Introduction

Biochemical studies

Insect haemolymph nutrients should be affected when treated with insecticide so difference in total haemolymph protein, lipids and carbohydrates Table (3) plus the two main enzymes of protein synthesis glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) Table (4) of S.littoralis 4^th instar larvae treated individually with median lethal time (LC₅₀) of the P. lilacinum and C. cyminum after five day post treatment was studied. The results demonstrated that, both P. lilacinum and C. cyminum caused reduction in haemolymph protein content (308±1.6 and 332±1.7 mg/ml) compared to that of the control (430±1.6 mg/ml), and stimulation in the total carbohydrates content (256±1.4 and 265±0.8 mg/ml) as compared to that of the control treatment (252±1.4 mg/ml) respectively. Our results also clarified that total haemolymph lipids of the untreated larvae is 372± 2.4 mg/ml, in the P. lilacinum treated larvae it showed significant increase, recorded 390 ±1.8 mg/ml while it decrease in the larvae treated with C. cyminum (384± 1.5 mg/ml). The mean level of GOT and GPT enzyme respectively in the untreated larvae is 44 ± 1.3 and 53 ± 0.9 mg/ml while great inhibition noticed in both enzyme level in the larvae treated with P. lilacinum and C. cyminum recorded a 28 ± 0.6 and 39 ± 0.3 mg/ml and 43 ± 0.4 and 43 ± 1.1 mg/ml respectively.

Table 3: Effect of Purpureocillium lilacinum and Cuminum cyminum on the total lipid, protein and carbohydrates content (mg/ml) in the haemolymph of the 4^th instar larvae of Spodoptera littorlais.

Table 4: Effect of Purpureocillium lilacinum and Cuminum cyminum on the Glutamic oxaloacetic transaminase (GOT) and glutamine pyruvic transaminase (GPT) (mg/ml) in haemolymph of the 4^th instar larvae of Spodoptera littorlais.

Discussion

Insect pathogenic fungi are the most common organisms that infect several orders of the insect pests (Majeed, et al., 2017) ^[34], there is a great interest for using them for the bio control of insect pests, as they play significant role in suppression of the insect population (Smith, et al., 1999) ^[59]. Entomopathogenic fungi have a unique place among other entomopathogens (Richard, et al., 2010) ^[50] in which they should not be swallowing like virus and bacteria and can attack their hosts directly by binding to their integument. Among the insect pathogenic fungi, Purpureocillium which considered essential natural bio-control agents and source of mycopesticides for pests management all over the world (Sanjaya, et al., 2016) ^[52].

Plants produce several secondary chemical compounds to protect themselves from any pathogens attack, these chemical compounds can be used in the pest control, (Pavela, 2016) ^[46] so recently, researchers focused on these plant natural products to be used as safe insecticides, C. cyminum is an essential oil belonging to Apiaceae Family which considered the most family that well known of their insecticidal activity (Ebadollahi, 2013) ^[14]. In the current study the tested fungal isolate P. lilacinum and tested essential oil C. cyminum were tested against the second and the fourth instar larvae of S. littoralis and the results data proved the toxic effect of both against the two tested larval instar. The results clarified that there is positive relationship between the concentration and the mortality rate and also the younger instar larvae is much more sensitive than the older one Purwar and Sachan (2005) ^[47]. Obtained data in the present study are agree with several authors examined the potential use of P. lilacinum and C. cyminum as biological control agents against several insects. Kepenekci, et al., (2015) ^[29] reported a potential infectivity of P. lilacinum against the last larvae instar of Phthorimaea operculella (Zeller) and Leptinotarsa decemlineata (Say) and recorded mortality percentage 43.3% and 33.2% respectively at the 10^th day of treatment with the fungal concentration of 10⁸ spore/ml, also Lopez, et al., (2014) ^[31] proved the potential pathogenicity of P. lilacinum against herbivores and aphid of cotton under greenhouse and field conditions. In parallel Hoang Chinh NGUYEN, et al., 2017 ^[28] isolated six Purpureocillium species and evaluated their pathogenicity against Plutella xylostella and Spodoptera litura and confirmed the strong virulence of the six isolate while P. lilacinum isolate showed the highest infectivity against the two tested insects. In addition, P. lilacinum has been used as an effective nematocide against nematodes (Meloidogyne spp.) (Sharma, et al., 2014) ^[54].

