


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3countries to a serial infection of at least one of the four serotypes that have evolved (Halstead,2008).

2. Materials and methods

2.1. Test plant and chemicals

Seeds of celery,A. graveolensL. (Umbelliferae) were authenticated and therby obtainedfrom Mr. Arshad Gabol, Department of Botany, University of Karachi, Pakistan. The voucherspecimen named HEJ-14 was deposited in the Medicinal Plant Unit, International Center forChemical and Biological Sciences, University of Karachi, Pakistan. The followings chemicalswere from commercial sources indicated: HPLC grade ethanol (Tedia Company, Fairfield, USA),dimethylsulphoxide (Fisher Scientific Company, UK).

2.2. Mass rearing of mosquitoes

The dengue fever mosquito, A. aegypti larvae was collected in July, 2010 throughdipping method from the natural breeding sites located in the Karachi City, Pakistan to establishthe mosquito colony in our laboratory. Larvae were then reared in 250 ml of glass beakers(ILDAM)) containing ca. 200 ml of distilled water (Millipore, USA). 2% (w/v) brewers yeastsolutions was routine wise supplied them as a food source. Pupae was transferred to glassbeakers (100 ml) containing 80 ml of distilled water and maintained in the standard Gerbergmosquito cages for adults emergence. Adults were maintained by providing with cotton circlessoaked with 10% honey solution (Khalis Honey, Pakistan). Two days after emergence, femalemosquitoes were allowed to blood feed periodically from chicks. A few days after having a bloodmeal, gravid mosquitoes laid their eggs on the filter paper strips in the glass cups. The filterpaper stripe with eggs was brought to dry under laboratory condition for overnight and then keptin the same condition until use them for larval hatching. Mosquito rearing and all experimentswas conducted under laboratory conditions at 271C; 12h L: D phase.

2.3. Solvent-accelerated-solid-liquid extraction

Fresh seeds were dried under room temperature. The dried seed materials was thenground through a manual grinder and passed through a 25-mesh sieve to obtain a fine powder.The powdered (ca. 1 Kg) material was then extracted with 3L of HPLC grade ethanol in amodified glass jar through percolation and then left the preparation to stand for 48 hours. Theextracts was then collected by decantation and filtered through whatman No 1 filter paper andevaporated to dryness at 40C by using a rotary evaporator (Bchi Rotavapor R-200, BchiLabortechnik, AG, Switzerland). After complete evaporation of solvent, the ethanol extract wasweighed and kept in amber colored vials; sealed with parafilm PM-996 (Pechiney plastic packacing, Menasha, USA) and stored the sample in refrigerator until use for experiments.

2.4. Preparation of the test concentrations by using PSMs enriched seed extract of A.graveolensto determine their toxicological and biological activities on mosquitoes

Plant secondary metabolites enriched ethanol extract of the seed of celery was tested toinvestigate their various activities against the A. aegypti mosquitoes by using WHO standardmethods with slight modifications. Initially 1% stock solution of extract was formulated with asmall volume of distilled water and dimethylsulphoxide (DMSO) and the resulting preparationwas then diluted in such a way which gave the final test concentrations ranging from 100 to 500


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4ppm (w/v) in 50 ml of test water. Then a total of 20 larvae were separately released in fivereplicates for each tested concentration. A two type of control was raised with a similar volumeof DMSO used for extract formulation in distilled water and distilled water only. The number oflarval knockdown (KD) was then counted at 4 hours after treatment. Larval mortality relateddatas due to the toxic effect of the extract were counted at 24 and 48 hours after treatmentrespectively, during which time no food was offered to the larvae. Larvae were considered dead

if they failed to move after probing with a blunt needle in the siphon or thoracic segments. Alldead larvae were then discarded and all surviving larvae in treatment and in controls were keptunder the same condition to estimate the pupation and adult emergence rate respectively at 72and 96 hours after treatment respectively.

