RESEARCH ARTICLE Published Online: September 22, 2020 https://doi.org/10.31893/jabb.21002 Toxic effects of Drimia maritima (Asparagaceae) ethanolic extracts on the mortality, development, sexual behaviour and oviposition behaviour of Drosophila melanogaster (Diptera: Drosophilidae) Fatma Zohra Saadanea* | Wafa Habbachia | Sarra Habbachia | Nour El Imene Boublataa | Abderachid Slimanib | Abdelkrim Tahraouia aApplied Neuroendocrinology Laboratory. Department of Biology, Faculty of Sciences, University Badji Mokhtar Annaba 23000, Algeria. bDepartment of Biology, Faculty of Science, University Badji Mokhtar BP 12, 23000 Annaba, Algeria. *Corresponding author: [email protected] Abstract Drimia maritima is a toxic Mediterranean plant and is considered a commercially important drug by its medicinal values and pharmacological properties. In this work, we seek to evaluate the direct and delayed effects of the ethanol extracts of this plant on vinegar fly (Drosophila melanogaster), an excellent laboratory model. The treatment was carried out by ingestion on second-stage larvae (L2) and then we evaluated the impact of this treatment on mortality, development, sexual behaviour, on female's oviposition choice, fecundity, and fertility of adults. The results showed that mortality rates can reach 100% after 15 days of treatment as we recorded an acceleration of the flies' development. After treatment, we observed incomplete nuptial courtship and a significant decrease in eggs laid number and larvae number of the first generation treated. This study indicates that D. maritima ethanolic extract has a neurotoxic property, our results confirmed the presence of toxic secondary metabolites in the extract studied. Keywords fruit fly, mating, Mediterranean plant, reproduction, toxicity 1. Introduction this aspect, and more particularly on the use of spontaneous species in traditional medicine (Hammiche and Gueyouche In recent years, natural products have drawn the 1988). researchers’ attention to find new solutions alternatives Our study is based on the medicinal plant Drimia where the aims are to reduce the excessive use of synthetic maritima which is very widespread in the Mediterranean pesticides. Among these alternatives in nature, products of region, Africa and India their medicinal part is mainly plant origin are, in particular, more interesting because they composed of fresh or dried bulbs (Ghahreman 1997; Zargari are less toxic, biodegradable, and target-specific (Dua et al 1996). The oldest writings on these plants date back to 1500 2010; Subramaniam et al 2012). B.C. and mention the medicinal Drimia properties (Stoll Recently, the plant extracts with insecticidal 1954). To date, many studies have been conducted to identify properties have continued to develop and some non- the chemical constituents and to understand the European countries have regulatory requirements for pharmacological properties of this species. extracts and essential oils use (Isman 2019). Plants are rich in It is a Mediterranean plant, perennial by an enormous bioactive secondary chemical metabolites and have proven bulb whose diameter reaches, 20 to 30 cm and weight their insecticidal activity by killing or repelling insects between 5 to 7 kg (measures of Algerian species). It’s formed (Sukumar et al 1991; Ghosh et al 2012). of nested scales that are also called tunics or scales, whitish Medicinal plants are valuable resources for the color called Scille d'Italie or Scille "female" or reddish Scille majority of rural and urban populations in Africa and are the d'Espagne or Scille "male", depending on the variety. The main means by which people treat themselves (Badiaga external scales are plain and membranous, the medium 2011). Several species are known for their remarkable scales are thick and fleshy (Hammiche et al 2013). The therapeutic properties (Quezel 1978). Algeria, by the richness flowering stem (about 1 m) bears at its end a cluster of small and diversity of its flora, constitutes a real phylogenetic white flowers with a star-shaped corolla (Joly 2010). reservoir, with about 4000 species and subspecies of vascular This work purpose to test the direct and indirect toxic plants (Dobignard and Chatelain 2013). The study of effects of D. maritima ethanolic extract on fruit fly, traditional medicine and herbal treatment is therefore of Drosophila melanogaster, especially, the effects on the particular interest because little research has been done on mortality, development, sexual, and oviposition behaviour. J Anim Behav Biometeorol (2021) 9:2102 Received: August 09, 2020 | Accepted: August 29, 2020 www.jabbnet.com Saadane et al. (2021) 2 D. melanogaster, the world's most widely distributed species, 2.5. Effect of the plant extract on sexual behaviour