Insect pathogenic fungi enter the insect body directly through the integument (Sevim, et al., 2015) ^[53], by physical and enzymatic process. Many factors combined in the action mechanism of entomopathogenic fungi, firstly, the fungus spores attach on the insect cuticle, then germinate and enter the cuticle by forming appressorium. Hyphae grow and reproduce continuously in the insect body and haemolymph and finally lead to the insect death. The most fundamental factor in entomopathogenic mechanism is toxin secretion by fungi, for example paecilotoxin (Leucinostatins) sectrated from P. lilacinum Abbas H. Burhan and Mohammed R. Annon., 2019. These toxic substances can cause insect death even before spread of spores in tissue of parasitic fungus (Charnley, 2003) ^[13].

 N.E. BEN-KHALIFA, et al., 2018 ^[42] tested the efficacy of six Apiaceae extracted oils on S. littoralis and showed that C. cyminum produced 100% larval mortality against the tested third instar larvae. Mukesh Kumar Chaubey, 2017 ^[41] evaluated the effectiveness of C. cyminum and black piper oils on greater grain weevil and revealed that both oil repelled the weevil adults, induced high mortality and exhibit median lethal time (LC₅₀) 0.2 and 0.1 µl/cm2 and 0.2 and 0.1 µl/cm² for the two essential oil respectively, also they inhibit both AchE activity and oviposition of S. zeamais adults. Toxic effect of C. cyminum related to monoterpense (main constituents) which have effective insecticidal toxicity (Abdelgaleil, et al., 2009) ^[2], penetrate very rapidly in the body of the insects and intervene with the insect physiological function (Haouas, et al., 2012). Another main components of C. cyminum is Cuminaldehyde that effectively inhibit actylcholinestearse enzyme activity. Abdelgaleil, et al., (2009) ^[2]. Yeom, et al., (2012) ^[65] tested some Apiaceae family extracted oils including C. cyminum and reported that at a concentration of 5 mg/filter paper they have approximatly 90 % fumigant toxicity against German cockroaches adult male. (Tunc¸ et al., 2000) ^[61] mentioned that C. cyminum oil produce 100 % mortality on T. confusum and Ephestia kuehniella Zeller eggs.

The present study indicated the influence of insect haemolymph nutrients due to pathogen attack. Proven results in this study showed difference in total lipid, protein, carbohydrates, GOT and GPT of treated S. littoralis 4^th instar larvae with both P. lilacinum and C.cyminum when compare to control and these data are agree with El-Badawy, S. S, et al., 2018 ^[16] who investigated the effect of some entomopathogenic fungi including P. lilacinum on biochemical content in haemolymph of S. littoralis and found that P. lilacinum isolate caused significant increase in total carbohydrates and total lipids and reduction in total protein content compared to control at fourth day post treatment. Nirupama, (2015) ^[44] detected that total protein content of silkworm, Bombyx mori was reduced gradually at the end of 4^th and 5^th day due to the fungi infection. Also our results were proved by Vidhya, et al., (2016) ^[62], they mentioned that infection of the army worm S.litura (Fabricius) by three fungal pathogens B. bassiana, M.anisopliae and Verticillum lecanii showed significant decreased of total protein content at fourth day post treatment as compared to control. In parallel, Meshrif, et al., (2010) ^[36] revealed that, there was significant increase in plasma carbohydrates of S. littoralis 5^th instar larvae when injected by fungi (Nomuraea rileyi and B. bassiana). Gabarty, (2011) ^[23] showed significant increase in the total content of lipids in the greasy cut-worm Agrotis ipsilon (Huf.) larvae treated with B. bassiana and M. anisopliae as compared with untreated one. In contrast to our results the obtained data of Nada, (2015) ^[43] showed that total lipids decreased significantly when adults of N. virdula treated with M. anisopliae. In the same line Marei, et al., 2009 ^[35] evaluated the influence of Sesame oil on the biochemical aspects of S.littoralis and found great reduction in total lipids. Abou El-Ghar, et al., 1996 ^[3] observed high inhibition of both total lipid and total protein of Agarotis ipsilon treated with ethanol extract of Melia azedarach. Also, some results of Amal S. Sobhi, et al., 2020 ^[9] are agree with our results as they recorded inhibition of total lipid and total protein of S.littoralis treated with essential camphor oil while reduction in total carbohydrates they observed are in contrast to our findings as we reported stimulation of total carbohydrates after treatment with essential C. cyminum oil.