2.5. Determination of the behavioural and malformation effects of PSMs enriched seedextract of A. graveolenson mosquitoes

Behavioral and morphological malformation symptoms on mosquitoes due to the toxicextract mediated disruption on the biological functions were simultaneously monitored andphotographed immediately and at time-intervals by using Cannon PC1234 Digital Camera(Cannon Inc., Japan), equipped with Kenko close up lens (Kenko, Japan) and compared withthose of the control assays. Behavioural symptoms of larvae includes: incapability of rising tothe water surface, Unnatural postures, trembling movement, lack of neuro-muscularcoordination. Any abnormal shape, size, discoloration or failures to pupate were indicated asmorphological malformation of larvae. Pupal malformation was recorded by any abnormalchange in shape, size or failure to develop to adult stage (pupal-adult intermediate). Adultmalformation was recorded by any abnormal change in colour, shape and sizes.

2.6. Calculation and statistical analysis

The percentage of larval mortality was estimated using the equations: Larval mortality %= DL / TL 100, where DL corresponds to the number of dead larvae and TL to the number oflarvae treated. The percentage of pupation was estimated using the equation: Pupation % = PN/ TL 100, where PN corresponds to the number of pupae formed and TL to the number oflarvae treated. The number of emerged adults was counted and the total number of adultemergence percentage was calculated by using the following equation: Adult emergence % =NEA / TL 100, where NEA = number of emerged adults and TL = number of larvae treated.The theoretical lethal knockdown of 50% (KD50) and lethal concentrations of 50%, (LC50), aswell as the corresponding 95% confidence intervals and chi-square values were determined byusing a computerized log-probit analysis program (Finney, 1971).

3. Results

3.1. Test plant and extraction

The analytical grade ethanol was used as solvent for extraction because it can extractvarieties of bioactive compounds from a single plant sample. Extractions was performed inairtight glass jar through cool percolation method in the dark to preserve the activity of thebioactive compounds in the extract fraction. Solvent was evaporated from the extract at lowtemperature to a certain volume, counted as a 100 percent crude ethanol extract. Ethanolextract of the seed ofA. graveolens, with a yield of ??? % (w/w), was semi-solid, ??? incolour,slightly aromatic. The solvent dimethylsulphoxide was used because it can completelydissolve the extract in the aqueous medium and it reportedly found safe when used similarly to


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5other organisms in vitro. In test water solutions seed extract gave olive colour. The bioactivitiesof the extract sample included (i) behavioural effects, (ii) the larvicidal, pupal and adultemergence inhibition effects, (iii) morphological growth disruption effects.

3.2. Behavioural effects and larval knockdown

Behavioural observations monitored under laboratory conditions, revealed thatimmediately after exposure to ethanol-extract of A. graveolens in test solution, all larvae wereexhibited a natural behaviour with the siphon pointed up through the water surface and headhung down. The process of larval feeding, both collecting filtering in the water column beneaththe surface by beating their head brushes (lateral palatal brushes) towards the pre-oral cavitywere observed and found normal. Interestingly between 5 to 10 min after treatment with theextract at concentration ranged between 200-500 ppm, all of the larvae found restless and theyperformed aggressive self biting to their anal papillae with their mouth parts and form a ringshape (head to siphon), this exclusive behavioural events was photographed for the first timeand presented in Fig. 1A.Both controls showed normal activity (Fig. 1C).Again at 15-30 minafter treatment, most of the larvae became irritated, wriggled up and down erratically andviolently. This restlessness behavioural patterns persisted, larvae slowed down their movement,failed to reach the water surface, followed by high level larval knockdown due to chronicparalysis was clearly seen onto the bottom of the glass beaker at 4 hours after treatment (Fig.1B) and compared with controls, shows normal behavior (Fig. 1D). The percentage of larvalknockdown is presented in Table-1, clearly shows that increasing the extract concentration levelfrom 100 to 500 ppm, knockdown rate was gradually increased. The theoretical KD50 value is238.15 ppm. More and more larvae at the tested concentrations results the acute toxicsymptoms morphologically and subsequently, most of the larvae found dead when mortality wasrecorded at two descending time intervals in vitro.

