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It has been found that plants and animals produce various organic compounds known as secondary metabolites. These compounds have found applications as insecticides, biochemical tools or fragrances. Even though secondary metabolites from marine organisms are sectors which are expanding very rapidly (Murti, 2006), terrestrial organisms represent the richest source of natural drugs, such as plants (e.g. paclitaxel (TaxolÂ®) from Taxus brevifolia which was later found to be produced by the endophytic fungus Taxomyces andreanae (Stierle et al. 1993)), or microorganisms (e.g. penicillins from Penicillium notatum) (Putz, 2009). In 2004, Samuelsson defined secondary metabolites as substances that are formed in organisms but that do not participate in those metabolic processes which are necessary for the life and development of the organism.
New eco-friendly collection methods should be used so as to provide sufficient biomass to researchers as marine resources should be used sustainably (Brümer and Nickel, 2003; Hedner, 2007). With respect to this, genetic engineering has made prospecting for new drugs much more environmentally friendly, it being routine to collect as little as 1kg of living material. DNA is being extracted from this and cloned into host bacterial cells which produce quantities of the chemical in the laboratory (Blunden, 2001; Murti, 2006).
According to research, marine organisms such as sponges, soft corals, algae, ascidians, bryozoans, and mollusks produce secondary metabolites that are not similar than those found in terrestrial organisms (Davidson, 1995; Newman and Cragg, 2004; Murti, 2006). Marine sponges are a rich source of structurally novel and biologically bioactive metabolites (Purushottama, 2009). Therefore, marine sponges have been a goldmine to chemist and also found their way in to biotechnological applications as they produce various novel chemical molecules (Kumar Jha and Zi-rong, 2004).
A research by Hay (1996 cited Hedner 2007) showed that the various secondary metabolites produce by chemically defended organisms have a synergic or additive effects among the different metabolites. According to Murti (2006), any newly produced secondary metabolites that offered an evolutionary advantage to the producing organisms would contribute in the survival of the new strain.
Biosynthesis of secondary metabolites
Primary metabolites are the precursor of secondary metabolites such as alkaloids, glycosides, flavonoids, volatile oils etc (Khanan, 2007). Due to the high investment in energy and carbon, it follows that these compounds probably have important ecological roles, either as protection against biotic factors such as herbivory, predation and competition or abiotic factors such as UV light. Whatever their specific functions they probably play some role that benefits the producing organism (Ronagni, NA).
The border between primary and secondary metabolism has not been well defined (Ronagni, NA). Metabolites are the intermediates and products of metabolism. The term metabolite is normally reserved to small molecules. Compare to primary metabolites, secondary metabolites are not directly involved in the normal growth, development, and reproduction but they usually have important ecological function. Secondary metabolism is associated to both environmental conditions and development stages (Khanan, 2007). In 2003, Hanson stated that the producers of most primary metabolites experience the biological effects of these metabolites within the cell or organism compare to secondary metabolites which exert their biological effect on other organisms.
Secondary metabolites are often synthesized using repeated monomers, as in the case of terpenoids, or by using a mixture of different, but structurally simple, building blocks. This expands structural diversity and allows for different types of substitution. The primary pathways involved in the production of secondary metabolites are the acetyl-polymalonyl (polyketide), shikimate and mevalonate pathways (Ronagni, NA).
Figure Interrelationship of biosynthetic pathways leading to secondary constituents
Marine secondary metabolism
The uppermost biodiversity is found in ecosystems, such as rocky coasts, kelp beds, and coral reefs, where species diversity and population density are particularly high (Haefner 2003; Hedner, 2007). Therefore, the marine environment is composed of an exceptional and various source of natural products, principally from invertebrates such as sponges, soft corals, tunicates, bryozoans and mollusks and from bacteria and cyanobacteria (Donia and Hammer, 2003; LI Kam Wah et al., 2006).
According to (Purushottama, 2009), one of the few de novo sources of drug discovery which is a marine natural is of global interest. Paul and Puglisi (2004) stated that a lot of attention was laid in marine chemical ecology focusing on chemical defense mechanism of microalgae and invertebrates. This defense mechanism controls competition between predator and prey. There are 3 parallel tracks in marine natural products chemistry, marine toxins, marine biomedicinals and marine chemical ecology. Combination of the three fields of study gives marine natural products chemistry its unique character and strength (Murti, 2006).
