The Effect of Chitosan on the Greater Wax Worm Galleria Mellonella L

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Int. J. Agricult. Stat. Sci. Vol. 16, Supplement 1, pp. 1743-1747, 2020 www.connectjournals.com/ijass DocID: https://connectjournals.com/03899.2020.16.1743 ISSN : 0973-1903, e-ISSN : 0976-3392 ORIGINAL ARTICLE

THE EFFECT OF CHITOSAN ON THE GREATER WAX WORM GALLERIA MELLONELLA L.

Hind I. AL-Khazraji* and Kameela Word Shaher Department of Plant Protection, College of Agricultural Engineering Sciences, University of Baghdad, Iraq. E-mail: [email protected]

Abstract: The study was conducted to evaluate the efficacy of the prepared chitosan from two different sources and its biological effects on greater wax worm larvae under laboratory conditions. Larvae of the third and fifth instar of the insect were fed by bee’s wax treated with chitosan prepared from the bodies of dead honey bees workers, called Beetosan and commercial chitosan. The results indicated the superiority of Beetosan in causing the highest effects on the biology of the greater wax worm, as the treatment led to an increase in both the duration of the larvae and the pupa. There was also noticeable decrease in the weights of the pupa resulting from the treated larvae, as well as the percentage of adult emergence. The number of eggs laid by the adults decreased to 300 and 525 eggs / female from the adults resulting from the treatment of the third and fifth instar larvae, respectively, at a concentration of 2 g/piece of Beetosan. Whereas, the number of laid eggs for the control treatment, reached 742 and 703 eggs / females from the third and fifth instar of larvae, respectively. The percentage of total fat for greater wax worm larvae decreased in both Beetosan and commercial chitosan treatments. Key words : Honeybees pests, Greater wax worm, Chitosan, Beetosan.

Cite this article Hind I. AL-Khazraji and Kameela Word Shaher (2020). The effect of Chitosan on the greater wax worm Galleria mellonella L. International Journal of Agricultural and Statistical Sciences. DocID: https://connectjournals.com/03899.2020.16.1743

