Effect of Few Marine Sponges and its Biological Activity against Aedes aegypti Linn. Musca domestica (Linnaeus, 1758) (Diptera: Culicidae)
Aim of the present study was planned to assess the biodiversity of the marine environment around the Muttom coastal region and then to isolate and characterized the secondary metabolites from the eleven sponges and screen them for their mosquito larvicidal effect. Due to increasing resistance of the vectors mosquitoes causing diseases of importance in public health, to chemical insecticides, is necessary the searching for alternative control methods, as the use of marine sponges extracts with insecticide activity, owing to its capacity of biodegradation and generation of minor environmental damage. In this work the insecticide activity is evaluated for the mixture of chloroform with methanol extracts of the eleven marine sponges on stadium V instar larvae of Aedes aegypti (L.) and Culex quinquefasciatus (L.) in conditions of laboratory (25°C and 75% Relative Humidity). The sponge extracts of Clathria gorgonoids and Callyspongia diffusa was found to be the most effective against A. aegypti larvae showed LC50 values at <50 ppm. A result demonstrates that Ircinia campana extract is most active against two insects, as long as the extract of Sigmadocia carnosa has a higher toxic effect on Aedes aegypti than on Culex quinquefasciatus. Between the sponges Clathria gorgonoids and Callyspongia diffusa were found to be more active towards both larvicidal and insecticidal properties. In view of both these activities, the subsequent sponges Haliclona pigmentifera, Sigmadocia carnosa, Petrosia similes and Ircinia fusca could be used to obtain novel pesticidal molecules.
Received: March 04, 2010;
Accepted: May 25, 2010;
Published: August 13, 2010
Researches have recently concentrated their efforts on the search of active
natural products derived from marine sponges as alternatives to conventional
insecticides. The results of these developments are new approaches to pest control
that differ fundamentally from conventional chemical control. New pest control
strategies were needed to eliminate the effects of injudicious pesticide use
and the concept of the Integrated Pest Management (IPM) found strong advocates
(Vasanthraj, 2008). As sponge produces various novel
chemical molecules, they have been a goldmine to chemists and also found their
way in to biotechnological applications. Due to increasing resistance of the
vectors mosquitoes causing diseases of importance in public health, to chemical
insecticides, is necessary the searching for alternative control methods, as
the use of marine sponges extracts with insecticide activity, owing to its capacity
of biodegradation and generation of minor environmental damage (Martinez
et al., 2007). Marine sponges are shown to exhibit antibacterial,
insecticidal, antiviral and antiplasmodial activities (Volk
et al., 2004; Rao et al., 2003a).
Van Wagenen et al. (1993) described seventeen
years ago investigating the potential of marine natural products to serve as
insect control agents via mechanisms of toxicity, interference with moulting
or metamorphosis and feeding deterrence. Previous reports of sponge sesquiterpenes
and diterpenes from gorgonians and sea pens. These marine invertebrates have
evolved chemical defense mechanisms against other invading organisms, which
involve the production of secondary metabolites (Blunt et
al., 2005; Wah et al., 2006). Among the
marine invertebrates, porifera (sponges) remain the most prolific phylum concerning
novel pharmacologically active compounds. It has been known for centuries, that
sponges contain bioactive compounds that are of potential medical importance
(Bokesch et al., 2002). The current thrust of
the investigations were involves identifying newer drugs and other pharmaceuticals
from marine origin, where as comparatively little attention has been made with
respect to the discovery of pesticide molecules (Morlan,
1966). The Secondary metabolites isolated from marine sponges may be an
alternative source for vector control agents to replace existing and highly
toxic synthetic insecticides and will play an important role in future insecticide
This term adopts rationalization of pest control techniques to minimize harmful
environmental effects. New and more specific target compounds appeared from
marine bioactive compounds to have potential role in IPM (Thakur
and Muller, 2004). In terrestrial environment Recently, studies have also
suggested that some bioactive compounds isolated from marine organisms have
been shown to exhibit anti-cancer, anti-microbial, anti-fungal or anti-inflammatory
and other pharmacological activities (Venkateswarlu et
al., 1993; Proksch et al., 2002; Donia
and Hamann, 2003; Haefner, 2003;
Kumar and Zi-rong, 2004; Mayer and Hamann, 2005).