SK Mirhaghparast, et al., 2013 ^[58] tested the effect of two entomopathogenic fungi on the metabolic enzyme of S.littoralis and found high activity of all tested enzymes including GOT and GPT enzyme, there is also noticeable elevation in the two enzyme of S.littoralis treated with four plant essential oils Mona, K. Elhadek, et al., 2015 ^[39], these results disagree with what has been accessed in the present study as there is reduction in the two enzyme observed after P.lilacinus an C.cyminum treatment.

Many studied revealed the ability of Neemazal (a neem preparation) and N. sativa extracts (Hamadah, 2009) ^[26] in disruption of GPT activity of S. gregaria.

Proteins are the most important compounds found in every cells of all living organisms, including many substances like enzymes and hormones that necessary for the main function of the living organisms (Fagan, et al., 2002) ^[21]. Carbohydrates are contributed to the structure and function of the insect tissues and organs, they providing the energy wanted for the growth and development of the cell (Lee, et al., 2002) ^[30]. Lipids are significant source of energy compared to carbohydrates, they can supply as much as eight time more energy per unit weight (Ali, 2011) ^[7]. Several species store lipids which are used during starvation Panizzi and Hirose 1995 ^[45]. They occupy a central place in insect physiology. They are considered to be a fundamental source of metabolic energy for the cell protection and proliferation.

In the present study change in the level of some biochemical parameters of S. littoralis larvae haemolymph may be due to physiological disturbance which are induced by the presence of a the physiological confrontation in the body like microorganism infections, tissues impairment or being a toxic material (Giboney, 2005) ^[25].

The significant reduction in the total protein may be due to binding with foreign substances, as any insecticides. This was revealed by Ahmed, et al., (1985) ^[6] when investigated dieldrin insecticides against Periplaneta americana, Or maybe due to the repression of DNA and RNA synthesis, as proposed by Mitlin, et al., (1977) ^[37] for boll weevils treated with chitin synthesis inhibitors and by Qadri and Narsaih (1978) for P. americana last nymphal instar injected with the plant extract, azadirachtin. In addition, the reducing in total protein may be due to the cracking of the protein into amino acids. GOT and GPT are the major enzymes in the protein metabolism (Mordue & Goldworthy, 1973). So, the reduction in total content of protein may be related to the reduction in both GOT and GPT that were resulted in the present study, and vice versa, the reduction in those enzymes may be due to the reduction of total protein as the enzymes are protein in nature ( Mitlin, et al., 1977) ^[37]. In other way the disturbance in these two enzymes is closely related to utilization of protein and amino acids hence damage many physiological functions and eventually lead to insect death (Ezz and Fahmy, 2009) ^[20]. Lipids are important structural constituents of the cell membrane and cuticle. Inhibition of total lipid can be attributed to the toxic stress induced by the tested insecticides stimulate the utilization of lipid and rapid conversion of total lipid contents to protein occurred to produce supplementary energy required by the insect body (Abuldahab, et al., 2011) ^[4]. Also, carbohydrates are important compounds as they are utilized by the insect body for the production of energy or conversion to lipids or proteins. Utilization of carbohydrates is controlled by amylase, trehalase, and invertase enzymes which play the main role in the digestion and utilization of carbohydrates by insects (Wigglesworth, 1972) ^[63]. Stimulation in total carbohydrates due to two tested insecticides noticed in this study may be due to disruption in the enzyme responsible for utilization of them. Generally biochemical changes in S.littorlais haemolymph nutrient content (Protein, Lipid and carbohydrates) recorded in this study either by increasing or by decreasing may be due to alteration or mutation of the genes responsible for biosynthesis of polypeptide chains constructing the enzymes regulating the metabolism of them and this affect these enzymes synthesis and function.