1. Introduction Several methods were used to control the insect, The greater wax worm Galleria mellonella L. is including the use of chemical pesticides and fumigation one of the most important pests affecting the wax using phostoxin and methyl bromide tablets. Research frames of beehives. The damage is caused by the is being done on alternative methods of pest control to feeding of the larvae on the wax, the brood of bees and reduce the phenomenon of environmental pollution and the remains of honey as well as pollen stored inside the the appearance of the characteristic of resistance in wax frames and in the event of severe injury, it turns some insects. These methods include the use of into a fragile black mass. The larvae also dig the beehive biological control, plant extracts and essential oils that wood to settle there before pupation, as well as are environmentally friendly materials, which were used secreting silk threads impede the flow of bees within to control many insects [Szabo and Heikel (1997), the beehive, ruining the hexagonal eyes and causing Abddel Jabbar (2001), Hood et al. (2003), AL-Khazraji the destruction of weak beehives and weakening the et al. (2016)]. However, the search for such alternative active beehives, and the silk threads that the larvae strategies is still ongoing and researchers are seeking produce while feeding on wax frames, which they use to find biologically active and non-toxic materials that as a means to protect them from attacking honeybee do not have side effects to control insect pests. workers. Despite the widespread prevalence of the greater wax worm in all areas of beekeeping, it is more Polymers have been used in many agricultural active in warm regions [Charriere and Imadorf (1997), applications, including the use of Chitosan, which is a Abddel Jabbar (2001)]. non-toxic, biodegradable, natural polymer that is a *Author for correspondence Received February 17, 2020 Revised June 17, 2020 Accepted July 10, 2020 1744 Hind I. AL-Khazraji and Kameela Word Shaher polysaccharide consisting of N-acetyl glucosamine units method mentioned by Nemtsev et al. (2001) and and is derived from chitin after the removal of the acetyl Tyliszczak et al. (2016). groups (CH3-CO) deacetylation. Chitin and chitosan Naturally dead working bees were collected from are mainly produced from shrimp shells, crabs and dead bees from the apiary of the College of Agricultural oysters. Chitosan has gained increasing commercial Engineering Sciences for several months, the sample attention due to its excellent properties including bio- was washed with water to remove dust and soil and compatibility, biodegradability, miscibility, bioactivity and was left to dry under laboratory conditions. After that polycationicity, so it has been used in a wide range of it was ground and then the process of the fields such as medicine, agriculture, food packaging and demineralization using HCl solution was carried out. biotechnology, etc. Because of the increasing demand The first step is to add 10 ml HCl to every 1 g bee /and for chitosan, the search for new sources through which leave for 24 hours with stirring after that the material is chitin and chitosan could be obtained as insects which filtered and washed thoroughly with distilled water represent a great source of chitin because of their rapid several times and treat with acid several times using reproduction and short life cycle, as the percentage of different concentrations of HCl and at different time chitin in the insect cuticle ranges (30-50%), One of periods and then wash well until the PH becomes 6.5- these insects is a honey bee insect, bees’ body is covered 8, it is then allowed to dry under laboratory conditions. with hard cuticle, which works to protect the internal The second step is to remove the proteins from the organs from external influences [Tolaimate et al. material (Deproteinization) obtained by treatment (2000), Kashyap et al. (2015), Tyliszczak et al. (2016)]. NaOH at 80°C and for different time periods (10, 20, Nemtsev et al (2001) indicated that as a result of 30 and 40 hours) after which the filtration, washing extensive development in beekeeping in Russia there with distilled water and drying is carried out. is an opportunity to obtain more raw materials for chitin The third step is to remove the acetyl group as large quantities of dead bees are obtained annually (Deacetylation), treat the resulting Chitin with the so it can be considered as a new source for obtaining previous step with 48% NaOH solution for two days, chitosan. The researcher also indicated that the bodies then wash it well with distilled water and dry. The last of dry dead bees contain 2-3% mineral materials, 50- step is s for the purpose of getting rid of the pigment 80% protein, 10-12% ketene and 20-30% melanins. melanin and the chitosan obtained from bees is called While the percentage of chitin in the bodies of dead Beetosan. bees after the extraction of carbon dioxide 20-22%. 2. Commercial chitosan (CS) produced by German This study aims to evaluate the effectiveness of chitosan company Direvo Industrial Biotechnology, which is and its role in controlling the greater wax worm and isolated from crustaceans. studying its effect on biological aspects of the greater wax insect. 2.2 Treatments of larvae 2. Materials and Methods For the purpose of studying the effect of treating the food of larvae of the third and fifth instar of the 2.1 Laboratory culture of the Insect greater wax worm with different concentrations of Honeycomb infested with the greater wax worm chitosan, clean and dark wax frames were prepared larvae were obtained from the apiary of the Department cut the wax into pieces of dimensions 10 x 10 cm2, of Plant Protection, College of Agricultural Engineering some wax pieces were treated with 1 g and the other Sciences, University of Baghdad during 2018-2019. The pieces were treated with 2 g powder of chitosan affected honeycomb were placed in wooden boxes prepared from two sources. Each treated piece of wax containing dark beeswax frames and covered with a was transferred to a glass dish with a diameter of 19 wooden lid. Cages were placed in the laboratory to cm and a height of 4 cm, then larvae were transferred obtain the different instars of the insect for the purpose to the dishes of the third instar of the wax worm and of subsequent experiments. the same treatments were returned to the fifth instar 2.2 Preparation of chitosan larvae. Each experiment consisted of three replicates 2.2.1 Chitosan was obtained from two sources at a rate of 10 larvae per replicate and per each 1. Dead honey bee workers, depending on the concentration. This is in addition to the control treatment Effect of Chitosan on the Greater Wax Worm G. mellonella L. 1745 in which wax pieces were treated with distilled water reached 14.8, 13, and 9 days for the Beetosan, the only and left to dry under laboratory conditions, after commercial chitosan and control treatments, that the larvae were transferred to the dishes. On the respectively. edge of the dish, Vaseline medical jelly was applied to The results also indicate a decrease in pupal weight, prevent the larvae from leaving the dish. especially in the treatment of Beetosan at a The dishes were transferred to an incubator at a concentration of 2 g/piece. The percentage of adult temperature of 30±2°C and a relative humidity of 70±5% emergence, average longevity and number of eggs laid/ and it was followed up daily to see the effect of the female was affected at concentration 2 g/piece for prepared chitosan from different sources in the biology Beetosan treatment, as it reached 46% and 5 days and of the greater wax worm larvae .The duration of the 300 eggs/female, respectively. As for the control larval and pupa instar, the pupal rate, pupa weight, adult treatment, it reached 97%, 12.33 days and 742 eggs / emergence, adult longevity, number of eggs laid/female, females, respectively. Whereas, the effect was low with and hatching ratio were recorded. regard to the percentage of eggs hatching in both 2.3 Estimate the total fat percentage treatments. Larvae of the third and fifth instar of the greater The results also indicate an increase in the duration wax worm were fed on beeswax pieces treated with of the larval period of the greater wax worm larvae concentrations 1 and 2 g/piece for each of the prepared treated in the fifth instar, which amounted to 31.6 days chitosan from the two sources in addition to the control when treated with a concentration of 2 g/piece of treatment. The larvae were transferred, before being Beetosan, while in the control treatment it reached 24.6 pupa, to tagged plastic boxes and kept in freezer for 24 days (Table 2). The results also showed that the highest hours for the purpose of the larvae death, after which increase in the duration of the pupa instar was for the they were left to dry under laboratory conditions. The pupa resulting from the feeding of the fifth instar larvae experiment was conducted at a rate of 3 replicates per of the greater wax worm on beeswax treated with treatment and at a rate of 15 larvae in each replicate. concentrations 1 and 2 g/piece of Beetosan, as the A weight of 1 g/piece was taken from the bodies of duration of the pupal period reached 12 and 12.6 days, dried and crushed larvae and placed in a filter paper respectively, while the duration of the pupa period in folded inside a glass beaker and then added a hexane The control treatment is 9.33 days. We also note a solvent for 16 hours, then transferred to the soxhlet for decrease in the weights of pupa and the rate of the the purpose of extracting fats and estimating the total emergence of adults resulting from the treated larvae. fat percentage in the treated and untreated larval bodies As for the longevity of the adult, the average [A.O.A.C. (1975)]. longevity of the adults ranged between 8.33-10 days 3. Results and Discussion when treated with different concentrations of Beetosan, while the adults longevity ranged between 3.1 The effect of chitosan on the biology of greater 9.50-10.67 days when treated with different wax worm larvae concentrations of commercial chitosan, while the control The results indicate that chitosan has an effect in treatment reached 13.67 days. As for the average some aspects of the biology of the greater wax worm number of eggs laid, it reached 525 eggs / female and (Table 1), as feeding the larvae of the third instar on the hatching rate reached 90.6% at concentration 2 g/ food treated with chitosan led to an increase in the piece in Beetosan treatment. In the control treatment, duration of the larval period and was the highest increase the average number of eggs laid was 710 eggs / female, in the treatment of wax cut treated with chitosan isolated and the percentage of hatching reached 97%. The from the bodies of dead bees (Beetosan) at a reason for the affected longevity and productivity of concentration of 2 g/piece it reached 35 days, while it adults resulting from larvae treated with chitosan was reached 29.67 days at a concentration of 2 g/piece in due to the effect of the rate of food consumption and the treatment of commercial chitosan, whereas, the the decrease in the metabolism efficiency of the treated larval period reached 25 days for the control treatment. larvae, which caused a decrease in the overall growth The treatment at concentration 2 g/piece caused the rate and thus the formation of small-sized pupa with highest increase in the duration of the pupa instar and low food stocks and this is directly related to the fertility 1746 Hind I. AL-Khazraji and Kameela Word Shaher

