Castor oil

Phlebotomine sand fly specimens used herein were collected at different occasions, in studies conducted in Apulia, Sicily and Basilicata regions, southern Italy [6 (link)-8 (link),18 (link)]. As a rule, collection sites were selected based on their characteristics, including presence of animals, type of vegetation, and degree of urbanization. Phlebotomine sand flies were collected using ordinary collection methods, such as sticky traps (white paper sheets coated with Castor oil), light traps (model IMT, Byblos per l’Igiene Ambientale di Wehbe Nasser, Cantù, CO, Italy) or mouth aspirators. Phlebotomine sand flies collected with light traps and mouth aspirators were directly preserved in 70% ethanol. Those caught with sticky traps, however, were firstly washed with 90% ethanol, in order to remove excess of oil [5 ] and then kept in labelled vials containing 70% ethanol.
Before proceeding with species identification, phlebotomine sand flies were examined using a stereomicroscope (Leica Microsystems, MS5, Germany), separated from other insects and according to sex. For mounting on slides, specimens were cleared with 10% potassium hydroxide solution at room temperature for 2 h. The material was then washed with water for 1–2 min, immersed in 10% aqueous solution of glacial acetic acid for 30 min, washed again with water for 30 min and, finally, slide-mounted in Hoyer’s solution as described by Lewis [24 ]. Species identification was made according to different morphological keys, species descriptions and other identification resources [14 (link),16 (link),17 ,25 ].
Out of about 16,500 phlebotomine sand flies examined over the past 10 years, representative specimens of each species were selected and further studied morphologically. Specimens of both sexes (i.e., 233 males and 186 females) were selected based on conservation status and quality of the clarification. In some cases, all insects of a given species (e.g., P. sergenti) or of a specific sex (e.g., P. neglectus female) were used, due to the limited number of specimens available. Several morphological characters were examined, but only key characters (e.g., pharynx and spermathecae of females and terminalia of males) were considered during the preparation of the identification keys. Incidentally, these characters were those reported in the keys proposed by Lewis [24 ].
Representative phlebotomine sand fly specimens for each species available were selected and relevant characters were drawn with the aid of a camera lucida (Leica Microsystems, L 3/20, Germany). The pencil drawings were scanned, the resulting files were imported into Adobe Illustrator C6 and the line drawings were made using a digitiser board (WACOM Intuous 5 touch PTH-650, Wacom Europe GmbH, Germany). Voucher phlebotomine sand fly specimens are deposited in the Laboratory of Parasitology and Parasitic Diseases at the Department of Veterinary Medicine, University of Bari, Italy.

To predict genes in the assembled genome, we used both homology-based and de novo methods. For the homology-based prediction, A. thaliana, grapevine, castor, and potato proteins were mapped onto the assembled genome using Genewise [56 (link)] to define gene models. For de novo prediction, Augustus [57 (link)] and GlimmerHMM were employed using appropriate parameters. Data from these complementary analyses were merged to produce a non-redundant reference gene set using GLEAN [58 ]. In addition, RNA-Seq data from multiple tissues (young roots, leaves, flowers, developing seeds, and shoot tips) from our previous study [17 (link)] were also incorporated to aid in gene annotation. RNA-Seq data were mapped to the assembled genome using TopHat [59 (link)], and transcriptome-based gene structures were obtained by cufflinks [60 ]. Then, we compared this gene set with the previous one to get the final non-redundant gene set of sesame (Tables S8 to S10 in Additional file 1). The non-coding gene predictions and gene function annotations were conducted as described in Supplementary Note 3 and Table S11 in Additional file 1.

The method described by Shoba and Thomas [9 (link)], was followed for this study with slight modification. The animals were all screened initially by giving 0.5 ml of castor oil one week before the actual experiment. Only those showing diarrhoea were selected for the final experiment. Twenty five mice fasted for 24 h were randomly allocated to five groups of five animals each. Group I (received 1% tween 80 at a dose of 10 ml/kg) served as control group, Group II received the standard drug loperamide 3 mg/kg, p.o. Group III, IV and V received the methanol leaf extract of C. aurea at the doses of 100, 200 and 400 mg/kg p.o., respectively. One hour after administration, all animals received 0.5 ml of castor oil and then they were individually place in cages the floor of which was lined with transparent paper. During an observation period of 4 h, the time of onset of diarrhoea, the total number of faecal output (frequency of defecation) and weight of faeces excreted by the animals were recorded.