Acta agriculturae Slovenica, 117/2, 1–6, Ljubljana 2021 doi:10.14720/aas.2021.117.2.2029 Original research article / izvirni znanstveni članek Euphorbia bupleuroides Desf. latex as biopesticide against the red flour beetle (Tribolium castaneum [Herbst, 1797]) and khapra beetle (Trogo- derma granarium Everts, 1898) Imene BRIK 1,2 , Naama FRAH 1 Received January 05, 2021; accepted March 26, 2021. Delo je prispelo 5. januarja 2021, sprejeto 26. marca 2021. Euphorbia bupleuroides Desf. latex as biopesticide against the red flour beetle (Tribolium castaneum [Herbst, 1797]) and khapra beetle (Trogoderma granarium Everts, 1898) Abstract: Laboratory evaluation of Euphorbia bupleuroi- des latex as biopesticide against the red flour beetle (Tribolium castaneum) and khapra beetle (Trogoderma granarium) were evaluated at ambient temperature. The insecticidal activity of latex was determined by direct contact application. Different concentrations were prepared by dilution of 2.5, 5.0, 7.0 and 10.0 µl of latex into 0.1 ml acetone. 1 µl was pumped regularly in the thorax of different insects. The latex of E. bupleuroides showed insecticidal activity against T. granarium and T. casta- neum. High levels of mortality were associated with the increase in the concentration and time of exposure as well. T. granarium adults are generally more prone to latex insecticidal effects than T. castaneum adults. After 6 days of exposure to E. bupleuroides latex, the LC 50 recorded was 14.12 µl for T. granarium adults, and 14.7 µl for T. castaneum. LC 90 numbers, on the other hand, were 38.8 µl for the former, and 51.44 µl for the latter. Key words: Euphorbia bupleuroides; biopesticide; latex; Tribolium castaneum; Trogoderma granarium; mortality 1 Laboratory for Improving Agricultural Productions and Protection of resources in arid zones, Department of Agronomy, Institute of Veterinary and Agricultural Sciences, University of Batna 1, Hadj Lakhdar, Algeria 2 Corresponding Author, e-mail: Imenebio5@gmail.com. Mleček prerastolikega mlečka (Euphorbia bupleuroides Desf.) kot bioinsekticid za zatiranje riževega mokarja (Tribolium castaneum [Herbst, 1797]) in indijskega žitnika (Trogoderma granarium Everts, 1898) Izvleček: Laboratorijsko vrednotenje mlečka iz prera- stolikega mlečka (Euphorbia bupleuroides Desf.) kot bioin- sekticida za zatiranje riževega mokarja (Tribolium castaneum (Herbst, 1797)) in indijskega žitnika (Trogoderma granarium Everts,1898) je bilo izvedeno pri sobni temperaturi. Insektici- dna aktivnost mlečka je bila določena z neposrednim nanosom. Različne koncentracije so bile pripravljene z razredčenjem 2,5; 5,0; 7,0 in 10,0 µl mlečka v 0,1 ml acetona. 1 µl raztopine je bil previdno vbrizgan v oprsje hroščev. Mleček prerastolikega mlečka je izkazal insekticidno delovanje na oba preučevana hrošča. Velika smrtnost hroščev je bila povezana s povečanimi koncentracijami in daljšim časom izpostavitve. Odrasli osebki indijskega žitnika so bili na splošno bolj dovzetni za strupeni učinek mlečka kot odrasli osebki riževega mokarja. Po šestih dnevih izpostavitve mlečku je bila LC 50 za indijski žitnik 14,12 µl in 14,7 µl za riževega mokarja. LC 90 vrednost je bila za prvega 38,8 µl in 51,44 µl za drugega. Ključne besede: Euphorbia bupleuroides; bioinsekticid; mleček; Tribolium castaneum; Trogoderma granarium; smrtnost Acta agriculturae Slovenica, 117/2 – 2021 2 I. BRIK, N. FRAH 1 INTRODUCTION Pest insects can potentially be carriers of pathogens, and are a substantial contributing source of allergens. That is because of their large cosmopolitan population and high numbers at homes and other buildings (Chang and Anh, 2002). Interest in developing safer alternatives to potentially replace toxic chemicals in pest control is increasingly growing. Bioactive substances and plant in- secticides serve different functions in pest control: act as repellents, impact oviposition or feeding, disrupt de- velopment, or serve as pest insecticides (Isman, 2017, as cited in Bohinc et al. (2020). The use of plant extracts is one of the most desirable pest control methods (Salvadores et al., 2007). Plants sec- ondary metabolites are recognized for their crucial role in pest control due to the selective, biodegradable, non- toxic nature of their products, as well as possessing fewer harmful side-effects on non-targeted organisms and the environment (Wink, 1993). Approximately 250,000 spe- cies of plants on earth have been labelled as possessing compounds with insecticidal properties (Rafael, 2001). Euphorbiaceae family is one of the largest and most diverse family in the plant kingdom; comprising of 7800 species in 300 genera (Webster, 1994). Diterpenoids and triterpenoids secondary metabolites are substantially present in Euphorbia species (Giner et al., 2000). They are endowed with striking biological anti-cancer qualities; for instance, they can serve antitumor purposes (Tanaka et al., 