Investigations on the Larvicidal Effect of Red Algal Seaweeds of Rameswaram Coast Against Tobacco Caterpillar Spodoptera Litura (Fab.)

Spodoptera litura (Fab.) (Lepidoptera: Noctuidae) commonly Tobacco caterpillar as a polyphagous pest has caused considerable damage in economically important crops such as cotton, groundnut, chilli, tobacco, castor, pulses, etc. in India, China and Japan (Kamaraj et al., 2008; Ahmad et al., 2013) and has estimated to cause crop losses between 10 to 30 per cent in major crops (Ferry et al., 2004). Numerous insecticides have been used for decades for the management of tobacco caterpillar. The indiscriminate use of chemical pesticides not only caused hazards to soil micro flora, animals and human life but also contribute to the development of resistance by pest resurgence and secondary outbreak of pests, toxicity to non target organisms etc. (Kannaiyan, 2002 and Tripathi et al., 2015).  These problems paved way for the search for alternatives which would reduce the use of insecticide, negate the resistance, resurgence and residual problems of insecticides (Sable and Rana, 2016). Marine algae as renewable living resources are rich in biologically active metabolites have been shown to have bactericidal (Cordeiro et al., 2006); fungicidal (Rajesh et al., 2011) and insecticidal activities (Sahayaraj and Jeeva, 2012; Yu et al., 2015; Kannan and Bharathkumar, 2016). In this context, red algal seaweeds collected from Rameswaram has been utilized to investigate their toxicity against S. litura.

Collection of seaweeds
 
Seaweeds belonging to Rhodophyta, Red algae G. corticata, L. ceranoides and A. taxiformis were collected in different seasons, from the coastal region and deep sea of Rameswaram, India. The seaweeds were collected by hand picking method and immediately washed with fresh sea water and were thoroughly washed three times with tap water to remove the excess salt, sand and epiphytes. To drain off the water, the algae were wiped with a blotting sheet and air – dried under shade and stored under dry condition in environmental chamber (Kannan and Bharathkumar, 2016). The collected seaweed’s identity was confirmed at Centre of Advanced Studies in Marine Biology, Faculty of Marine Sciences, Annamalai University.
 
Mass culturing of Spodoptera litura
 
Spodoptera litura egg masses were collected from groundnut field at Regional Research Station, Tamilnadu Agricultural University, Virudhachalam, Cuddalore district of Tamil Nadu were mass cultured using the natural diet (castor leaves) under laboratory conditions in sterilised plastic trays (surface sterilised with formalin 1%). The collected eggs were sterilised with 0.02% Sodium hypochlorite solution, dried and placed in sterilised plastic trays covered with a muslin cloth. The eggs were allowed to hatch and the neonates were fed with fresh tender castor leaves (washed with water and shade dried). First and second instar larvae were fed once in a day in the morning hours and subsequent instars were fed twice in a day in morning and evening hours.
         
The larvae were allowed to grow till pre-pupal stage. For enhancing pupation, sterilised soil was placed at the plastic tray’s bottom. After pupation, they were collected and placed inside the oviposition chambers (40 x 25 x 25 cm) for adult emergence. The emerged out adults moths were collected in test tubes and six pairs (sex ratio 5:1) were allowed inside the oviposition chamber for mating and oviposition. Adults were provided with a food containing 10 per cent honey solution and vitamin drops (cotton soaked with 10 per cent honey solution and few drops of multivitamins) to increase fecundity. Nerium leaves were kept inside adult emergence cage for egg laying. The petioles of leaves were immersed in water kept in a conical flask to maintain freshness.
        
Adult moths after three days of release laid the egg masses on Nerium leaves. Egg masses were collected and placed inside the Petri plate. After hatching, the neonates were transferred to plastic trays, fed them with castor leaves and reared. Batches of 25 larvae each was maintained per bucket and the culture was maintained. From the culture uniform aged third instar larvae were used for the Bioassay experiments (Kannan and Bharathkumar, 2016).
 
Preparation of Seaweed powder
 
Collected seaweeds, G. corticata, L. ceranoides and A. taxiformis were spread on filter paper and the dusts, debris, contaminants and other impurities were removed and allowed to shade dry until the moisture content was reduced. Shade drying process was continued until the plant materials were dry enough to be powdered. The dried plant materials were taken individually and ground to powder with the help of electric blender. Whole plant powder preparations were stored in glass containers and were used for the evaluation experiments (Indhumathi, 2017).
 