The Indian Ocean with a unique species of flora and fauna is one of the biologically
richest coastal regions in all of mainland of India. The biology of bacterium-sponge
relationship has elicited considerable interest among researches, as marine
sponges have been considered as a rich reservoiur of bioactive compounds (Bokesch
et al., 2002). The general objectives of this study were to evaluate
the biodiversity of the marine environment around the seas of Cape Comorin (Indain,
Ocean), to isolate and characterize secondary metabolites from sponges and screen
them for potential larvicidal and insecticidal properties. This paper reports
the taxonomic identification of some potential sponges as a source for further
exploration to obtain the pesticidal molecules. These bioactive principles either
may be produced from associated microbes or sponge itself. Hence, it is much
more important for greater cooperation and well co-ordinated efforts has been
seen between the bacteriologists, mycologists, natural product chemists and
entomologists for exploring the possibilities of developing newer pesticidal
molecules from marine sponges. There is a lot of scope to obtain of pesticidal
molecules especially new toxophoric groups, which can be appropriately incorporated
in molecules to obtain potent synthetic products with targeted features.
MATERIALS AND METHODS
Collection of sponges: Samples of sponges (n = 15) were collected in
the Cape Comorin coasts of Indian Ocean at depths varying from 10-15 feet by
snorkeling and SCUBA-diving during the year of April-2009. Sponges were gently
removed from the substratum and cut into small pieces then soaked in methanol
for preparing crude extracts. The intact sample specimens were sent to the Central
Marine Fisheries Research Institute (CMFRI), Trivandrum and Kerala, India for
collect the necessary details only identification purpose.
Preparation of crude extracts: The initial aqueous methanol extract was concentrated in the laboratory under reduced pressure and lyophilized. The lyophilized powder was extracted with 1:1 mixture of methanol and dichloromethane. At the same time the methanol soaked cut pieces (100 g) were further diced and extracted with the same mixture of solvents. The extracts were pooled and the organic portions were evaporated for obtaining solvent free crude extract. Then the test solutions with desired concentrations were prepared by mixing the known amount of crude extract in a carrier solvent, acetone (w/v) and after these preparations were subjected to larvicidal and insecticidal screening.
Biological screening: The cyclic colonies of Culex quinquefasciatus
were reared in our insectary at 27±1°C and 80±5% RH with a
photo period of 11:13 h light and dark cycles followed by the methods of Newman
and Cragg (2004) and Keiding et al. (1991),
respectively with little modifications.
Data analysis: The Musca domestica larval mortality in each dose/concentration
and control was recorded after 24 h of exposure. Percentage mortalities were
corrected for the natural mortality observed in the negative controls using
Abbott (1925) formula; P = PI C / 1 C,
where PI denotes the observed mortality rate and C means the natural mortality.
The median lethal concentration or dose (LC50 or LD50)
was calculated using Probit analysis (Finny, 1971)
that has been recommended by OECD guideline as appropriate statistical method
for toxicity data analysis. After linearization of response curve by logarithmic
transformation of concentrations, 95% confidence limits and slope function were
calculated to provide a consistent presentation of the toxicity data.
The use of marine natural products is an alternative pest control method, which helps to minimize the usage of toxic pesticides and their deleterious effects on non target insect species, livestock, wildlife and on the environment. The sponge extracts of Clathria gorgonoids and Callyspongia diffusa was found to be the most effective against A. aegypti larvae showed LC50 values at <50 ppm. However, other extracts of Dendrilla nigra, Petrosia testudinaria, Petrosia similes, Haliclona pigmentifera, Ircinia fusca, Sigmadocia fibulata showed LC50 values at <100 ppm (Table 1). The above eleven crude extracts were also screened for insecticidal properties using housefly lethality test against M. domestica and their LD50 values are presented in Table 2. Among the extracts, Clathria gorgonoids and Callyspongia diffusa sponges were proved to be the most promising extracts with insecticidal properties against female adult M. domestica at LD50 values at <50 μg/insect. Meanwhile extracts of the Ircinia fusca, Clathria reinwardti, Spirastrella inconstans and Sigmadocia pumila did not show either larvicidal or insecticidal activities even at higher concentrations or doses (400 ppm for larvicidal and 300 μg/insect for insecticidal activity).