In 2007, Hedner mentioned that secondary metabolites are produced by marine organisms as a response to physical conditions, such as lack of light, low temperature, high salinity or extreme pressure. These adaptations have given rise to a vast diversity both at biological and genetic levels. They have found a way in the sphere of biotechnology and are associated for the production of drugs, to remediate the environment, to assure food security and safety, to control over new resources and industrial processes. (Mayekaret al., 2010). In order for marine organisms to reproduce, communicate, protect them against predators and competitors, they have evolved biochemical and physiological mechanisms for these purposes (Halvorson, 1998; Murti, 2006).
In the marine environment, most of the bioactive metabolites that are used in clinical and preclinical trials are of invertebrates origin such as sponges, tunicates, bryozoans or mollusks which in contrast to the terrestrial environment where plants are the main producers of natural products (Proksch et al., 2002). The composition and type of compounds involved in the chemical defense can vary dramatically among geographic regions, habitats and between individuals in the local habitat, and even within a single individual (Harvel et al., 1993; Hay 1996; Hedner, 2007).
We have already passed the discovery phase in the field of marine natural products and we are now moving to the second phase where research to synthesize new drugs from marine sources is being driven by the understanding of relationships and processes. Plant, animals and microorganism grown under marine environment will be used for the development of new products and services which will have technological importance in the future. The presence of estuaries, creeks, deep seas and continental shelf renders Indian coast biodiversity and various marine resources. Therefore, the possibility for research for production of marine drugs is there (Mayekar et al., 2010).
Figure Marine natural product distribution
Source: Parvatkar, 2011
According to Bergquist (2009), sponge is a sessile, filter-feeding metazoan which utilizes a single layer of flagellated cells (choanocytes) to pump water in only one direction through its body. They can be found everywhere, in tropical and subtropical benthic marine habitats but are also found at higher latitudes and even in freshwater lakes and streams (Purushottama, 2009). Sponges have evolved antagonistic effect against other invading organisms, which evolve the production of secondary metabolites (Wah, L.K et al., 2006; Gehan M. Abou-Elela et al., 2009).
1.4.1 Marine sponge structure
The sponge body is organized around a system of pores, ostia, canals, and chambers, which conduct water current from the inhalant sponge surface to the exhalant apertures, the osculum (Murti, 2006). In 2011, Stone et al mentioned that some cells are also very pluripotent (i.e., capable of differentiating into other cell types) and sponges are capable of easily remodeling cell-cell junctions. These features probably allow sponges to adapt to diverse and extreme habitats and are largely responsible for the extreme phenotypic displayed by some sponges.
Nowadays, four classes of Porifera have been recognized namely: Calcarea, Hexactinellida, Demonspongiae and Sclerospongieae respectively. The Calcarea are exclusively marine sponges with a skeleton of calcium carbonate, organizes either as discrete spicules, or as a fused mass. The Hexactinellida are again marine sponges which are more common in deep water, their skeleton is siliceous, made up of megascleres and microscleres, both of which can have a hexactine structures. Dermospongiae is the most diverse class of sponges. There skeletons are composed of siliceous spicules, spongin fibers, or both. The skeleton of Sclerospongiae consists of siliceous spicules and spongin on a thick basal layer of calcium carbonate (Bergquist, 1978).
Figure Organisation of sponge body
1.4.2 Secondary metabolites from marine sponges
A research by Wallace (1997 cited Murti 2006) demonstrated that thousands of species of sponges have now be identified and each species produces a different set of secondary metabolites to help them occupy their particular niche. Marine sponges are a rich source of structurally novel and biologically active metabolites (Purushottama, 2009). Since marine invertebrates, especially sponges, often house a large number of microorganisms in their tissues, it appears tempting to assume that in many cases, associated microorganisms are the true producers of bioactive natural compounds (Tachibana et al., 1981; Putz, 2009). The symbiotic microbial community is highly novel and diverse, and species composition shows temporal and geographic variation (Webster and Hill 2001; Yoo Kyung Lee et al., 2001).
The use of chemical pesticides have become so alarming that nowadays conventional agriculture means using chemicals (Moazami, NA). In 2008 Miller stated that the application of synthetic pesticide has increase significantly since 1950 more than 50-fold, and at the present time pesticides are 10-100 times more poisonous than those used in 1950s.
Carson (2007) point out the consequence of the hazardous and careless use of synthetic pesticides in agriculture meaning polluting the natural environment causing a disbalance in the ecology, presence of pesticides in fruits and vegetable, soil, fodder, elimination of biological agent, pest developing resistance against chemical pesticides. This is why, it is essential to develop a new approach to tackle pests in a more eco-friendly, economically viable and socially acceptable for the farmers (Vasantharaj, 2008).