3.3. Larvicidal, pupal and adult emergence inhibition effects due to the toxicity of theseed extract of A. graveolens

Larvicidal effect due to the toxic activity of celery seed extract in ethanol against fourthinstar larvae ofA. aegypti is shown in Table 2.The susceptibility ofA. aegypti larvaeto variousconcentrations of the seed extract of A. graveolens was concentration and time dependent.Increasing the ethanol extract level from 100 to 300 ppm increased the larval mortality ratebetween 28 to 99% at 24 hours after treatment and 37 to 99% at 48 hours after treatmentrespectively, indicated that the activity of the photoactive compounds in the ethanol extract aretime dependent. High mortality (> 85%) values were observed at 200 ppm at both time frames.The 100% mortality was generated from the two highest concentration tested (400 and 500ppm) at only 24 hours after treatment. No mortality was observed in both control groups. At thelowest concentration, 100 ppm, only gave mortality 28% at 24 hours and 37 % at 48 hours aftertreatment. The ethanol-extract derived from the seed ofA. graveolens showed larvicidal activitywith a theoretical LD50of 126.13 (24 h) and LD50 of 112.53 (48 h) ppm respectively.

3.4. Morphological growth disruption effects due to the toxicity of the seed extract ofA.graveolens

The result presented in Fig. 2.clearly indicated that lower concentration of the extract ofcelery can effectively produced clear morphological growth disruption/malformations in thetreated mosquito larvae, pupae and adults compared to controls, showed normal structuralfeatures. Several forms of morphological malformations resulted from treatment of larvae,


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6pupae and adults with the extract. The apodous larvae shows several type of morphologicalmalformations: deforned mouth brushes, melanized and scleoritized cuticles (Fig. 2B), lightyellowish-albino coloured abdominal structures with a lack of peritrophic membrane outlines(Fig. 2C), compared to controls shows prominent mouth brushes and healthy cuticles and thedigestive tract structure shows clear PM outlines (Fig. 2A). The last abdominal segment ofAedes larvae which ended in two pairs of transparent sausage like anal papillae, showed

abnormal pigmentation with shrinkage (2B-C). Most of the treated larvae died as highlypigmented forms and some other died with PM disrupted forms. Most of the comma shapedpupae died as yellowish-albino coloured intermediates (Fig. 2F), others are died as a melanizedform with blackish cephalothorax (Fig. 2E). No visual malformations were seen in a pair offlattened paddles in the died pupae, transformed from the extract treated larvae. Control treatedgroups shows intact pupal structure (Fig. 2D). The adults emergence were 100% stopped whenextract used at concentrations ranged between 200-300 ppm. Although at only 100 ppm someadults emerged but died as deformed adult, had abnormal reduced wings (Fig. 2H) comparingwith DMSO treated control (Fig. 2G). Larval malformations which presented in Fig. 2.hampered the further transformation of larvae into pupae and only 63% of larvae couldsuccessfully pupate at 100 ppm concentration which directly proportional to the reduction inpupation rates. This was further highly reduced to 4% at 200 ppm. Larval deformities which also

shows in Fig. 2B-C, further influenced the total adult emergence time and rate: 45% recorded at96 hours after treatment.

4. Discussion

4.1. Test plant and their toxic phytochemical properties

Eco-friendly products are recommended in larviciding mosquito breeding sites. In thisstudy, a plant species commonly known as celery which have been used by the human sincelong as a promising vegetable is selected to investigate its potential toxicological and biologicalactivities against the dengue fever mosquito. Plants being a natural source of bioactivesecondary compounds, are known to potentially active as like synthetic larvicidal agents,

which may act in combination or independently hence necessity to carry out studies to find outthe possible effects of crude ethanol extracts derived from the seed of celery plant. Seeds areprimarily targeted in our investigation as because it reportedly contains phototoxic linearfuranocoumarins (LFC) which are thought to be restricted to schizogenous canals in seeds ofcelery (Berenbaum, 1991). Phototoxins are promising and unique among plant compoundsbecause most of them when excited by absorbing light energy their toxicity are greatlyincreased and make them as acute toxins with little organism-specificity (Downum, 1992). Onceactivated, they are capable of reacting with, and damaging, a variety of important cellularcomponents including DNA and enzymatically active proteins such as superoxide dismutase(Nivsarkar et al., 1991a). As such, they are promising to assay for the development of novelphotoactive bioinsecticides which might be capable of killing a wide-range of potentially harmfulorganisms.