is considered a dreaded nuisance both for the inconvenience caused by the parasitic diseases it can inoculate (Jolivet 1980; Sexual parade in fruit flies is an eventful behavior Joly 2006; Habbachi et al 2013). It is also an important vector (Clynen et al 2011; Chardonnet 2013), goes through six essential, predetermined and invariable steps (Terhzaz 2003; for various infectious microorganisms, including Dickson 2008; Revadi et al 2015) and requires many sensory phytopathogenic yeasts and bacteria that attack not only vegetable and fruit crops but also cereal and sunflower crops signals, of which chemical signals (cubic hydrocarbon (Kloepper et al 1979; Corby-Harris et al 2007; Nadarasah and pheromones) play an important role (Greenspan and Ferveur Stavrinides 2011; Becher et al 2012). 2000; Sokolowski 2001). The male first walks up to a potential female and taps her on the cuticle with his front legs; if the 2. Materials and Methods female moves, the male follows and vibrates a wing. Then, when the female stops, the male runs in circles around her, 2.1. Elevage des insects lick her genitals with his proboscis, and tries to hang her up until mating (Revadi et al 2015). Drosophila melanogaster was described by Johann In this work, we treated the larvae group with the Wihelm Meigen in 1830. Its reproduction is very fast. Its life sublethal concentration of D. maritima extract (0.12 µg/ml) cycle is very short and includes three larval instars and a and then recovered the adults as soon as they emerged. 48 pupal stage from which an adult emerges that can fly and hours after emergence, these adults were used for sexual reproduce. A wild strain harvested from rotten apples in behaviour tests where we note the time and number of Annaba region (Algeria), was used. The culture was carried contacts, the time and number of wing vibrations, the time out in vials (250 ml) capped with a foam pad and containing and number of licks, the time and number of mating attempts an agar-based nutrient medium of cornmeal and brewer's as well as the time and duration of mating if successful. These yeast. The culture was maintained at 25±1 °C, a humidity of tests are carried out according to four types of crossings: 70 to 80%, and a 12-hour scotophase. control male X control female, treated male X treated female, 2.2. Drimia maritima (Asparagaceae) control male X treated female and treated male X control female. It is a bulbous plant, which grows in the Mediterranean region. The main properties of this plant 2.6. Effect of the plant extract on oviposition behaviour and considered in traditional medicine are dropsy, cough and reproduction: respiratory diseases, jaundice, skin problems, leprosy, To the purpose of determining the egg-laying site baldness, gastric disorders, cancer, epilepsy, joint pain, and choice, we observed mated females and we noted the site use as an antidote (Stannard 1974; Aliotta et al 2004). The D. eggs hatching, as well as eggs and larval numbers. maritima bulbs had significant insecticidal activity against several species (Pascual-Villalobos and Fernandez 1999; 2.7. Data analysis Metin and Burun 2010). For the present study the plant is collected in the region of Seraïdi (Annaba, Algeria) The toxicological parameters (LC50%, LC90%, LT50%, and LT90%) were calculated according to Finney's (36°54'27.02" north latitude, 7°39'49.95" east longitude). mathematical methods (Finney 1971). Regarding sexual and 2.3. Preparation of the ethanolic extract of D.maritima oviposition behavior tests, results were analyzed statistically by descriptive metric methods then an analysis of variance For the extract, we macerated the dry bulb powder (ANOVA) was performed on XLSTAT 2009 software (300g) in ethanol (250 ml at 70%) for 24 hours in laboratory (Addinsoft, New York, NY). conditions and on the shade. After filtration using Whatman filter paper, the liquid obtained was evaporated using a 3. Results magnetic stirrer heated to 45 °C to remove ethanol. The 10g leg recovered was stored at 4 °C until use. 3.1. Effect on mortality and development 2.4. Treatment of larvae with D. maritima extract The results showed that D.maritima ethanol extracts act on the duration of larval development and larval mortality We prepared three different concentrations 0.12 depending on the concentration applied. The three µg/ml, 0.25 µg/ml, and 0.5 µg/ml. The treatment is done by concentrations used showed high larvicidal activity at the end ingestion; each concentration is mixed with food (40g) which of mortality monitoring (15 days after treatment) (Table 1); will be distributed in four different tubes. In these tubes, 20- and 100% of the population was killed after 15 days of second instar larvae (L2) from the mass rearing are placed. In treatment