Evaluation of seaweed powder for larvicidal action against third instar larvae of S. litura
 
The prepared seaweed powder of Red algae @ 1, 3, 5, 7, 10 and 20 per cent concentrations were evaluated against uniform aged third instar larvae of S. litura. Leaf powder was treated on the castor leaf discs of 5cm diameter by dusting and the treated leaf discs were placed in the Petri dish (5 discs per plate). Six hours pre-starved third instar larva were released @ one larva per leaf disc and allowed to feed on the treated leaves. The experiments with seven treatments were replicated three times with five larvae per replication under completely randomized design. As subsequent feeding, after feeding the treated leaves, larvae were provided with untreated leaf discs. Data on the larval mortality of the test insect was recorded every 12 hours up to pupation and the adult emergence data were statistically analyzed and documented (Gomez and Gomez, 1976).

Investigations in the laboratory to assess the repellent, antifeedant and larval mortality effect of three red algal seaweed’s powder on S. litura observed with the following results. None of the concentrations exhibited repellent and antifeedant activity. The G. corticata leaf powder exhibited larval mortality after 36 hours of treatment whereas the other two seaweeds’ powder displayed larval mortality only at 60 hours of treatment. G. corticata influenced larval mortality between 20 and 100 per cent within 36 and 72 hours of treatment in all the treatments. The larval mortality data at 36 hours of treatment in the highest concentration (20%) was 40.00 per cent and with the succeeding times i.e., 48 and 60 hours the per cent mortality gradually increased with 80.00 and 86.66 per cent respectively and at the 72 hours the larval mortality reached 100 per cent (Table 1). The larval mortality data collected at 72 hours after treatment revealed that all the concentrations encountered cent per cent death of larva (Table 1).
 

       
The A. taxiformis leaf powder demonstrated larval mortality only after 72 hours of treatment whereas after 72 hours of treatment, the mortality of treated larvae was between 6.66 and 53.33 per cent. At 96 hours of treatment, the maximum larval mortality (53.33%) was observed at 20 per cent concentration (T₆) followed by 10 and 7 per cent (46.66%) respectively compared to control where no mortality was occurred (Table 1). The experiment was continued after 96 hours and observed that the larvae were transformed into pupa and the data were furnished as below. The larval to pupal transformation was between 46.66 and 80.00 per cent with the maximum (80.00%) pupation exerted by one per cent concentration and the minimum (46.66%) was by 20 per cent concentration. Insect growth regulator activity viz., pupal malformation was observed in the developed insects wherein 20 per cent concentration resulted maximum malformation with 46.66 per cent of pupated insects were malformed followed by 20.00 per cent at 10 per cent concentration whereas no malformation at one per cent concentration and also in control (Table 1). All the transformed pupae other than malformed were emerged out with the lowest adult emergence was in  20 per cent concentration with 0 per cent followed by 10  and 7 per cent concentrations with 33.33 and 40.00 per cent adult emergence wherein 100 per cent adults were emerged out in control (Table 1).


       
Data from the studies on the influence of L. ceranoides for their larval mortality of S. litura observed that only at 72 hours of treatment the treated larva showed the signs of death and the mortality was between 0.00 and 26.66 with the maximum mortality at T₆. The data in Table  1 showed that the larval mortality gradually increased and it was observed up to 132 hours after which it has pupated. The larval mortality was observed at 96 hours of treatment showed that the mortality range was between 33.33 and 66.66 per cent with the maximum mortality (66.66%) exerted by the maximum concentration and the minimum by one per cent concentration (33.33%). The observed larval at 132 hours envisaged that the maximum per cent death (86.66%) was exhibited by 20 per cent concentration (T₆) followed by 10 per cent concentration (80.00%), 7 per cent concentration (73.33%), 5, 3 and 1, per cent concentration (66.66%)  compared to control where no mortality was noticed (Table 1).
        
The larva to pupal conversion is high (33.33%) at 1, 3 and 5 per cent concentration and low (13.33%) at 20 per cent concentration without any malformation. All the transformed pupae were emerged out as adults without any mortality (Table 1).
        
Comparing the larval mortality pattern exhibited by three red algae, G. corticata initiated the larval mortality at 36 hours of treatment and all the treated larva at all concentrations have died at 72 hours of treatment whereas A. taxiformis and L. ceranoides powder treatments showed larval death only at 72 hours of treatment and the larval duration extended up to 96 and 132 hours respectively (Table 1). The time taken to cause larval mortality by G. corticata was lesser compared to other two red algae and it has caused cent per cent mortality at 72 hours wherein the insects treated with other two algae extended the larval periods also.
        
Comparing the pupation data between A. taxiformis and L ceranoides the later found to be effective in reducing the survival of pupa than the earlier (Fig 2).
 

        
Adult emergence data given different inkling that A. taxiformis at the highest concentration only influenced cent per cent effect when compared to L. ceranoides, but in other concentrations, L. ceranoides powders exhibited decreased level of adult emergence over their rival A. taxiformis (Fig 2).
        