4.2. Behavioural effects and larval knockdown

The results obtained through behavioural observations in larvae suggest that ethanolextract derived from the seed of celery could be act as a cytolysin and affects the neuro-muscular coordination in the chemical synapses somewhere as evident by aggressive selfbiting, trembling movement, spinning and uncoordinated behavioural activity and paralysis. Thesymptoms observed in treated larvae were similar to those caused by synthetic nerve poisons,i.e. excitation, convulsions, paralysis, and death. Although it is not clear to us from the


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7symptomatic observation that why the ethnaol extract treated larvae specifically performed suchviolent bites to their anal papillae. It probably appeared due to the fatal electrolytes dischargefrom the anal region resulted from the photo enhanced cytotoxic activity of the extract. And itcould be the primary reason concerned with the mechanism of toxicity and linked with themorphological disruptions of the test insects, matter discussed elaborately in later sections.When it was known to us those anal papillae in the larval mosquito regulates the electrolyte

levels (Beker et al., 2010) and is essentially required for the sustainability of their life functions.Interestingly similar behavioral observations also have been reported from the same plant fromChoochote et al. (2004). Observation of the poisoning symptoms of insecticides is not only ofpractical importance for insect control but can also contribute to the elucidation of mode ofaction of insecticides.

4.3 Larvicidal, pupal and adult emergence inhibition effects due to the toxicity of theseed extract of A. graveolens

The present studies in which ethanol extract from the seed of ,A. graveolens whenapplied into the artificial test water medium containing the fourth instar Aedes aegypti larvaeexhibited a potent toxicity to the mosquito larvae and resulted high level larval knockdown andhigh mortality. The result presented in Table-2 revealed that time is an essential factor foractivity generation because the seed of this plant mainly contains the phototoxic compoundsand they definitely need a continuous light exposure through time for their photo-activation. Thelarval mortality was observed to increase as the concentration of the test samples was raised.This toxic activity trend was found correlated in the case of time elapsed mortality. These wereattributed to the likely photoactiivity mechanisms that regulate the action of the phototoxin. Theuse of phototoxin rich extracts and photochemical processes as a tool to control the populationof insects have been recently gained interest to examine in both laboratory experiments andfield studies. Ben Amor and Jori (2000) found that after 1 h irradiation of Ceratitis capitata,Bactrocera oleae or Stomoxys calcitrans which ingested a few nanomoles of porphyrin per flywith light intensities of the order of 1000 E s -1m-2causes about 100% death in laboratory tests.In fact, UV/visible light can penetrate to a depth of about 1 cm in most biological tissues; as theextent of penetration is wavelength dependent (Svaasand et al., 1990). The result in this studyfound similar and in the same time in some case found controversial with few other studiesreported earlier: The ethanol-extractedA. graveolens seed possessed larvicidal activity againstfourth instar larvae ofA. aegypti with LD50and LD95values of 81.0 and 176.8 mg/L, respectively(Choochote et al. 2004).Their used concentration found higher and reported time found muchnarrower for the 100% mortality. This is in contrary to our results because we not observedsimilar scale mortality even within 7 hours after the treatment other than the complete paralysis.However Momin and Nair (2000) isolated 4 compounds from the hexane extract of celery seedand found that the three among them: b-selinene, 3-nbutyl- 4, 5-dihydrophthalide, and 5-allyl-2- methoxyphenol were larvicidal with 100% mortality of fourth instarAe. aegypti at 50, 25, and200 g/ml respectively. Different phototoxic compound, alpha-terthienyl which found in the rootsof Tagetes species also showed promising toxicity to the mosquitoes (Arnason et al., 1987) and

suggested that it could be a good candidate for the development of photo-activated insecticides.

