Insecticidal Potential of Botanicals from Red Seaweeds against Stored Grain Pests, Rice Weevil (Sitophilus oryzae L.) and Cowpea Weevil (Callosobruchus maculatus Fab.)

Rabia Bibi1, R. Muhammad Tariq2, Samir A.M. Abdelgaleil3 and Munawwer Rasheed1, 4* 1Centre of Excellence in Marine Biology, University of Karachi, Karachi-75270 2Department of Zoology, University of Karachi, Karachi-75270 3Department of Pesticide Chemistry, Faculty of Agriculture, Alexandria University, Egypt-21545 4Department of Chemistry, University of Karachi, Karachi-75270 Article Information Received 19 August 2019 Revised 12 march 2020 Accepted 20 June 2020 Available online 22 October 2021


INTRODUCTION
A part from insufficient food production needed for completing the human population demand, food security is also a fundamental problem in many developing countries including Pakistan. Advanced technologies have not been introduced in various under developing countries. These technologies include grain dryers, seed pelleting, sensors, nano-techniques, genetic manipulation of cowpeas against bruchids, sealed bags such as Purdue Improved Cowpea Storage 'PICS' and hermetic bags and/or modern storehouses that built on scientific design (Adedire and Ajayi, 2003;Mutungi et al., 2014;Afzal et al., 2019;Mahto et al., 2019;Kpoviessi et al., 2019). Considerably, more than 1600 insect species have been known to attack O n l i n e

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as the poor man's meat which contains a rich source of protein and carbohydrate fulfilling the human nutritional requirement (Ahmed et al., 1991). However, by the last couples of year, crop production is affected by the climate change, drought condition, shortage of water and damages caused by the insects (Rehman et al., 2015). Infestation by coleopteran beetle reduces the quantity of the harvested grain by feeding and also seeds' productivity by reducing its germination ability. Their rapidly growing population contaminates grains with eggs, dead bodies, webbing, toxins, and hairs or by casting skins which increases the grain susceptibility for fungus growth, a further source of diseases. Moreover, proteins secreted by insects are involved in producing allergic reactions to human (Arlian, 2002). Ultimately, the grains damaged by the beetles with holes lose its cultural and commercial values, becoming unfit for the human and animal consumption (Tripathi et al., 2009).
Sitophilus oryzae L. also known as rice weevil (Coleoptera: Curculionidae) is the most damaging cereal pest. Both the immature and adult stages are the internal feeder and very difficult to recognize. It lefts grains broken and hollow form inside. Extensive feeding on grains is reported to reduce about 75% of the grain's total weight .
Callosobruchus maculatus Fab. (Coleoptera: Bruchidae) trivially called cow-pea weevil is the serious, multivoltine pest, causing serious damages to stored pulses in Pakistan and many other tropical countries. Bruchid beetles feed on the grains producing holes, which are then used to lay eggs. Often whole grain and these holes are found covered and filled with eggs. Heavy infestation resulting in the severe weight loss and powdering of stored commodities. In a period of 3 to 6 months storage, up to 90% damage can be caused by the C. maculatus alone (Ofuya, 1986;Boeke et al., 2004).
Control strategy is still based on the fumigation and synthetic pesticides, an environmental hazards. Residues of pesticides cause risk to health of living organisms and real danger to human. To combat the increasing resistance, rate the demand of novel and effective bioactive insecticidal compound has been increased (El-Wakeil, 2013). Therefore, some quick action is needed to be taken to control the destruction of food crop, especially by the stored pests. Thus only by controlling crop pests it can be possible to get the healthy nutritional food. A number of botanicals touted as an antifeedant, toxicants, and repellent against stored grain pests have been reported broadly (Jayasekara et al., 2005;Abdelgaleil et al., 2009Abdelgaleil et al., , 2016. Marine algae are rich source of novel bioactive and secondary metabolites possess diverse biological activities (Kolanjinathan et al., 2014). More than 2400 natural products have been reported from seaweeds and the largest varieties of natural products are found to be present in Rhodophyta (Red algae) (Domettila et al., 2013). Bio-potential of seaweeds against dengue mosquito population (Bibi et al., 2020), root rotting fungi and nematodes (Sultana et al., 2018), antimicrobial (Ambreen et al., 2012), antileishmanial (Sabina and Aliya, 2011), antioxidant (Sabina et al., 2006) larvicidal (Hira et al., 2017) have been reported. However, few citations are available concerning the toxicity to stored grain pests (Fukuzawa and Masamune, 1981;Abou-Elnaga et al., 2011;Rizvi and Shameel, 2003). Large biomass of seaweeds, enriched with bio-active compounds, is wasted every year along the Karachi coast (Dawn News, 2020). Therefore, current study is an approach to utilize red seaweeds as an alternative safer source of bio pesticides to replace synthetic pesticides.

