Evaluation of novel natural product insecticide from The Egyptian Red Sea coast

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(8)pp: 99107(2017) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
The marine environment is the greatest biome of bioactive natural products, which produce several novel structures with special biological properties.These products may not be found from terrestrial origins.The Red Sea is one of the most marine bio-diverse ecosystems on the earth with high numbers of endemic of marine organisms.The Red sea has many types of soft corals which may reach up to 180 species.Many Known soft corals species worldwide, approximately 40% are endemic to the Red Sea (Edwards1987).Soft corals of the genus Sarcophyton (family Alcyoniidae) are particularly rich in cembranoids diterpenes, Triterpenoids, Tetratepenoids, Ceramide and Cerebrosides.Cembranoids contain 14-membered macro cyclic skeletons and exhibit a wide range of biological activities including anti-tumor, neuro-protective, antimicrobial, calcium-antagonistic, and antiinflammatory activity Hussien & Mohamed (2015).New and many specific target compounds appeared from marine bioactive products to have a potential role as insect pests management (IPM), (Thakur and Muller, 2004).Soft corals are marine invertebrates having a wide range of terpenoid metabolites.These terpenes, mostly cembranoids, represent the main chemical defense for coral against natural predators (Roethle and Trauner 2008).Soft corals of the genus Sarcophyton (family Alcyoniidae) are rich in cembrane terpenes, (Blunt et al 2008 ) .Cembranoids contain a 14-membered macro cyclic skeleton and show a wide range of biological activities including anti-tumor, neuro-protective, antimicrobial, calcium-antagonistic, and antiinflammatory activity (Sawant et al,. 2006, Wahlberg andEklund 1992).
Sarcophyton glaucum, known as rough leather coral, is a common leather coral found in the Red.It grows upon seaward slopes, reef flats and in lagoons (Lieske et al., 2004).Lin and Yue (2013) reported that eleven sequiterpenes with six skeletons from 1995 to July, 2011, (one is a novel skeleton) were isolated from six species of this genus, i.e. , Sarcophyton buitendijki, S. elegans, S. acutangulum, S. glaucum, S. ehrenbergi, and S. trocheliophorum.Moreover, in recent years several kinds of research had paid attention toward marine natural products and many studies have recommended that some of the bioactive compounds isolated from marine organisms exhibited insecticidal properties and its metabolic products may be considered alternative sources for vector and agricultural pest control agents.These bioactive metabolites may potentially replacing the existing and highly toxic synthetic insecticides and will play an important role in the future insecticide development program, (Venkateswara et al., 2003).While previous studies have also suggested that some secondary metabolite isolated from marine organisms had been shown to exhibit larvicidal, insecticidal, and IGR (insect growth regulator) activities (Handayani et.al. 1998, Ogamino andNishiyama 2005).Soft coral of the genus Nephthea (Alcyonacea, Nephtheidae) has yielded a variety of bioactive sesquiterpenes and diterpenes which exhibited insecticidal activities on polyphagous pest spodoptera littoralis (Handayani et al.,1997).Our study is shed light on the insecticidal effects of ethanol, chloroform and aqueous extracts of soft coral Sarcophyton glaucum against the storage pest Sitophilus oryzae.

Insect Culture:
Adults of S .oryzaewere collected from naturally infested Freeka (green durum wheat Triticum aestivum).Insects were reared in the laboratory on clean, uninfested and sterilized Freeka grains.Twenty adults of insects were released in 20 g of Freeka grains which kept in glass jars (21x10 cm) capped with a cotton cloth to ensure ventilation.All the jars were maintained at room temperature 27±10°C and 65 % RH and continuous darkness.All individuals used in the test were 5-7 days old.

Animal Material:
Soft coral samples Sarcophyton glacum were collected from the Egyptian Red Sea Gulf of Suez, Al-ain Elsokhna, August, 2015 at a depth of 4-6 m.The soft coral was identified by Dr. Abdel Hamid Abdel-Rahman Ali Assistant professor at the National Institute of Oceanography and Fishers Suez branch.Samples of Sarcophyton glaucum were taken to Department of Entomology, Faculty of Science, Benha University.Freshly collected sample of S. glaucum was immediately frozen and kept at −20 °C until ready for solvent extraction and analysis.