4.4. Morphological growth disruption and malformation effects due to the toxicity of theseed extract of celery

Digital camera based macro-photographic technique was found easy for the cleardetection of the morphological growth disruption affects due to the photoactivated cytotoxicity ofsome compounds in the extracts on treated fourth instar larvae which possibly generated from


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8the neuro-muscular disturbance and subsequent cytological degenerations in the electrolytescontrol mechanisms located in the anal papillae of the mosquito larvae, probably further led toan interruption of the osmotic and ionic regulation and may be this phenomena intrinsicallyassociated with the death of mosquito larvae. This positively correlated in our behavioral andalso morphology observations on the treated mosquito larvae. Although the lower limits of ionconcentrations that permit survival of mosquito larvae have not yet been established, ionic

imbalance resulting from the interruption of ionic regulation is also a harmful condition. Ourfindings corresponded to those of earlier works that investigated the effect of plant naturalproducts on some species of mosquitoes. Chaithong et al. (2006) reported that pepper extractwhen tested to theAedeslarvae extensively damage and shrunken cuticle of the anal papillae.Similarly alpha-terthienyl, once introduced into the water medium containing mosquito larvae,enters into the anal gills and subsequently made halide leakage, releasing all the electrolytesinto the medium leading to death of the larvae (Downum et al., 1984). Nivsarkar at al.(1991a)observed an increase in the superoxide dismutase activity from 1st instar to 4th instar Aedeslarval stage. This increase seems to be a protective mechanism against hazardous oxygenderivatives generated by the actin of the phototoxin alpha-terthienyl . superoxide dismutase isfound in the entire gill, except in the tracheal network. Further studies conducted by Insun et al.(1999) revealed the severely morphological disruption of anal papillae observed in dead C.

quinquefasciatus larvae. After treatment with ethanolic extract of K. galanga, damaged analpapillae, with a shrunken cuticle border and destroyed surface with loss of ridge-like reticulumwere found under light and scanning electron microscopes, respectively. Green et al. (1991)reported distinct features of alteration such as highly swollen anal papillae of A. aegypti larvaeafter treatment with whole oil of Tagetes minuta. The two pairs of anal papillae are flexible, sac-like structures consisting of an epithelium covered by cuticle and situated on an extension of theterminal segment of mosquito larvae. In the fresh-water mosquito larvae, uptake and eliminationof most ions occur via the anal papillae, while the process of ion conservation is mainly locatedin the digestive tract (Garrett and Bradley 1984). The capacity to take up sodium, potassium,chloride, and phosphate ions from the medium was markedly reduced or lost in papilla-lesslarvae reported from Koch (1938). Other lipoidal membranes, in particular the neuromuscularsheath, become involved in the photoprocess as the photosensitizer diffuses to other sites

(Robinson, 1983). This is confirmed by the early photoinactivation of enzymes such asacetylcholinesterase (Ben Amor et al., 2000) which represents the neurotransmitter enzyme. Ageneralized oxidative modification of the membranes takes place, as suggested by ultrastructural studies (Callaham et al., 1977). Changes in membrane permeability are alsodemonstrated by the presence of altered potassium levels in the hemolymph (Weaver et al.,1976). The hemolymph volumes decrease significantly upon photosensitization and thehemocoel fluids undergo a rapid transfer from the body cavity to the alimentary canal with aconsequent increase in crop volume.

Result presented in this paper also observed that most of the larvae treating with celeryseed extract having no PM lines in the midgut lumen compared to controls, indicated that toxiccompounds in the extract can also act on peritrophic membrane degeneration after entering

orally and possibly act through the cytotoxic mechanisms as well. Dijoux et al. (2006) haveshown that Citrus aurantium dulcisand Cymbopogon citratus essential oils were phototoxic andcytotoxic. In other words, cytotoxicity seems rather antagonistic to phototoxicity. In the case ofcytotoxicity, essential oils damage the cellular and organelle membranes and can act asprooxidants on proteins and DNA with production of reactive oxygen species (ROS), and lightexposures do not add much to the overall Reaction. Obviously, cytotoxicity or phototoxicitydepends on the type of molecules present in the extracts and their compartmentation in the cell,producing different types of radicals with or without light exposure. However, such anantagonism is not quite a strict rule. Thus, when studying an extracts or essential oils it may be