MATERIALS AND METHODS
Insecticidal screening of four red seaweeds A. taxiformis, G. foliifera, L. karachiana, J. rubens extracts were experimented against two species of stored grain pests S. oryzae and C. maculatus

Test insects
For pest infestation, rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae) and cowpea weevil, Callosobrocus maculatus F. (Coleoptera: Bruchidae), were collected from the Toxicology Laboratory of Department of Zoology, University of Karachi. Pests were reared in laboratory under controlled conditions maintained at 29 ± 4 °C, 50 -70 % RH. Rice weevil, S. oryzae was reared in whole rice and C. maculatus was reared in whole lentils. First generation of adults (1 -4 days old) was used for the toxicity assessment.

Collection of seaweeds
Two species of red seaweeds Laurencia karachiana sp. nov (molecularly confirmed Bibi et al., 2019) Fig. 1). Samples were deposited in the Karachi University Herbarium (KUH), for voucher numbers as mentioned in parenthesis (vide supra).

Preparation of seaweed materials and extraction
Seaweeds were washed thoroughly with tap water to remove salt, sand, or epiphytes, and air-dried at room O n l i n e

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temperature. Samples were grounded and sieved (60 mesh size). Extracts were prepared using Soxhlet extraction method. Powdered seaweed (20g (x4)) was refluxed with solvent (500 ml) for 8 h in Soxhlet flask (250 ml) as per method described in AOAC (1995). Solvents of varying polarity i.e., hexane, dichloromethane, and methanol (Fisher Scientific, Leicestershire, UK) were used. Solvent was evaporated using rotary vacuum evaporator (Eyela, Tokyo, Japan). Extracts were refrigerated in amber vials at 4 o C till toxicity assessment. Details of the extracts' codes are given in the footnotes of tables.

Contact toxicity assay
For toxicity assessment contact toxicity method was applied (Auamcharoen et al., 2012). Briefly, 9 cm diameter filter paper discs (Whatman No. 4) were impregnated with five different concentrations (0.94, 1.26, 1.57, 2.89 and 2.2 mg/cm 2 ) of extract along with three replicates and placed on the glass petri plates (9 cm diameter). The solvent (acetone) was allowed to evaporate at room temperature. Twenty adults of S. oryzae and C. maculatus were introduced separately into the center of each petri dish and covered tightly with lid. Rice and lentils were added in petri dishes for food (not treated). Each experiment was conducted in triplicate. Two control groups (+ve) and (-ve) i.e., with or without acetone, respectively were run in parallel with the experiment. The number of dead and moribund insects was counted with 24 h intervals after treatment till 96 h. Knockdown effect was also determined at their respective doses. The inhibitory oviposition effect of treated extracts on C. maculatus (10 pair) was calculated by counting the number of eggs laid with the help of magnifying glass at 2.2 mg/cm 2 applied dose after 96 h.

Statistical analysis
Mortality in control was corrected using Abbot's formula (Abbott, 1925). Data were subjected to statistical analysis using SPSS 21 ® software. Probit ® analysis (Finney, 1971) was applied to calculate the LC 50 values at 95% confidence interval. Bonferroni test was applied to check the statistical difference (P = 0.05) on the oviposition inhibitory effect of extracts on the C. maculatus (SPSS version 21 ® Statistical Package for Social Sciences, USA).