It makes no doubt that we must turn ourselves in the selection of broad spectrum biopesticides and improvements in the production, formulation and application technologies (Moazami, NA). Biopesticides contribute largely when used in line with Integrated Pest Management (IPM) programs (Gupta and Dikshit, 2010). A research by Kogan (1998 cited Bailey et al., 2011) show that pest management should be done efficiently in such a way that the impact on other components of the agroecosystem are minimized simultaneously. Hence, the need of producers, wider society and the environment must be taken into consideration.
Biopesticides fall into three categories that is, biochemicals pesticides, microbial pesticides and plant incorporated protectants (Muraleedharan and Elayidom, 2008). Although many pesticides are designed to kill pests, some may only inhibit their growth, or simply attract or repel them (Joshi, 2006). Biopesticides are often complex in their activities and modes of action, offering new tools in the quest to develop programs that can manage resistance (Manker, 2012).
1.5.1 Benefits of biopesticides (Joshi, 2006)
Biopesticides are naturally less harmful than synthetic pesticides.
Biopesticides are designed to kill specific targeted pests or organisms while conventional pesticides may affect many different organisms which do not need to be controlled.
Biopesticides are often effective even when they are used in small quantities and also the decomposition rate is often high, therefore resulting in lower exposures and avoiding the pollution problems caused by chemical pesticides.
As part of Integrated Pest Management (IPM) programs, biopesticides contribute to decrease the use of synthetic pesticides without decreasing crop yield.
Biopesticides have the potential to reduce the risks associated with pesticides.
1.6 Biology of Plutella xylostella (Diamondback moth)
Belonging to the family Plutellidae and order Lepidoptera, the diamondback moth, with scientific name Plutella xylostella (Knodel and Ganehiarachchi, 2008), is a real threat to the Brassicaceae family throughout the globe, and can have a drastic effect on the economy if not monitored (Facknath, 1997). Development time to pupa ranges from 25-30 days is dependent to environmental conditions like temperature (Capinera, 2000). These moths got their name due to the diamond pattern form by the stripe on their wings when they are at rest. The eggs are around 0.5mm in length, oval and pale yellow. The larvae (caterpillars) are pale green, slightly tapered at each end can reach 12 mm throughout their four stages. During the first two stages they have a dark head. When disturbed, they wriggle and often drop from the plant on a silken thread. The presences of a gauze-like cocoon on the underside of the leaves into which they pupate detect their maturity stage. At first the colour of pupa is green and then turns to brown larvae before the adult moth emerges (Henry and Baker, 2008). Larvae go through 4 instars: the range of days per instar is 3-7, 2-7, 2-8, and 2-10 for the 1st-4th instars (Meischer, 2003).
The Diamondback moth (DBM), Plutella xylostella is a major problem throughout the earth as their larvae causes serious damage to crucifer crops such as cabbage, broccoli, cauliflower, collards, kale, kohlrabi, Chinese cabbage, and Brussels sprouts. All the different development stages of the plant from seedling to head are subject to attack (Cordero, 2009). Damage is caused by larval feeding. The young larvae feed on the internal tissue forming shallow mine which look like white marks. When they grow, they come out to feed on the underside of leaves leaving holes (Anon, NA). ).Even though the larvae are tiny they are quite numerous and they can feed on the entire leaf leaving the veins. They are particularly injurious to seedlings and may lead to disturbing formation of head in cabbage, broccoli and cauliflower. Larvae present in floret generally leads to complete rejection of produce (Capinera, 2001).
1.6.2 Plutella xylostella in Mauritius
The diamondback moth (DBM), Plutella xylostella, lies among the most damage causing pest on crucifers in Mauritius(Dunhawoor and Abeeluck, 1997).Pest attack severity determines the insecticidal dose (MANR, 1995; Facknath, 1997):
(MANR, 1995; Facknath, 1997):
Suntap (cartap hydrochloride),
Cascade (flufenoxuron) and
Figure Progression of pesticides in Mauritius due to the resistance of the Diamondback moth Source: Facknath 1997
Initiated in 1994 the DBM project with association of international atomic energy agency (IAEA) tried to develop measures to control the environment and that could be implemented in the IPM list to farmers so as approach an attempt to reduce pesticide load on crops (Dunhawoor and Abeeluck, 1997).Considerable research is being done to develop an IPM strategy for DBM control. The University of Mauritius and the Ministry of Agriculture have looked for new approaches (Facknath, 1997).