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9of interest to determine systematically its cytotoxic as well as its possible phototoxic capacity.Similarly It has been documented that the root extract Derris urucu affected peritropic matrixstructure of Aedes aegypti larvae causing damage to the midgut epithelium (Gusmao et al.,2002). In insects, the midgut is main sight of digestion and absorption (Beker et al., 2010). Themidgut lumen is lined by non-cellular membranous structure, peritrophic membrane, whichprotects the mid gut, cells from toxic substances and pathogens that enter the midgut through

food (Peters, 1992). Gut disruption by the activity of phototoxic Alpha-terthienyl was alsoobserved before in other insects (Champagne et al., 1986). Correspondingly, the toxic effect ofethanolic-extracted Magonia pubescens on Ae. aegypti larvae was mainly in the midgut,showing partial or total cell destruction, high citoplasmatic vacuolization, increasedsubperitrophic space and cell hypertrophy, and the epithelium did not maintain its monolayerappearance (Arruda et al. 2003).

The result suggest that ethanol extract containg growth regulatory compounds whichpossibly generated on the disturbance of hormonal control. The most important deformities,pupal-adult intermediates and ecdysal failure, seemed to be the second cause of the mortalities.Likewise, such abnormalities were noted following treatment of immature mosquitoes with

juvenile hormone (JH) analogues and chitin synthesis inhibitors (El-Barky, 1993). The plant

natural products that detrimentally affect insect growth development offer a continual source ofinspiration and challenge. Insect growth regulation properties of plant extracts are veryinteresting and unique in nature, since insect growth regulator works on juvenile hormone. Theenzyme ecdysone plays a major role in shedding of old skin and the phenomenon is calledecdysis. When the active plant compounds enter into the body of the insect, they may die due toabnormal regulation of hormone-mediated cell or organ development. Other insects may dieeither from a prolonged exposure at the developmental stage to other mortality factors or froman abnormal termination of a developmental stage itself.Photosensitized induction ofphysiological and morphological abnormalities was detected at the larval, pupal and adult stageof mosquitoes (Pimprikar et al., 1979). In particular, there often appears to be an incompleteextrication of the pupal stage from the larval cuticle, while several adults are stuck to the chitininner lining of the puparium (Fairbrother, 1978). Similarly treatment with phototoxin alpha

terthienyl on herbivorous insects shows dense sclerotization on pupae (Downum et al., 1984).Our results made also clear co-relation with the recent findings reported from Khater and Khater(2009) where the essential oil of Apium graveolens has been reported not only to causeblowfly, Lucilia sericata larval mortality but also produced clear morphological abnormalities inlarvae, pupae and adults. Similar observations were obtained by other plant extracts againstdifferent mosquito species in earlier studies. Saxena et al. (1984) who had noticed similarmorphological deformities, including darkening of the larval cuticle, during moulting anddevelopment of C. quinquefasciatus induced by Ageratum conyzoides extract. Sakthivadiveland Thilagavathy (2003) reported that the acetone fraction of the petroleum ether extract ofA.mexicana seeds exhibited larvicidal activity, formation of larval-pupal intermediates, formationof pupal-adult intermediates.

We concluded that the crude ethanol extract of the seed of A. graveolens whichreportedly possess phototoxic compounds offers potentials against Ae. aegypti, particularlythrough its toxic and growth disruption activities. Its promising toxicity to mosquitoes makes it asa promising candidate for commercial bioinsecticide development. The photoactivatableinsecticides, which act through photodynamic pathways, clearly appear from many studies topossess several favorable features and a broad scope of applications. The main advantage oflight-activatable phototoxins is certainly represented by their lack of toxicity towards mostbiological systems in the dark, which minimizes their impact on the environment. However, itsvertebrate toxicology and its effects non-target organisms need further study before it seriously


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10be considered alternative to conventional mosquitocides. In fact, while most phototoxins andacridines are fairly photostable, both porphyrins and xanthenes undergo a fast degradation ofthe aromatic macrocycle upon illumination with sunlight or equivalent artificial light sources, witha consequent loss of absorption in the near- UV/visible range (Philogene et al., 1985). Thisfeature adds further value to the use of these compounds rich celery plants and its products asphotoinsecticidal agents but field trials are recommended. Currently our group are in the final

stage to isolate and purify the active compounds from the different extract fractions of celeryand in the same time we are investigating the enzyme inhibition activities together with theultra-structural effects of celery on mosquitoes.