The results pertaining to larval mortality indicated that G. corticata was the best among the three seaweeds and to justify the results the phytochemicals were found out using GCMS anlaysis. The total number of compounds identified in ethanol extract was 13. The compounds identified in G. corticata through GC-MS are Hydroxylamine, O-decyl-, Decanoic acid, ethyl ester, Tetradecane, Octadecane, 6-methyl-, 4,7,10- Hexadecatrienoic acid, methyl ester, 1- Gala-1-ido-octose, 1-Dodecanol, 3, 7, 11- trimethyl-, Tetradecane, 2, 6, 10- trimethyl-, 7, 9 – Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, 1,2-Benzene- dicarboxylic acid, butyl octyl ester, 2-Bromotetradecanoic acid, Benzyldiethyl-(2,6-xylylcarbamoylmethyl)-ammonium benzoate and 6,9,12,15-Docosatetraenoic acid, methyl ester with their GC- MS Chromatogram of the number of peaks of the compounds detected was shown in Fig 3.
 

       
The reports evidenced in this study are in accordance with the findings based on the evaluation of a red algae Plocamimum cartilagineum for its insecticidal action against the sucking pest, Aphis fabae and D. cingulatus by  Argandona et al., (2000) and evidenced to have an insecticidal action against the sucking pest which demonstrated 91 per cent mortality against D. cingulatus after 48 hours exposure. The report by Rajesh et al., (2011) also in support of our findings that the algae have the insecticidal action wherein they have observed that the green algae Caulerpa scalpelliformis was found to be toxic to Dysdercus cingulatus and suggested that it can be utilized in the management of S. litura and D. cingulatus. Further, Holden and Ross (2014) made known that the commercial extracts of brown seaweed Ascophyllum nodosum suppressed Thrips and Persia mite colonies and this suppression was same as the suppression in number due to an insecticide abamectin treatment. Similar reports of botanical leaf powder’s efficiency in protecting the pulse grains against Callosobruchus chinensis was demonstrated by Suthar and Bharpoda (2015). Further evidences of green algal potentiality of Chaetomorpha antennina and Ulva fasciata against S. litura by Kannan and Bharathkumar (2016) and Brown algae G. edulis potentiality against  vectors Culex quinquefasciatus and Chironomus circumdatus by Madhiyazhagan et al., (2017) have  proved that seaweed algae either red or brown to have insecticidal action.
       
The higher larvicidal and IGR activity influenced by G. corticata may be due to the presence of phytochemicals and the GCMS analysis shown to have 13 chemicals inside of which some of them may be have greater influence on larval mortality. The present findings were in accordance with the reports of  Argandona et al., (2000); Zakaria et al., (2011; Neelamathi and Kannan (2016) and Deepak et al., (2017) wherein (Argandona et al., 2000) found that red algal seaweed Plocamium cartelagenium  inhabited Violacene, mertensene, Dibromomertensen, Dihydromertensen,-4-6-Trichloro-3-(2’-chlorovinyl)-1, 3-Dimethylcycohexane which are responsible for the insecticidal action against many insect. Presence of Oleic acid (58%), n – hexandecanonic acid (24.73%) and aromatic dicarboxylic acid in red algal seaweed A. spicifera was reported by Zakaria et al., (2011). The carbohydrate content was highest in Cystoseira indica, protein in Gracillaria corticata, phenol content in Padina boergesenii, flavanoid content was found greater in Cystoseira indica, chlorophyll content in Monostroma latissimum and carotenoid content was more in Dictyopteris acrostichoides and these phytochemicals were found to inhibit the growth and development of pathogenic micro-organisms like against Aspergillus niger and Penicillium janthinellum (Kumar et al., 2014).
       
The GC- MS analysis of crude methanolic extract of Turbinaria ornata revealed the presence of a mixture of volatile compounds of which Tetrachlorobenzene, n-Tetradecane, Isopropyl isothiocyanate, Heptanal and Humulene epoxide III were reported to have insecticidal action. (Neelamathi and Kannan, 2016). Similarly, the phytochemical screening of brown marine algae Turbinaria ornata revealed the presence of saponin, alkaloids, amino acids, fixed oil and fat and phenolic compounds (tannins, flavonoids and total phenol (Deepak et al., 2017). These reports supported the results in this present study that the phytochemicals might have caused the mortality and IGR activities against the insect and would be investigated in future to exploit their potential to become a new approach in Integrated pest management.

Red algal seaweed Gracilaria corticata powder at all the concentration tested have demonstrated cent per cent larval mortality at 72 hours of treatment whereas the other two seaweeds have initiated their mortality only at 72 hours and the phytochemicals present in G. corticata confirmed the presence of insecticide chemicals.

Authors are grateful to Professor and Head, Department of Entomology for extending facilities for the conduct of research work.