Table 1: The effect of chitosan food treatment on some biology aspects of the 3rd larval instar of the greater wax worm. Beetosan Commercial CS Biological Aspects Conc. Conc. Control L.S.D (0.05) 1g/piece 2g/piece 1g/piece 2g/piece Larval period (day) 33.33 35 29 29.67 25 4.756 Pupal period (day) 13.67 14.80 12 12.50 9.00 1.984 Pupal weight (g) 0.110 0.1000 0.140 0.123 0.183 0.044 Adult emergence % 50 46 63 59 97 4.732 Adult longevity(day) 6.33 5 8 6.33 12.33 1.112 No. of eggs /female 450 300 510 490.0 742 198.6 % egg hatching 83 81 89.33 85.64 98 9.74

Table 2: The effect of chitosan food treatment on some biology aspects of the 5th larval instar of the greater wax worm. Beetosan Commercial CS Biological Aspects Conc. Conc. Control L.S.D (0.05) 1g/piece 2g/piece 1g/piece 2g/piece Larval period (day) 29.33 31.67 27 28 24.67 4.589 Pupal period (day) 12 12.67 11.33 11.67 9.33 2.522 Pupal weight (g) 0.150 0.143 0.163 0.160 0.180 0.041 Adult emergence % 62.50 57 70 66.87 92 2.951 Adult longevity(day) 10 8.33 10.67 9.50 13.67 3.169 No. of eggs /female 580 525 654 605 710 173.0 % egg hatching 91 90.60 94.33 92.30 97 4.905