The present preliminary investigations are helped us to short list the bio-active sponge crude extracts, which possess larvicidal and insecticidal activities. These active extracts could be used for obtaining new leads to isolate bioactive pesticidal molecules from marine origin. The percentage of active crude extracts (about 40%) identified in the present experiments is high in comparison to other terrestrial natural products. Use of these invented novel products in mosquito control instead of synthetic insecticides could reduce the environmental pollution. The present results may be useful as blue prints to isolate the active principles from these active crude extracts.
||LC50 values (ppm) for 24 h with their 95% fiducial
(lower and upper) limits, regression equation, Chi-square (χ2)
and p-levels of certain marine sponges against 5th instar larvae
of Aedes aegypti
||LD50 values (μg/fifth instar larvae) for 24
h with their 95% fiducial (lower and upper) limits, regression equation,
of certain marine sponges against 3-4 day old of female Musca domestica
The larvicidal activities of sponge extracts were evaluated against the fourth-instar larvae of Culex quinquefasciatus. The bioassays were performed at a room temperature of 30±1°C by exposing 25 larvae in each concentration of the extract. Triplicates for each concentration and the control (Distilled water), were tested for fifth instar larval bio-control potential. Table 1 show the relative activity among the eleven experimental sponges denoted the highest as well as lowest relative activity showed 4.67 and 1.0 responsible sponge species such as Ircinia campana and Clathria gorgonoids respectively. Though minimum and also with average relative activity observed with Petrosia similes 2.97 as well as moderate activity reflected as 3.40 particularly with Haliclona pigmentifera sponge.
Healthy female houseflies of three to four day old were treated topically on the dorsal surface of the thorax with five to six different doses (ranging from 100 to 500 μg/insect) of test solutions (1 μL/insect) with the help of a microapplicator.
A minimum of three replicates (35 flies each) were used for every test concentration. Simultaneously, control groups were also run with zero concentration of the test substance. After treatment the flies were transferred into observation jars and the mortality was recorded after 24 h. Marine sponge terpenoids have dominated the subject of chemical ecology since they have been studied for their activities against a variety of insect models. The relative activity showed that the Ircinia campana sponge having maximum activity 7.19 and moderate relative activity shows in Petrosia similes 3.67 also Clathria gorgonoids having the almost lowest relative activity.
Among the extracts evaluated, five of the sponge extracts were C. diffusa, C. longitoxa (Henschel), C. diffusa (Ridley) S. carnosa (Dendy), H. pigmentifera (Den.) and D. nigra (Dend.) showed significant activity in both larvicidal and insecticidal assays. Based on the results the most promising extracts are from C. longitoxa (Hen.) and C. diffusa that showed both larvicidal activities with LC50 at <50 ppm and <50 μg per insect, respectively. From this study, over all results were clearly indicated the better performance on both larvicidal and pesticidal activities shown marine sponge mainly belongs to the family of Microcionidae as well as Chaliniidae and the order of Poeciloscleridae and Haplosclerida. Based on the present findings envisaged that the above said sponges (crude extracts) consisted secondary metabolites could be useful for searching new novel pesticidal molecules from marine origin.