Table Research strategies presently being explored for the control of Plutella xylostella in Mauritius
At The University of Mauritius
Pathogens (Bacillus thuringiensis)
Intercropping and use of trap crops
Combinations of above
Botanical and cultural control
At the Ministry of Agriculture
Synthetic pesticides and growth regulators
F 1 sterility
Source: Facknath 1997
1.7 Insecticide resistance
Resistance, from the scientific perspective, is a heritable, statistically decrease in sensitivity to a chemical in a pest population relative to the response of susceptible populations that have never been exposed to pesticides. When resistance occurs, the efficiency of a pesticide is decreased significantly (Dennehy and Dunley, 1993).
IRAC (NA) mentioned that the repetitive failure of an insecticide to control a pest population at a certain level reflects insecticide resistance even though the insecticide has been used by following the instruction on the product label and problems like storage, application, and unfavorable climatic or environmental conditions can be eliminated as a cause of failure. Dennehy and Dunley (1993) stated that the chemical provide less control of the pest at locations where the proportion of population that is resistant is high compare to locations where it is low and where there is no resistance at all as the relative efficacy of the insecticide has been reduced.
1.7.1 Development of resistance
Pesticide resistance is a genetically based phenomenon. When a pest is exposed to a pesticide, a few individuals will survive. This is because they are genetically predisposed to be resistant to the pesticide. The increase in application rate and dose of the pesticide will not only kill a large proportion in the pest population but will also cause an increase in pesticide resistance. The parents will pass their genetic makeup to their offspring. Consequently, the ability of the pest to be resistant to pesticide will continue to increase from generation to generation (Bellinger, 1996).
Both the frequency and intensity of the resistance determine the level of resistance and the relative efficiency of the pesticide. Frequency refers to the proportion of the pest population that is resistant; intensity is the strength of the resistance in each resistant pest. It makes no doubt that as the frequency of resistant individual increases in a population resistance will be a real problem. The intensity of a resistance can also affect the pesticide's usefulness in the field. The efficacy of a pesticide on the field is not affected drastically when a pest population has a resistance of low intensity even though the frequency is high. In contrast to this, the efficiency of a chemical can be reduced if the level of resistance is high despite the fact that the frequency in the pest population is low (Dennehy and Dunley, 1993).
Figure Frequency and intensity of resistance Source : (Dennehy and Dunley, 1993)
It has been shown that resistance is not the only reason why the relative efficiency of a chemical is decreased. Some other reasons are (Dennehy and Dunley, 1993):
The breaking down of the pesticide by soil microorganisms.
The pH of the spray water is too high.
Poor application procedures of the pesticide.
Insects have developed numerous ways in which they have been able to become resistant to crop protection products, and they can display more than one of these mechanisms simultaneously (IRAC, NA).
1.7.2 Mechanisms of resistance
Insects have developed numerous ways in which they have been able to become resistant to crop protection products, and they can display more than one of these mechanisms simultaneously (IRAC, NA).
â€¢ Behavioral resistance
The resistant insects may detect or identify a hazard and avoid the toxin. This type of mechanism has been reported for several classes of insecticides, as well as organochlorines, organophosphates, carbamates and pyrethroids. The insects can bring an end to their feeding, or leave the target area (for example, they can move to the underside of a sprayed leaf, move deeper in the crop canopy or fly away to another area where insecticide has not been applied) if they come across certain pesticide.
â€¢ Penetration resistance
This type of resistance depend on the absorption rate of the toxin by the insects that is, the susceptible insects absorb the toxin more rapidly than the resistant one. The reason behind this is that the insect's exoskeleton develops barriers which prevent the chemicals to get into their bodies rapidly and this allows them to be protected from a broad range of insecticides. Penetration resistance often works in collaboration with other forms of resistance and this produce a synergic effect to the other mechanisms.
â€¢ Metabolic resistance
It is the most frequent mechanism and represents one of the greatest challenges. When the insecticide is applied, those insects that are resistance can detoxify or break down the toxin faster than non-resistant insects, or they can quickly free their bodies of the toxic molecules. The insecticides are broken down by the internal enzyme systems of the insects. Resistant strains may possess higher levels or more efficient forms of these enzymes. Furthermore, these enzyme systems may also have a wide spectrum of activity (i.e., they can degrade many different insecticides).
â€¢Altered target-site resistance
The site where the toxin generally binds in the insect are altered in order to reduce the insecticide's effects. This is the most common mechanism after metabolic resistance.