RESULTS
LC 50 values obtained in seaweeds treatment to C. maculatus and S. oryzae are presented in Tables I and II respectively indicate the toxic effects of red seaweeds. Mortality in the control group was found considerably below 5%. Probit ® analysis showed that C. maculatus was more susceptible with high mortality than that of S. oryzae. Although, increasing concentrations and exposure duration decreases LC 50 values. Detailed data is presented as supplementary data (Supplementary Tables S1 and S2).
In general, toxic effects of different botanicals from seaweeds were found least effective after 24 h exposure. S. oryzae and C. maculatus showed low susceptibility to different test extracts at lower applied doses. Meanwhile, the dichloromethane extract of A. taxiformis (DA) was significantly effective against C. maculatus LC 50 1.14 mg/ cm 2 after 24 h with complete control i.e., 100% mortality at 2.2 mg/cm 2 obtained after 48 h treatment with LC 50 0.75 mg/cm 2 (Table I). HL and ML showed toxicity next to DA extract with LC 50 1.24 mg/cm 2 and LC 50 1.55 mg/cm 2 respectively after exposure of 24 h.
Knockdown effects of DA and HL were noted for C. maculatus prior to the mortality with sever paralysis and jerking effects. These extracts were also effective in inhibiting the oviposition rate. Significantly lower numbers of eggs were laid by C. maculatus on grains (Fig. 2).
In contrast, S. oryzae was found tolerant at all the higher applied doses of dichloromethane, methanol, and hexane extracts after 24 h. However, after 48 h only DA achieved significantly higher mortality (88 ± 4.4%) at 2.2 mg/cm 2 and Anti Pest Activity of Red Seaweeds O n l i n e (56 ± 2.9%) at 1.57 mg/cm 2 (LC 50 1.43 mg/cm 2 ) (Table II). Complete mortality obtained after 96 h at a dose of 2.2 mg/ cm 2 (LC 50 0.93 mg/cm 2 ) ( Table II). In addition to the DA, profound toxic effects against S. oryzae was also determined in the MA (LC 50 1.16 mg/cm 2 ) and in the HA (LC 50 1.27 mg/ cm 2 ), HG (LC 50 1.32 mg/cm 2 ) and HL (LC 50 1.43 mg/cm 2 ) after 96 h (Table II). HJ also provided significant toxicity (LC 50 1.64 mg/cm 2 ) with >50% mortality achieved after 72 and 96 h exposure at higher applied doses (Table II).

DISCUSSION
Pest damage in store commodities, including cash crops adversely affects their quality and quantity. Chemical pesticides are the common practice. Fumigants such as aluminum phosphide, sulfur dioxide, ethylene dichloride, methyl format, methyl bromide are widely used in storehouses. Some countries reported genetic resistance in insects against phosphine, an alarming situation (Afzal et al., 2019). In the current study red seaweeds have proven significant contact and fumigant effects against S. oryzae O n l i n e

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Anti Pest Activity of Red Seaweeds 5 and C. maculatus. Results showed that botanicals of red seaweeds attribute towards strong activity against both the species tested. These active compounds should be isolated and identified. In general, red seaweeds are reported to be enriched with the terpenes, alkaloids and polyphenolic compounds (Perez et al., 2016). Wide range of secondary halogenated metabolites has been isolated from seaweeds showed significant antifeedant and repellent effects (McClintock and Baker, 2001). However, isolation of bioactive compounds and toxicity assessment of the seaweeds inhabiting coast of Karachi (northern Arabian Sea) have not been carried out.
Systematically, the only study tested the toxic effects of red, brown, and green seaweeds against the five species of stored grain pest i.e., Callasobrocus analis, Rhyzopertha dominica, Sitophilus oryzae, Tribolium castaneum and Trogoderma granarium by applying only one dose (200 mg) of one extract (methanol) in filter paper (Rizvi and Shameel, 2003). Authors reported that the red seaweed Osmunda pinnatifida was active on number of insects tested with 20% mortality while the brown seaweed Jolyna laminariodies exhibited moderate effects (with 40% mortality) after 24 h of application, rest of seaweeds showed no effects.
Results of the current study also supported these observations determining low mortality after 24 h of application. In the present study, both the species (S. oryzae and C. maculatus) showed low susceptibility to most of the extracts. However, Rizvi and Shameel (2003) did not determine the dose dependency and time related effects of seaweeds. Thus, current experiment encompassed and studied the contact or fumigant toxicity varies with insect species, extract's concentration and exposure time. The contact toxicity tests of the seaweed extracts against S. oryzae showed very low susceptibility in all treatments within 24 h. In such course of time no any extract achieved >40% mortality (LC 50 >2.2 mg/ cm 2 ) (Table II and Supplementary Table II). Maximum mortality (>90%) was achieved in most of the extracts at higher doses and/or exposing insects for longer duration of time (48-96 h) especially in the case of DA, MA and HL. The mortality was found decreasing in following order; HA, HG (>70%) and HJ and ML (>60%). C. maculatus was more susceptible towards the seaweed extracts with studied mortality. Knockdown effect was also noted prior to the mortality. Among four red seaweeds A. taxiformis showed significant toxic effects towards C. maculatus adults along with marked knockdown effect.
Exposure of C. maculatus with the DA (1.14 mg/cm 2 ) and MA (1.73 mg/cm 2 ) extracts of A. taxiformis induced 50% mortality after 24 h application. In general, treatment with L. karachiana extracts significantly reduce the C. maculatus population, while treating with J. rubens was not effective with the exception of hexane extract (HJ) inducing 50% mortality after 48 h (Table II). Fukuzawa and Masamune (1981) in their study also reported the toxic effect of two isolated acetylenic sesquiterpene compounds from red seaweed Laurencia pinnata on the bean beetle Callosobruchus chinensis. Similarly, the non-polar crude extract and isolated acetogenin(s) from Laurencia papillosa also exhibited high insecticidal activity against the 3 rd instar larvae of Tribolium confusum (Abou-Elnaga et al., 2011).