Acknowledgments

The first author KEK express his sincere thanks to the Higher Education Commission,Pakistan for offering him a short term visiting Professorship under the Foreign Faculty HiringProgram (Phase-III).Authors are grateful to Professor Dr. Parvez Hassan, Institute of BiologicalSciences, The University of Rajshahi, Bangladesh for providing Bio-statistical softwares.

Author contribut ions and conf licts o f interest statement

KEK conceptualized and designed the research, performed the research and did macro-photography, analyzed the data, drafted the manuscript and made final revisions. RMT providedinstrument for mosquito rearing and organized the field activities. SA provided laboratory benchand equipments and MIC participated in study design and made critical revision of themanuscript as a group leader. The author(s) declare that they have no conflicts of interestregarding this article.

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Table 1

Knockdown effect due to the toxicity of the ethnaol extract derived from the seed of A.graveolenson the the fourth instarA. Aegypti larvae+.

Test conc.in ppm

% Larvalknockdown

KD50( 95% C.I., ppm)

LCL UCL

2values

(d.f.)

100200300400500

Control -1(DW+DMSO)Control-2 (DW)

144768717500

238.15(208.21-261.08 7.00(3)

+Values are based on five concentrations (100, 200, 300, 400 and 500 ppm) and five replications with 20

insects each.


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Table 2

A potent mosquito larvicidal effect due to the toxicity of the phototoxin enriched ethnaolextract derived from the seed ofA. graveolens+.

ExoposurePeriods

(h)

Test Conc.in ppm

% Larvalmortality

LC50( 95% C.I., ppm)

LCL UCL

2values

(d.f.)

24 100200300

Control 1(DW+DMSO)Control-2 (DW)

28889900

126.13(116.49 -136.57 0.20 (1)

48 100200300

Control -1(DW+DMSO)Control-2 (DW)

37969900

112.53(103.83 -121.96 1.33 (1)

+Values are based on three concentrations (100, 200, and 300 ppm) and five replications with 20 A. aegypti (4

th

stage ) larvae, each.


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A B

C D

Fig. 1. Digital photomicrograph of the 4th stage A. aegypti larvae: (A)

showing the aggressive anal gills biting behaviour of the A. graveolens

seedextract treated larvae at concentration of 200 ppm recorded at 10 minafter the treatment; (B), expressing high level larval knockdown recorded at

4 hours after the treatment; (C) and (D), showing completely normal

behavioural patterns generated from the control treatment ( distilled water

plus DMSO).


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Normal larva

TH

Malformed larvae

TH

A B CAP -S

Normal pupa

Sclerotized larvae

-

I-LDTDarkly pigmented

cuticleAlbino plus pigmented

cuticle

Malformed pupae

TH

AP -S

AP -S

Pupal- adult intermediates

Darkly pigmented

RT

RT

A-MP

CT CT

CT

Normal adult Malformed adult

G HMW

NW

Fig. 2. Morphological malformations which were observed in the Aedes aegypti

larval (B) and (C); pupal (E) and (F); adult ( H) stage after the treatment with the

ethanol extract of the seed ofA. graveleonsat concentration of 100 ppm. Aedes

Larva under control (A); pupa under control (D) and adult under control treatment

G . I-DT Intact larval di estive tract D-LDT disru ted di estive tract TH

thorax; RT, respiratory trumpet; CT, cephalothorax; A-MP, adult like mouthparts;

NW, normal wing; MW, malformed wing.

apium paper_khondkar ehteshamul kabir_ report_b6

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