Extraction and Separation:
The Marine Sarcophyton glaucum was cut into small pieces, blended and extracted sequentially with Ethanol, chloroform, and distilled water in Botany Department, Faculty of Science ,Benha University.Initially, the S.glaucum (100 g) was soaked in 200 ml .Ethanol for 72 h. and filtered through filter paper.The residue was dried and then extracted sequentially with ethanol, chloroform and distilled water solvents, after 72 h. of soaking in each solvent separately.The extracts were condensed separately under reduced pressure by using vacuum evaporator, and the solvent-free crude extracts were collected in glass vials and stored in 4 °C until use.Stock solutions of the three solvent extracts were prepared and then subjected to bioassay screening.The chemical analysis was performed for screening the active materials by using stander of the different solvent procedures (Trease andEvans, 1989 sofowra 1993) Table (1) .

Insects Bioassay:
Five concentrations were prepared 20, 200, 500, 1000 and 2000 ppm of ethanolic, chloroform and aqueous extracts of S. glaucum added to 20g. of Freekeh (green durum wheat, Triticum aestivum ) which kept in glass jars (21x10 cm).The grains in the control contained solvent only no extract .The jars with their contents were gently shaken to ensure thorough admixture of the freekah and treatment extracts.Twenty adult S. oryzae were introduced to each of the jars and covered with cotton cloth and tight with rubber bands.Three replicates of the treatments and untreated controls were prepared.The adult mortality was assessed daily by counting the dead adults.Adults were considered dead when probed with sharp objects and there were no responses Statistical Analysis: The mortality percentage of S. oryzae in each concentration and control was recorded after 24 h of exposure and the mean ± SE for each triplicate were calculated.The data was then subjected to one way ANOVA test using SPSS program version 21.LC 50 and LC 95 were calculated by Probit analysis (Finny, 1971).

RESULTS AND DISCUSSION
This work is focusing on marine natural products as safe pest control method which may help in minimizing the applying of toxic pesticides and their dangerous effects on non-target insect species, livestock, humans and on the environment (Fatope et al., 1993).Previous studies concluded that the marine natural products may act as toxicant and insects growth regulator.These extracts of marine natural products could be marketable due to their low cost, biodegradability, and safety to the environment as pest control agents.Natural product especially, marine products can support chemical control methods or can play role in integrated pest control programs (IPCP).The current study is conducted to evaluate the efficacy of different concentrations of soft coral S. glaucum extracts with the time of exposure of insecticidal activity on Sitophilus oryzae.Screening analysis results showed that the active materials of soft corals S. glaucum were extracted by ethanol, chloroform and distilled water are terpenes, alkaloids and tannins, respectively, table (1).This result was in agreement with the findings of Kita et al.,(2010) & Fukatsu et al.,(2007) by applying soft coral S. glaucum, which have Secondary metabolites are often derived from symbiotic algae and/or symbiotic bacteria.(20, 200, 500,1000 and 2000 ppm.) were at values 16.66, 30.00, 36.66,50.00 and 53.33%.respectively, with LC 50 at Value 1502.0 ppm.and LC 95 at value 5611.0 ppm In addition, the mortality percentage after 24h. of exposure at concentration was (20, 200, 500,1000 and 2000 ppm) were 10.00,16.66,20.00, 36.66 and40.00.respectively, with LC 50 at Value 2273.0 and LC 95 at value 6200 ppm.The mortality percentage between groups were significant p ≤ 0.05 in relation to control.This toxicity may be revealed to the extract contain active ingredient terpenes.These results are in a good agreement with the previous report by (Gross et al.,2004, Sawant et al., 2006), they stated that some of the typical terpenoids are known as chemical defense tools to protect soft corals from its natural enemies, as feeding deterrents or act as a toxicant.Moreover, most of the secondary metabolites extracted from soft corals have been reported to exhibit a wide spectrum of biological activities such as anti-microbial, anti-fungal, anti-viral, anti-fouling (Wei et al., 2013).These secondary metabolites have ecological importance to soft corals (Ishii et al., 2010a).In addition (Blunt et al., 2008, Hussien & Mohamed, 2015), reported that soft corals of the genus Sarcophyton (family Alcyoniidae) are rich in cembranoids, diterpenes Triterpenoids, Tetratepenoids, Ceramide and Cerebrosides.Cembranoids contain a 14-membered macro cyclic skeleton and exhibit a vast range of biological activities including neuro-protective, antimicrobial and calcium-antagonistic.In the current study, we recorded the mortality percentage increased with increasing the concentration and exposure time.So, the mortality of insects increased with increasing concentration level and time of exposure from 24 h to 96 h (Fig. 2) The Data in (Fig. 2) cleared that the high mortality percentage was achieved at 83.33% and insecticidal activity of chloroform extracts of S. glaucum on S.oryzae adults may be due to its active ingredient at concentration 2000 ppm followed by the values 70.00, 63.33, 56.66 and 36.66%respectively, at concentrations1000 ,500, 200 and 20 ppm with LC 50 and LC 95 values 166.9 ppm and 3200 ppm after 96 h While the mortality percentage after 72 h of treatments were 20.00 , 46.66, 56.66 , 63.66 and70.00%, at concentrations 20, 200, 500,1000 and2000 ppm. respectively, with LC 50 and LC 95 values 659.7 ppm and 4078.0 ppm respectively.The mortality percentage after 48h. of treatment at the same concentrations were 13.33, 23.33 ,33.33,40.00and 43.33% respectively, with LC 50 and LC 95 values 2119.0 and 6887.0 ppm respectively The mortality percentage after 24 h of exposure were 6.66 ,13.33, 23.33 and 26.66 % respectively, with LC 50 and LC 95 at Value 2771.0 and 6985.0 ppm respectively (Table .1).