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R. Bibi et al. It was also observed that owing to the extractability some of the extracts were least active against stored grain pests. The polar solvent (methanol) extracts more constituents than a polar solvent (hexane). Dichloromethane has an intermediate polarity. A. taxiformis and L. karachiana possess potent toxic, knockdown and oviposition effect. The constituents present in applied botanicals derived from A. taxiformis using dichloromethane solvent exhibited knockdown effects, showing the presence of fumigant and/ or contact toxicants in the extract which possibly inhibit the neurotransmitter or alter the insect physiological phenomenon. Volatile compounds enter the insect body through spiracle resulting in accumulation of CO 2 which in turn suffocates insects. Suffocation keeps spiracle open and more fumigants enter attach with hemoglobin causing extra cellular fluid and/or hemolymph acidosis (Sugiura et al., 2008).
Inhibition of neurotransmitters e.g. acetylcholinesterase (AChE) induces damages by blocking sodium-potassium channel and disturbing nerve-membrane potential effecting CNS and PNS (Rattan, 2010). Consequently, knockdown effect which was observed in the C. maculatus while exposing insects to the dichloromethane extract of A. taxiformis (DA) and hexane extract of L. karachiana (HL) induced high mortality within 24 h of application. In addition, the marked decline in egg laying by C. maculatus in these treated extracts was also determined (Fig. 2). Studied extracts from L. karachiana and A. taxiformis significantly reduced the number of eggs laid by C. maculatus (P < 0.001) subsequently reducing adult emergence. Current results are also in agreement with Schmidt et al. (1991) who reported the significant effects of vapours of Acorus calamus oil in adult mortality and in the declining the egg laying after 96 h of treatment. Sahayaraj and Kalidas (2011) also studied the toxic effect of brown seaweed Padina pavonica against cotton sucking pest Dysdercus cingulatus (Fab) (Pyrrhocoridae). Chloroform and benzene extracts of P. pavonica induced high nymphal mortality, along with the reduction in the egg hatchability and abnormalities in embryonic development. The eggs were found varying in color, shape, and size. In the current study, all treatments of A. taxiformis and L. karachiana that significantly reduce the eggs laying by C. maculatus counted after 96 h of treatment ultimately reduces seed damage in turn become beneficial in making the grains viable for the human and animal consumption.

CONCLUSION
To conclude dichloromethane extract of A. taxiformis (DA) was found insecticidal. C. maculatus was being more susceptible. Hence, suggesting possible use in the further experiments and stored product pests control. Additional experiments are required to understand the mode of action of the particular compounds involved in the mortality, knockdown effect, reduced oviposition, and effects on the progeny, egg hatchability, adult emergence, and embryonic development in order to understand and to make the use of botanicals of red seaweeds at large scale in controlling pests in stored grain commodities.

ACKNOWLEDGMENTS
Authors are thankful to Dr. Lubna Ghazal, Department of Geography, University of Karachi for map assistance, Dr. Sarwat Ghulam Rasool for statistical analysis and Dr. Aisha Khan, Department of Botany, University of Karachi for voucher specimen.

Statement of conflict of interest
The authors have declared no conflict of interest O n l i n e 5±2.9 5±2.9 5±2.9

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(-control) 0±0 0±0 3.3 ± 3.3 3.3 ± 3.3 *H, D, and M in codes represents hexane, dichloromethane, and methanol extracts while J, L, A, and G represents J. rubens, L. karachiana, A. taxiformis, and G. foliifera. Superscript a to d indicate significant difference among variables (Bonferroni text; P < 0.05). No mortality observed in "MJ" in any applied concentration.