2000), anti-proliferative (Cateni et al., 2010), anti- oxidant and cytotoxic (Aslanturk et al., 2013), and modu- lators of multidrug resistance (Vasas et al., 2012). Euphorbia bupleuroides Desf. is labelled as an her- baceous plant, characterized by plain and simple leaves. It is commonly found in mountain rock areas (Quezel and Santa, 1963), and is utilized in Algeria as an endemic medicinal plant in traditional medicine with varied uses ranging from the extirpation of thorns to the treatment of warts, as well as the use of the decoction of roots for anti- inflammatory purposes. Furthermore, two of the major chemical compounds of E. bupleuroides are diterpenoids and triterpenoids (Aichour et al., 2014). They are the most relevant in the insecticidal nature of E. bupleuroides and similar plants (Singh, 2012; Vimal and Das, 2014). This study is conducted to assess the toxicity of Eu- phorbia bupleuroides latex against grain pests, red flour beetle (Tribolium castaneum [Herbst]) and khapra beetle (Trogoderma granarium Everts). 2 MATERIAL AND METHODS 2.1 PLANT MATERIAL In April 2018, samples of Euphorbia bupleuroides were collected from their natural habitat of mountain rock areas around Tazoult, east of the city of Batna in the North East of Algeria (35° 28’ 54” N, 6° 15’ 39” E).  2.2 LATEX PREPARATION The stems of the collected samples of Euphorbia bu- pleuroides were cut using a knife, allowing the latex to come out into a container. Latex was then collected in beakers, which were then corked tightly to prevent both evaporation and solidification. They were, after that, la- beled and preserved in a refrigerator to maintain fresh- ness. The whole procedure took approximately three days overall. 2.3 INSECTS Cultures of the red flour beetle (Tribolium castane- um) and khapra beetle (Trogoderma granarium) were maintained at 27 °C and 65 % relative humidity (RH) on a wheat flour of a growth culture room in the dark. Adult insects, 1–7 days old, were used for bioassay. 2.4 BIOASSAY METHODS 2.4.1 Insecticidal Activity The insecticidal activity of latex was determined by direct contact application. Different concentrations were prepared by dilution of 2.5, 5.0, 7.0, and 10.0 µl of latex into 0.1 ml acetone. For each preparation, 1 µl was pumped regularly in the thorax of different insects; 10 adult insects were enclosed in a Petri dish. Controls were treated similarly but exposed only to acetone. Each concentration and control were replicated four times. Mortality percentage was determined at 2, 4 and 6-days following treatment. Upon the observation of no leg or antennal movements were, insects were considered dead. The percentage of insect mortality was calculated using Abbott’s correction formula for natural mortality in un- treated controls (Abdel-Sattar et al.,2010). Acta agriculturae Slovenica, 117/2 – 2021 3 Euphorbia bupleuroides Desf. latex as biopesticide against the red flour beetle ... and khapra beetle (Trogoderma granarium Everts, 1898) 2.5 STATISTICAL ANALYSIS Probit analysis of concentration-mortality data was conducted to estimate the LC 50 , LC 90 values, their 95 % confidence intervals and related parameters (Finney, 1971). Probit analysis was fitted using the ‘’LC_probit’’ function in the ‘’ecotox’’ package in R (Robertson et al., 2007). 3 RESULTS AND DISCUSSION The insecticidal activity of E. bupleuroides latex was tested against T. granarium and T. castaneum (Table 1). Data on the accumulative mortality of the two stored products insect species along 6 days of exposure to la- tex revealed that E. bupleuroides latex was significantly toxic for both species, though at different concentrations, all slope regressions were qualified significant (p < 0.05) (Table 1). The mortality rates of T. castaneum are summarized in Figure 1. At a concentration of 5 µl, mortality rates remained negligible from day 2 up to day 6. However, after 6 days, mortality rates increased from 5 to 90 %. The higher the concentration, the less time is required to achieve high levels of toxicity. At 7.5 µl and 10 µl, 10 and 42.5 % mortality rates are observable after 2 days, 7.5 and 20 % after 4 days, and 57.5 to 90 % after 6 days. The highest concentration (10 µl) caused 42.5 % a mortality rate just after 2 days of exposure (Figure 1). The results on Figure 2 demonstrate that the mortal- ity rate of T. granarium adults varied with concentrations and time. In fact, after 6 days, the recorded mortality rate was 15, 22.5, and 40 % using 5.0, 7.5, and 10.0 µl concentrations respectively. Lethal concentrations of latex were calculated af- ter 2, 4, and 6 days of exposure for both species. Results showed that T. granarium adults are generally more sensitive to latex insecticidal effects than T. castaneum adults (Table 1). Evidently, after 6 days of exposure to E. bupleuroides latex, the LC 50 recorded was 14.12 µl for T. granarium adults, and 14.7 µl for T. castaneum. LC 90 