Conclusion

Results of the current study clarified the capability of the two tested biocides P. lilacinum and C.cyminum to induce high mortality rate to the two tested instar larvae of S.littoralis and caused sharp disturbance in lipid, carbohydrates, proteins and both GOT and GPT enzymes. Toxicity and biochemical changes of S.littoralis treated individually by the two biocides used are prompted as a result of the physical invasion of P. lilacinum vegetative growth, sporulation, enzymatic degradation and toxin productions, also due to the main active constituents of C.cyminum (Cuminaldehyde, β-pinene, γ-terpinene and p-cymene) which characterized by high insecticidal potential including toxicity, repellent, antifeeding and disturbance ability of insect growth and development. Consequently these two biocides agents can be used as a promising eco-friendly alternatives of the chemical pesticides against this detrimental, harmful and injurious pest S. littoralis.

References

1. Abbas H Burhan, Mohammed R Annon. Pathogenesis of Paecilomyces lilacinus against the immature stages of Musca domestica L. Journal of Pharmaceutical Sciences and Research. 2019; 11(4):1595-1601.
2. Abdelgaleil SA, Mohamed MI, Badawy ME, El-arami SA. Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase activity. Journal of chemical Ecology. 2009; 35:518–525
3. Abo El‐Ghar GES, Khalil ME, Eid TM. Some biochemical effects of plant extracts in the black cutworm, Agrotis ipsi/un (Hufnagel) (Lep., Noctuidae). Journal of Applied Entomology. 1996; 120:477-482
4. Abuldahab FF, Abozinadah NY, Al- Haiqi NS. Impact of Bacillus thuringiensis β– exotoxin to some biochemical aspects of Musca domestica (Diptera: Muscidae). Journal of Bacteriolgy Research. 2011; 3(6):92-100.
5. Addy ND. Rearing the forest tent caterpillar on an artificial diet. Journal of Economic Entomology. 1969; 62:270-271.
6. Ahmed MS, Ali FA, Shakoori AR. Effect of dieldrin on the whole-body protein content of Periplaneta americana. Pakistan Journal of Zoology. 1985; 17(1):105-109.
7. Ali Rehab MS. Combined effect of gamma radiation and entomopathogenic nematoda on some store product pests. Ph. D., Thesis, Fac. of Agric., Ain Shams Univ, 2011.
8. Alves SB, Moraes AS. Quantificac¸ ão de inóculo de patógenos de insetos. In: Alves, S.B. (Ed.), Controle Microbiano de Insetos. FEALQ, Piracicaba, 1998, 765-777.
9. Amal S, Sobhi, El kousy SM, El-Sheikh TAA. Some Toxicological and Physiological Aspects Induced by Camphor oil, Cinnamomum Camphora on the Cotton Leafworm, Spodoptera littoralis (Boisduval). (Lepidoptera: Noctuidae). Egyptian Academic Journal of Biological Sciences. 2020; 12(2):63-73
10. Bakr EM. Ldp line 3. (Site of internet), 2000. http://www,Ehab soft, com.
11. Baronos H, Blackstock J. Estimation of lipids in marine animals and tissue: Detailed investigations of the sulphophosphovanillin method for total lipids. Journal of Experimental Marine Biology and Ecology. 1973; 12:103-118.
12. Chandler D, Davidson G, Pell JK, BALL BV, Shaw K, Sunderland KD. Fungal biocontrol of Acari. Biocontrol Science and Technology. 2000; 10:357-384. ISSN 0958-3157
13. Charnley AK. Fungal pathogens of insects: cuticle degrading enzymes and toxins. Advances in Botanical Research. 2003; 40:241-321.
14. Ebadollahi A. Plant Essential Oils from Apiaceae Family as Alternatives to Conventional Insecticides. Ecologia Balkanica. 2013; 5(1):149-172.
15. EI-Defrawi ME, Toppozada A, Mansour N, Zeid M. Toxicological studies on Egyptian cotton leaf worm Prodenia litura (F.). I: Susceptibility of different larval instars to insecticides. Journal of Economic Entomology. 1964; 57:591-593.