Fig. 1: The effect of chitosan treatment on the percentage of total fat in greater wax larvaes and longevity of adults. reduced the activity of the alanine aminotransferase A study was conducted by Said et al. (2011) to enzyme. While the treatment resulted in an increase in investigate the effect of chitosan prepared in several the urea level and aspartate aminotransferase enzyme different chemical methods on the larvae of the third activity. The treatment also caused a decrease in the instar of the wax worm when larvae are fed on percentage of hatching, which was 4% in the treatment industrial food treated with a lethal half-concentration chitosan and 99% in the control. LC50 of chitosan led to the occurrence of clear 3.2 The percentage of total fat in the greater wax histological effects on cells of the middle gut for larvae, worm larvae the treatment also reduced the total protein content and Treating food with Beetosan resulted in a decrease Effect of Chitosan on the Greater Wax Worm G. mellonella L. 1747 in the percentage of fats, especially at third larval instar A.O.A.C. (1975). Official methods of analysis. Association of the greater wax worm with a concentration of 2 g/ of official analytical chemists. 13 m jed. Washington D.C. piece as the total fat percentage reached 19.14%, while AL-Khazraji, H.I., R.F. Ahmed and R.S. Al-Jorany (2016). in the control treatment it reached 40.38% (Fig. 1). In Effect of feeding treatment with some extracts of black the Commercial (CS) treatment, the fat percentage for pepper on some biological aspects of cotton leaf worm. the third larval instar ranged between 27- 31.53%. As The Iraqi Journal of Agricultural Sciences, 47(3), 856- 864. for the fifth larval instar, the total fat percentage ranged between 29.99 - 35.84%, while the total fat percentage Charriere, J. and A. Imadorf (1997). Protection of honey combs from moth damage (Swiss Bee Research Center, Federal in the fifth larval instar in the control treatment was Dairy Research Station. Liebfeld, CH- 3003 Bern. 40.94%. Communication No. 24. Li et al. (1992) indicates that chitosan possesses a Hood, W.M., P.M. Horton and J.W. McCreadie (2003). Field positive ion charge (the amino group) that has the ability Evaluation of the Red Imported Fire Ant (Hymenoptera: to chemical bond with many compounds, so we can Formicidae) for the Control of Wax Moths (Lepidoptera: say that despite the difficulty of determining the cause Pyralidae) in Stored Honey Bee Comb. J. of Agricultural of the negative impact of chitosan on the life of the and Urban Entomology, 20(2), 93-103. insect, it may be due to the role of the amino group and Kashyap, P.L., X. Xiang and P. Heiden (2015). Chitosan its association with fats, mineral ions and proteins nanoparticle based delivery systems for sustainable Existing in the insect’s food and converting it into agriculture. Int. J. Biol. Macromol., 77, 36-51. compounds that interfere with physiological or chemical Li, Q., E.T. Dunn, E.W. Grandmaison and M.F.A. Goosen activities related to growth, development and (1992). Applications and properties of chitosan. J. Bioactive and Compatible Polym., 7, 370-397. reproduction in the insect, which leads to many changes in the insect’s life, especially those related to the vital Nemtsev, S.V., O.U. Zueva, R.G. Khismatoullin, M.R. construction of body tissues. Khismatoullin and V.P. Varlamov (2001). Bees as potential source of chitosan. Proc. 37th Int. Apic. Congr.1-3. Based on the previous results, it can be concluded Szabo, I.T. and D. Heikel (1997). Fumigetion with soz control that chitosan possesses a biological activity that heralds wax moth in honey bee comb. Bee. World, 68, 37-38. new uses of safe and non-polluting materials that can Said, S.M., S.M. EL-Sayed, H.E.A. Farid and M.M. Abozid serve as a basis for manufacturing chemical compounds (2011). Insecticidal effect of Chitosan Prepared by that have mechanisms of effect that differ from the different Chemical Processing Sequences against mechanisms and methods of chemical pesticides. Galleria melleonella. Az. J. Pharm. Sci., 43, 123-132. Acknowledgements Tolaimate, A., J. Desbrières, M. Rhazi, M. Alagui, M. Vincendon and P. Vottero (2000). The influence of The authors would like to thank Editor and learned deacetylation process on the physicochemical referee for their fruitful comments and suggestions characteristics of chitosan from squid chitin. Polymer, which led to a considerable improvement of the paper. 41, 2463-2469. References Tyliszczak, B., A. Drabczyk, S. Kudlacik, K. Bialik-Was and Abddel Jabbar, T.M. (2001). Effect of Eucalyptus plant A. Sobczak- Kupiec (2016). Beetosan/Chitosan from bees Eucalyptus camaldulensis Dehnhon the biology of the -preparation and properties. International Journal of Greater Wax Moth Galleria mellonella (L.). M.Sc. Thesis Advances in Science Engineering and Technology, 4(2), of Science in Agriculture. University of Baghdad, Iraq. 118-120.