numbers, on the other hand, were 38.8 µl for the former, and 51.44 µl for the latter (Table 1). Mortality rates increased with rising concentration levels of E. bupleuroides latex. Additionally, mortality rates of T. castaneum and T. granarium differed in ac- cordance with the different concentration levels of E. bu- pleuroides latex. Figure 1: Mortality rates in T. castaneum treated with different concentrations of latex. Acta agriculturae Slovenica, 117/2 – 2021 4 I. BRIK, N. FRAH Figure 2: Mortality rates in T. granarium treated with different concentrations of latex. Table 1: Latex toxicity on T. castaneum and T. granarium adults (LC 50 and LC 90 ) after 2, 4, and 6 days. Species Assay time (days) LC 50 (µl) 95% LCL – UCL LC 90 (µl) 95% LCL – UCL Slope ±SE P Tribolium castaneum 2 14.7 9.76 – 9.81E+3 38.8 17.02 – 1.93E+8 3.04 ± 0.860 0.0004 4 56.03 17.98 – 5.23E-42 482.05 53.42 –3.77E-84 1.37 ± 0.715 0.049 6 14.7 9.76 – 9.81E+3 38.8 17.02 – 1.93E+8 3.04 ± 0.860 0.0004 Trogoderma granarium 2 22.12 13.26 – 323.8 78.12 28.54 – 21222.5 2.33 ± 0.835 0.005 4 40.74 16.10 – 3.613E+09 320.08 48.72 – 2.040E+19 1.43 ± 0.663 0.031 6 14.12 9.44 – 105.5 51.44 21.14 – 9780.8 2.28 ± 0.625 0.0002 4 DISCUSSION Different plants belonging to the Euphorbiaceae family have been studied all over the world for their toxic constituents. For instance, according to Govindarajan et al. (2008), Leaf extract of Acalypha indica L. exhibits larvicidal and ovicidal activities against malaria vec- tor - Anopheles stephensi Liston, 1901. Acalypha alnifolia Klein ex Willd. extracted leaves demonstrated, in similar fashion to Acalypha indica L., larvicidal properties, but differed in having pupicidal effects—rather than ovi- cidal—against the same species (Murugan et al., 2011). De Silva et al. (2008) studied the insecticidal properties of Euphorbia antiquorum L. latex againt rice insect pests, whilst Euphorbia fischeriana Steud. had anti-feeding ef- fects on stored-product insects according to Geng et al. (2011). Euphorbia bupleuroides latex was tested several times against common species and pest insects that are widespread in houses, restaurants, and food stockages (Saito and Hama, 2000). For example, It was proven ef- fective and toxtic against Blattella germanica Linnaeus, 1767 adults and larvae (Azoui et al.,2016) . Vimal and Das (2014) confirmed that mortality rates increased with the increase in concentration of Eu- phorbia antiquorum L. latex extract. It was also found that latex was a strong pesticide against Aedes aegypti (Linnaeus and Hasselquist,1762) larvae, where LC 50 val- Acta agriculturae Slovenica, 117/2 – 2021 5 Euphorbia bupleuroides Desf. latex as biopesticide against the red flour beetle ... and khapra beetle (Trogoderma granarium Everts, 1898) ue was 14.34 ml dl -1 after 24 hours, 10.70 ml dl -1 after 48 hours, 6.62 ml dl -1 after 72 hours of exposure. The insecticidal effects of Sebastiania cornicu- lata Müll. against Laodelphax striatellus (Fallén,1826), Nilaparvata lugens ( Stål, 1854) and Sogatella furcifera (Horvath 1899), were evaluated by Lee et al. (2010). Results indicated that the chloroform fraction of S. cor- niculata possessed the highest potential for insecticidal activity against L. striatellus (DL50 = 1.09 µg/female), N. lugens (DL50 = .46 µg/female), and S. furcifera (DL50 = 2.32 µg/female). (LD stands for “Lethal Dose” . LD 50 is the amount of a material, given all at once, which causes the death of 50% (one half) of a group of test insects). Mwine et al. (2010) examined larvicidal properties of Euphorbia tirucalli L. latex against larvae of Anopheles mosquitoes. Results showed that the latex made total mortality at the highest dilution used of 1:250 in 5 days. Plant latex comprises of a substantial mixture of proteins and specialized products that include alkaloids, terpe- noids, cardenolides, and many other components, most of which are toxic against insects and pathogens (Agraw- al and Konno, 2009; Hua et al., 2015; Huber et al., 2015; Konno, 2011; Konno et al., 2006). Numerous ingredients have been isolated from the extracts of Euphorbia species (Jain et al., 2008). Al-Y ounis and Abdullah (2009) identified flavonoids and phenolic acids from several species of Euphorbia genus including Euphorbia granulata Forssk. and Euphorbia heliscopia L. Different triterpenoids and diterpenoids were isolated from various Euphorbia species (Sutthivaiyakit et al., 2000; Sun et al., 2011; Aichour et al., 2014). According to Lima et al (2006), terpenoids are the most significant ele- ment in the insecticidal property of several plant species. 5 CONCLUSION This study confirms the success of latex as a biope- sticide against certain stored products insect species; namely, T. castaneum and T. granarium. Hence, latex could serve as an alternative to synthetic insecticides for the protection of stored grain. 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