Fig.2. Mean Mortality percentages at different concentration levels of chloroform extract from Sarcophyton glacum on the adults of Sitophilus oryzae
The Data in cleared that the insecticidal activity of aqueous extracts of S. glaucum on S.oryzae adults .The mortality percentage ( above medium) it was 63.33% at concentration 2000 ppm.followed by 26.66%, at the lowest concentration 20 ppm.The other concentrations 1000 , 500 and200 ppm.were at values 50.00, 46.66 and 40.00% respectively, with LC 50 and LC 95 at values 1036.0 and 5550.0 ppm.respectively,.On other hands the mortality percentage after 72h. of treatments were 16.66, 23.33, 36.66 and43.33%, at concentrations 20, 200, 500,1000 and2000 ppm. respectively, with LC 50 and LC 95 at Values 2224.0 and 6982.0ppm .respectively (Table .2).The mortality percentages after 48h. of treatment at the same concentrations were10.00,16.66,23.33,26.66 and 36.66%respectively,with LC 50 and LC 95 values 27776.0 and 7610.0ppm.respectively,.The mortality percentage % after 24h. of exposure were 6.66, 10.00 ,13.33 ,16.66 and 23.33 % ,respectively with LC 50 and LC 95 at Values 3851.0 and 8708 ppm.The results were recorded the mortality percentages by using the three extracts ethanol, chloroform and aqueous from soft corals Sarcophyton glaucum, on Sitophilus oryzae adults.We concluded that the ethanolic extract was more effective and achieved the highest mortality rate on S.oryzae adults 100%.On the other hand, the aqueous extract was less effective and give the lowest mortality rate (above medium) on S.oryzae adults 60.33%.Meanwhile, chloroform achieved high mortality rate less than Ethanol 83.33% at the concentration (2000 ppm.) after 96h.Meanwhile, the mortality percentages at the lowest concentration (20 ppm.) of ethanol, chloroform and aqueous extracts after 96h.were recorded at 40.00, 33.33and 23.33%.respectively,.These results cleared that the ethanol extracts had potential insecticidal activity followed by chloroform, at last, came aqueous extracts.The Sitophilus oryzae is susceptible to the active materials of Sarcophyton glaucum extracts and more susceptible to ethanol followed by chloroform and distilled water.
The rate of mortality percentage increased by increasing the concentration and time.In all treatments, the mortality percentages between groups were significant p ≤ 0.05 in relation to control.This results may be revealed to the presence of terpene , alkaloids and tannins and other chemical contents.Moreover, Sesquiterpenes are secondary metabolites present in many marine organisms including soft coral e.g., Dendronephthya sp., Sinularia gardineri, Litophyton arboreum, Sarcophyton trocheliophorum, S. glaucum and Parerythropodium m fulvum ( Elkhayat et al., 2014, Ellithey et al., 2013;Al-Footy et al., 2015, Al-Lihaibi et al., 2014).
In addition some authors confirmed the presence of chemicals were isolated from soft corals Sarcophyton glaucum.Ne'eman et al., (1974) reported that new diterpene was isolated from soft corals Sarcophyton glaucum .This compound was given the name sarcophine.It was toxic to mice, rats and guinea pigs and showed strong anti-acetylcholine action on the isolated guinea pig.Sarcophine was also a competitive inhibitor of cholinesterase in vitro.Also, Feller et al.,(2004), explored, that the chemical content of S .glaucumcollected from many seas, was thoroughly resulting in the discovery of a large number of cembranoid ,biscembranoid ,sterols, and other secondary metabolites.Abdel-Lateff et al., (2015) investigated the S.glaucum collected from the Red Sea chemically and they found that eleven isoprenoidal metabolites,a new natural cembranoid, sarcophinediol, and seven known cembranoids.Soft corals, sponge and algae investigated by some researcher for bio-insecticides, antibacteria, , antifeedant and other bioactive compounds.Results showed extracts of sponges Hymanicidon sp., Placortis nigra, Theonella sp., and Ianthella sp., soft corals of Sinularia gravis and Sarcophyton cinereu had antibacterial activity, while those from Petrosia nigricans, Placs pongia melobesioides, Rhabdastrella of globostellata, Stylissa of Carteri, Theonella sp., were active against Aedes aegypti larvae.( Ekowati 2008).