16. El-Badawy SS, Sahar S Ali, AA El-Hefny, Gamila AM. Heikal. Study of Infection Impact by Entamopathogenic Fungi on some Biochemical Contents in Haemolymph of Cotton Leaf Worm, Spodoptera littoralis (Boisduval) Journal of Plant Protection and Pathology, Mansoura Univ. 2018; 9(9):605-610.
17. El-Gaied L, Mahmoud A, Salem R, Elmenofy W, Saleh I, Abulreesh H, Arif I, Osman G. Characterization, cloning, expression and bioassay of vip3 gene isolated from an Egyptian Bacillus thuringiensis against whiteflies. Saudi Journal of Biological Sciences. 2020; 27:1363–1367.
18. Elumalai K, Krishnappa K, Anandan A, Govindarajan M, Mathivanan T. Antifeedant activity of medicinal plant essential oils against Spodoptera litura (Lepidoptera : Noctuidae). International Journal of Recent Scientific Research. 2010; 2:62-68.
19. Emad M, Barakat S, Mohamed O. Abokersh. Characterization of the haemolymph from Schistocerca gregaria adults after infection with entomopathogenic fungus Beauveria bassiana. Life Science Journal, 2016, 13(3).
20. Ezz NA, Fahmy NM. Biochemical effects of two kinds of mineral oils and an IGR on adult female mealybug Ferrisia virgata (Cockerell). Egyptian Academic Journal of Biological Sciences. 2009; 1(1):33-40.
21. Fagan WF, Siemann ER, Mitter C, Denno RF, Huberty AF, Woods HA et al, Nitrogen in insects: implication for trophic complexity and species diversification. American Naturalist. 2002; 160:784-802.
22. Ferron P. Biological control of insect pests by entomogenous fungi. Annual Review of Entomology. 1978; 23:409-442, ISSN 0066-4170.
23. Gabarty A. Combined effect of gamma radiation and some fungal control agents on the greasy cut- worm Agrotis ipsilon (Huf.). Ph.D Thesis. Fac. Science (Girls branch).Al-Azhar University, 2011.
24. Ghulam A, van der Wopke W, Vlak JM. Biological and genetic characterization of a Pakistani isolate of Spodoptera litura nucleopolyhedrovirus. Biocontrol Science and Technology. 2017; 28:20-33.
25. Giboney PT. Mildly Elevated Liver Transaminase Levels in the Asymptomatic Patient. American Academy of Family Physician. 2005; 71:1105-1110.
26. Hamadah Kh. Sh. Some developmental, haematological and enzymatic effects of certain plant extracts on the desert locust Schistocerca gregaria (Orthoptera: Acrididae). Ph.D. Thesis, Al-Azhar, University, Cairo, Egypt, 2009.
27. Haouas D, Cioni P, Ben Halima-Kamel M, Flamini G, Ben Hamouda M. Chemical composition and bioactivities of three Chrysanthemum essential oils against Tribolium confusum (du Val) (Coleoptera: Tenebrionidae). Journal of Pest Science. 2012; 85:367-379.
28. Hoang Chinh NGUYEN, Thi Van Anh TRAN, Quoc Linh NGUYEN, Nhu Nhut NGUYEN, Minh Khiem NGUYEN, Ngoc Thanh Tam NGUYEN et al, Newly Isolated Paecilomyces lilacinus and Paecilomyces javanicus as Novel Biocontrol Agents for Plutella xylostella and Spodoptera litura. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2017; 45(1):280-286.
29. Kepenekci I, Oksal E, Saglam HD, Atay T, Tulek A, Evlice E et al. Identification of Turkish isolate of the entomopathogenic fungi, Purpureocillium lilacinum (syn: Paecilomyces lilacinus) and its effect on potato pests, Phthorimaea operculella (Zeller)(Lepidoptera: Gelechiidae) and Leptinotarsa decemlineata (Say)(Coleoptera: Chrysomelidae). Egyptian Journal of Biological Pest Control. 2015; 25:121-127.
30. Lee KP, Behmer ST, Simpson SJ, Raubenheimer D. A geometric analysis of nutrient regulation in the generalist caterpillar Spodoptera littoralis (Boisduval). Journal of Insect Physiology. 2002; 48: 655-665.
31. Lopez DC, Zhu-Salzman K, Ek-Ramos MJ, Sword GA. The entomopathogenic fungal endophytes Purpureocillium lilacinum (formerly Paecilomyces lilacinus) and Beauveria bassiana negatively affect cotton aphid reproduction under both greenhouse and field conditions. Public Library of Science. 2014; 5; 9(8):e103891.
32. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. Journal of Biological chemistry. 1951; 193:265-275.
33. Luangsa-Ard J, Houbraken J, van Doorn T, Hong SB, Borman AM, Hywel-Jones NL, Samson RA. "Purpureocillium, a new genus for the medically important Paecilomyces lilacinus". FEMS Microbiology Letters. 2011; 321(2):141-9. Doi:10. 1111/j.1574-6968.2011.02322.x. PMID 21631575.
34. Majeed MZ, M Fiaz, Chun-Sen Ma, M Afzal. Entomopathogenicity of Three Muscardine Fungi, Beauveria bassiana, Isaria fumosorosea and Metarhizium anisopliae, against the Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) Egyptian Journal of Biological Pest Control. 2017; 27(2):211-215.
35. Marie SS, Amr EM, Salem NY. Effect of some plant oils on biological, physiological and biochemical aspects of Spodoptera littoralis (Boisd.). Journal of Agriculture and Biological Sciences. 2009; 5(1):103-107.
36. Meshrif WS, Rohlfs M, Hegazi MAM, Shehata MG, Barakat EMS, Seif AI et al. Humoral response and plasma changes of Spodoptera littoralis (Lepidoptera: Noctuidae) following injection with the entomopathogenic fungi: Beauveria bassiana and Nomuraea rileyi Proc. 6th international conference on biological sciences. (Zool.). 2010; 6:96-102.
37. Mitlin NG, Wiygul, JW Haynes. Inhibition of DNA synthesis in boll weevil (Anthonomus grandis Boheman) sterilized by Dimilin. Pesticide Biochemistry and Physiology. 1977; 7:559-563.
38. Mohamed HA, Alkordy, Atta AA. Effect of host plants on biology of Spodoptera littoralis (Boisd.). Egyptian Academic Journal of Biological Sciences. 2019; 12:65-73.
39. Mona K Elhadek, Aziza H Mohamady, Reham E Ali. Toxicity and Biochemical Effects of Four Plant Essential Oils against Cotton Leafworm, Spodoptera littoralis (Boisd). Egyptian Academic Journal of Biological Sciences. 2015; 7(1):153-162.
40. Mordue W, Goldworthy GJ. St Transaminase levels and uric acid production in adult locusts. Insect Biochemistry. 1973; 3:419-427.
41. Mukesh Kumar Chaubey. Evaluation of Insecticidal Properties of Cuminum cyminum and Piper nigrum Essential Oils against Sitophilus zeamais Journal of Entomology. 2017; 14:148-154.
42. Ben-Khalifa NE, Chaieb I, Laarif A, Haouala R. Insecticidal ctivity of six Apiaceae essential oils against Spodoptera littoralis Biosduval (Lepidoptera: Noctuidae) Journal of new sciences, Agriculture and Biotechnology. 2018; 55(1):3603-3609.
43. Nada Maha S. Response of green stinkbug nezara viridula (linnaeus), to the activity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae Journal of Plant Protection and Pathology, Mansoura University. 2015; 6(12):1633-1644,
44. Nirupama R. Biochemical studies on total protein, carbohydrate and lipids content level during the infection by fungi white muscardine disease of silkworm, Bombyx mori L. Munis Entomology and Zoology Journal. 2015; 10(2).
45. Panizzi AR, Hirose E. Seasonal body weight, Lipid content, and impact of starvation and water stress on adult survivorship and longevity of Nezara viridula and Euschistus heros. Entomological Experimentalis et al. Applicaticata. 1995; 76:247-253.
46. Pavela R. History, presence and perspective of using plant extracts as commercial botanical insecticides and farm products for protection against insects – a review. Plant Protection Science. 2016; 52:229-241.
47. Purwar JP, Sachan GC. Biotoxicity of Beauveria bassiana and Metarhizium anisopliae against Spodoptera litura and Spilarctia oblique. Annals of Plant Protection Sciences. 2005; 13(2): 360-364.
48. Qadri SSH, Narsaih S. Effect of azadirachtin on the moulting process of last instar nymph of Periplaneta americana (L.). Indian Journal of Exprimental Biology. 1978; 16:1141-1143.