The use of marine natural products is an alternative pest control method, which
helps to minimize the usage of toxic pesticides and their deleterious effects
on non target insect species, livestock, wildlife and on the environment (Fatope
et al., 1993). Earlier, we have isolated a molecule, ethylene bisisobutyl
xanthate, from marine green alga, Dictyosphaeria favulosa (Kim
and Mendis, 2006) based on the larvicidal activity (LC50 value
at <50 ppm). The investigation further revealed that this molecule also exhibited
Insect Growth Regulator (IGR) properties against A. aegypti (Rao
et al., 1995). Based on the bio-active properties, several analogues
of alkylxanthates were synthesized and evaluated against lepidopteron pest,
Spodoptera litura and Helicoverpa armigera. Three of the analogues
i.e., methylene bis (tetrahydrofurfuryl xanthate), m-Fluorobenzyl n-butylxanthate
and m-Fluorobenzyl isobutylxanthate have shown antifeedent and IGR activities
against mosquitoes and agricultural pests (Rao et al.,
2003b; Taylor et al., 2005). During the last
decade, various studies on natural plant products against mosquito vectors indicate
them as possible alternatives to synthetic chemical insecticides (Thomas
et al., 2004; Dharmagadda et al., 2005;
Singh et al., 2005). Previous literature also
indicated that the marine organisms possess maximum percentage of bioactive
substances with novel biological properties than the molecules originated from
terrestrial origin (DOD, 1991).
Another findings relate this kind of sponge based pesticidal activity clearly
described by Balbin-Oliveros et al. (1998) and
marine biomolecules effect in rat brain enzyme synthesis level (Rao
et al., 1998) that is Philippine marine sponge of the genus Strongylophora
yielded a new meroditerpenoid-strongylophorine dimer (1) and the known meroditerpenoids,
strongylophorine-2 (2), strongylophorine - 3 (3) and strongylophorine-4 (4).
The structures of the compounds were established on the basis of NMR spectroscopic
also compound three was active against the phytopathogenic fungus Cladosporium
cucumerinum and also against the neonate larvae of the polyphagous pest
insect Spodoptera littura (Fab) demonstrated earlier by Okada
et al. (2006).
Further studies on identification of active compounds, toxicity and field trials
are needed to recommend the active fraction of these sponge extracts for development
of eco-friendly chemicals for control of insect vectors. Edrada
et al. (1996) demonstrated that the seven compounds were exhibited
insecticidal activity toward neonate larvae of the polyphagous pest insect Spodoptera
littura (with an ED50 of 35 ppm) when incorporated in artificial
diet and offered to larvae in a chronic feeding bioassay. Again Okada
et al. (2006) reported two novel insecticidal
metabolites, calyculin E (1) and F (2), which had insecticial activity against
the German cockroach and mosquito larvae, were isolated from a Japanese marine
sponge, Discodermia sp. Van Wagenen et al.
(1993) demonstrated that the validity of the premise that the marine biosphere
might be source of new insecticides, did not disclose compounds of commercially
significant potency against important insects pests. In case of that report
clearly described a simple, but novel sponge metabolite with striking activity
against not only our primary assay insect the tobacco hornworm, but also one
of the more intransigent insect pests the cockroach. Again Funda
(2007) described the insecticidal compounds derived from tropical marine
sponge A. carti demonstrated that the guanidine alkaloids, hymenialdisine
and debromo-hymenialdisine exhibited insecticidal activity towards neonate larvae
of the polyphagous pest insects Spodoptera littoralis (LD50
88 and 125 ppm, respectively).
Along with the eleven extracts evaluated seven of the sponge extracts, i.e., S. carnosa, H. pigmentifera, I. fusa, I. campana and C. gorgoniods, S. inconstans and Petrosia similis showed significantly higher activity on both larvicidal and pesticidal evaluation. Based on the results the most promising extracts are from C. gorgonoids and C. diffusa that showed both larvicidal and insecticidal activities with LC50 and LD50 at < 50 ppm and < 50 μg per insect, respectively. The results obtained from this study suggested that the above said sponges could be useful for searching new novel pesticidal as well as insecticidal molecules from marine origin. From the results, able to identify the secondary metabolites in sponges that were found to be effective pesticidal activity against lepidopteran pests and also effective insecticidal activity against the larvae of Aedes aegypti (Say).
Authors would like to tell the deepest gratefulness to our College Correspondent Rev. Fr. Premkumar (MSW) Malankara Catholic College, Mariagiri, for given sound support with persuade for preparing this manuscript.
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