Conclusions:
The present study is the first experimental evidence insecticidal activities of soft coral Sarcophyton glaucum marine extracts recorded against adults of Sitophilus oryzae.The mortality percentages by using these active extracts of the novel product of soft coral are promising in S.oryzae control.Moreover, are achieved high mortality percentage on S. oryzae adults control in comparison with other terrestrial pesticides.The using of these the marine natural product instead of synthetic insecticides could reduce the environmental pollutions.

Fig. 1 .
Fig.1.Mean Mortality percentages at different concentrations of ethanolic extract of Sarcophyton glacum on the adults of Sitophilus oryzae

Fig. 3
Fig.3 Means Mortality percentages of different concentrations levels of aqueous extract of Sarcophyton glacum on the adults of Sitophilus oryzae

Table . 1
: Screening analysis of active materials of Sarcophyton glaucum by different solvents extracts(Ethanol, chloroform and distilled water).denote to the presence of active ingredient --denote to not the presence of active ingredient This study of acute toxicity by using different concentrations(20, 200,  500,1000 and 2000 ppm)of ethanolic extract from soft coral S.glaucum on adults Sitophilus oryzae, after 96h ( Fig.1) shows that 100% mortality percentage was achieved at highest concentrations of ethanolic extracts from S.glaucum 2000 ppm.And 1000 ppm.Meanwhile, the mortality percentages of other concentrations 500, 200 and 20 ppm.were 80.0 ,76.66 and 43.0%respectively, after 96 h of treatments with LC 50 at value 63.18ppm.andLC95 at value 967.1 ppm While the mortality percentage after 72h. of treatments at concentrations20, 200, 500,1000 and 2000  ppm.were at values 26.66, 43.33, 56.66,76.66 and 83.33% respectively ,with LC 50 at Value 423.5 ppm.and LC 95 at value 2193.0 ppm Meanwhile, the mortality percentage after 48 h of treatment at the same concentrations was *