49. Reitman S, Frankel S. A colorimetric method for determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology. 1957; 28: 56-63.
50. Richard JS, Neal TD, Karl JK, Michael RK. Model reactions for insect cuticle sclerotization: participation of amino groups in the cross-linking of Manduca sextacuticle protein MsCP36, Insect. Biochemistry and Molecular Biology. 2010; 40:252-258.
51. Roberts DW, Hajek AE. Entomopathogenic fungi as bioinsecticides. In: Frontiers in Industrial Mycology. G. Leathan (Ed.). Chapman and Hall, New York. 1992, 144-159.
52. Sanjaya Y, Ocampo VR, Caoili BL. Pathogenicity of three entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana, and Paecilomyces lilacinus, to Tetranychus kanzawai infesting papaya seedlings. Arthropods. 2016; 5:109-113.
53. Sevim A, Sevim E, Demirbağ Z. General biology of entomopathogenic fungi and their potential to control pest species in Turkey (Entomopatojenik fungusların genel biyolojileri ve Türkiye’de zararlı böceklerin mücadelesinde kullanılma potansiyelleri). Erzincan Üniversitesi Fen Bilimleri Enstitüsü Dergisi. 2015; 8(1):115-147.
54. Sharma A, Sharma S, Dalela M. Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium. Microbial Pathogenesis. 2014; 75:16-20.
55. Shimaa M. Desoky, Yasmein A. EL Sayed, Suzan A. Ibrahim. Toxicological Studies and Histopathological Changes on Black Bean Aphid, Aphis craccivora Induced by Entomopathogenic Fungi, Metarhizium anisopliae and Purpureocillium lilacinum Egyptian Academic Journal of Biological Science. 2020; 12(1):185-196.
56. Shorey HH, Hale RL. Mass-Rearing of the larvae of Nine Noctuid species on a simple artificial medium. Journal of Economic Entomology. 1965; 58: 522–524.
57. Singh NB, Sinha RN. Carbohydrates, lipids and proteins in the developmental stages of Sitophilus oryzae and S. granarius (Coleoptera: Curculionidae). Annals of. Entomological. Society of America. 1977; 70:107-111.
58. SK Mirhaghparast, A Zibaee, J Hajizadeh. Effects of Beauveria bassiana and Metarhizium anisopliae on cellular immunity and intermediary metabolism of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) Invertebrate Survival Journal. 2013; 10:110-119.
59. Smith SM, Moore D, Karanja LW, Chandi EA. Formulation of vegetable fat pellets with pheromone and bassiana to control the larger grain borer, Prostephanus truncates (Horn). Pesticide Science. 1999; 55:711-718.
60. Tripathi AK, Prajapati V, Kumar S. Bioactivities of l-carvone, d carvone, and dihydrocarvone toward three stored product beetles. Journal of Economic Entomology. 2003; 5:594-601.
61. Tunc I, Berger BM, Erler F, DaglI F. Ovicidal activity of essential oils from five plants against two stored-product insects. Journal of Stored Products Research. 2000; 36:161-168.
62. Vidhya D, P Rajiv, Nalini Padmanabhan. Impact of entamopathogenic fungal infection on the detoxifying enzyme in cotton leaf worm, Spodoptera litura (fabricius). International Journal of pharma and bio sciences. 2016; 7(4):(b)943-948.
63. Wigglesworth VB. The Principles of Insect Physiology. The Analysis of Time Series: An Introduction with R (Chapman & Hall/CRC Texts in Statistical Science) 1972.
64. Wraight SP, Inglis GD, Goettel MS. Fungi, In: Field manual of techniques in invertebrate pathology, L.A. Lacey & H.K. Kaya, (Eds.), 223-248, 2nd edition, Springer, Dordrecht, ISBN. 2007; 978-1-4020-5931-5.

Yeom HJ, Kang JS, Kim GH, Park IK. Insecticidal and acetylcholine esterase inhibition activity of Apiaceae plant essential oils and their constituents against adults of German cockroach (Blattella germanica). Journal of Agricultural and Food Chemistry. 2012; 60:7194-7203.