Neem cake as a promising larvicide and adulticide against the rural malaria vector Anopheles culicifacies (Diptera: Culicidae): a HPTLC fingerprinting approach

Abstract Mosquitoes are insects of huge public health importance, since they act as vectors for important pathogens and parasites. Here, we focused on the possibility of using the neem cake in the fight against mosquito vectors. The neem cake chemical composition significantly changes among producers, as evidenced by our HPTLC (High performance thin layer chromatography) analyses of different marketed products. Neem cake extracts were tested to evaluate the ovicidal, larvicidal and adulticidal activity against the rural malaria vector Anopheles culicifacies. Ovicidal activity of both types of extracts was statistically significant, and 150 ppm completely inhibited egg hatching. LC50 values were extremely low against fourth instar larvae, ranging from 1.321 (NM1) to 1.818 ppm (NA2). Adulticidal activity was also high, with LC50 ranging from 3.015 (NM1) to 3.637 ppm (NM2). This study pointed out the utility of neem cake as a source of eco-friendly mosquitocides in Anopheline vector control programmes.


Introduction
Currently, mosquito-borne diseases, such as malaria, yellow fever, dengue, West Nile and Zika virus are of huge medical and veterinary importance (Benelli 2015a;Benelli, Lo Iacono, et al. 2016a). Despite the recent positive results in limiting malaria diffusion (reduction in malaria mortality rates by more than 25% globally since 2000 and by 33% in the WHO African Region), this plague still has predominant importance in number of infections (2013: about 198 million cases of malaria), deaths (an estimated 584,000 deaths) and public concern (WHO 2014; Benelli & Mehlhorn 2016). Anopheles culicifacies Giles is the most important malaria vector in rural and peri-urban areas of peninsular India, contributing to nearly 65% of total malaria cases per year. A. culicifacies is a complex of five sibling species, provisionally designated as A, B, C, D and e. Among these five, only three species (i.e. A, B and C) have been laboratory colonised. A and C are more competent vectors of Plasmodium spp. over B (Kaur et al. 2000;Amerasan et al. 2016).
In an eco-friendly perspective, botanicals recently gained attention as effective and cheap sources of mosquitocidal products (Benelli 2015a(Benelli , 2015bMurugan et al. 2015). In particular, we focused on neem (Azadirachta indica A. Juss, Meliaceae) due to the large utilisation of its seed oil as insecticide (ePA 2012;Nicoletti et al. 2016). For its potentiality, neem is considered one of the most important plants for humankind future, as recently reported by WHO/UNeP, who recognised neem as one of the most promising tree of the twenty-first century (Ruskin 1992;Puri 1999;Brahmachari 2004;Nix 2007;Pankaj et al. 2011;Del Serrone et al. 2015). Traditional medicines report neem specific uses against insects and other pests (Bhownik et al. 2010;Nicoletti et al. 2016). Cold-pressed neem kernel oil has attracted worldwide attention for its toxicity against more than 400 arthropod pests (Benelli, Bedini, et al. 2015;Benelli et al. 2016b;Nicoletti et al. 2016). The absence of environmental toxicity of neem oil products, as well as antifeedant and repellent activities has been certified by US environmental Protection Agency (ePA, 2012). Neem-based products could be used as a more environmentally friendly alternative to traditional mosquito larvicides, being azadirachtins biodegradable by the action of sunlight, which do not accumulate in the field (Isman 2006;Chandramohan et al. 2016a). Furthermore, neem preparations, including nanoformulations, do not heavily harm the beneficial insects and aquatic invertebrates (Awad & Shimaila, 2003;Peveling & ely 2006;Ruiu et al. 2008;Chandramohan et al. 2016b). However, the utilisation of neem oil is limited by the high cost and the degradation of the limonoids exposed to the sunlight. Therefore, we worked on the hypothesis that by-products of neem extraction could be interesting for mosquito control, in particular that the neem cake, currently considered of low interest and actually used in agriculture as fertilizer or as animal feed (Benelli et al. 2014;). An important aspect concerns the variability of neem products, whose chemical composition must be determined. In fact, several pre-harvesting (i.e. geographical origin, cultural practices, treatment by insecticides and chemicals, peaking) and post-harvesting factors (i.e. exsiccation, conservation, storage) can influence deeply the composition of neem-derived products. Thus, the limonoids' presence in neem cake is quite different from that of the oil, with salannin largely dominant against azadirachtin A. It is necessary to consider that although more than 30 limonoids have been identified in neem, their structures vary in skeleton and functional groups. Furthermore, the multiple activity of neem products should be assigned to the phyto-complex of neem extracts instead of single constituents. As a confirmation, despite the large phytochemical study already performed on neem, every year other new constituents are reported. The low cost and the availability of neem cake makes it a potential important raw material for developing new eco-friendly insecticidal products (Nicoletti & Toniolo, 2012;Benelli et al. 2016b;Nicoletti et al. 2016).
By the metabolomic fingerprint approach, it is possible to compare efficiently, side-byside in the same conditions, two extracts of the same raw material, evidencing also little differences. When applied in the determination of the constituents of neem cake extracts, previous HPTLC (high performance thin layer chromatography) analyses highlighted a low presence of several limonoids in neem cake extracts, with high amount of salannin (in n-hexane, methanol and ethyl acetate extracts) and nimbin (n-hexane extract), as confirmed by the HPLC analysis (Nicoletti & Toniolo, 2012). Considering the differences in chemical composition of neem-marketed products, in the present study, two different marketed products of neem cake were studied, in order to evidence possible bioactivity differences. To better evidence differences in composition and activity, the two raw materials were separately extracted using methanol and ethyl acetate. Methanol was chosen as general solvent, whereas ethyl acetate more selectively dissolves azadiractins and similar nonpolar constituents. Hereafter, the following abbreviations were used: NM1 = neem cake methanol extract, commercial sample 1. NA1 = neem cake ethyl acetate extract, commercial sample 1; NM2 = neem cake methanol extract, commercial sample 2; NA2 = neem cake ethyl acetate extract, commercial sample 2. The resulting extracts were examined and compared for differences in composition and the insecticidal activity on A. culicifacies was evaluated in ovicidal, larvicidal and adulticidal experiments.

Results and discussion
HPTLC fingerprints presented the same sequence of spots, as expected in case of the same raw material (Figure 1). As expected, NA extracts resulted richer in the strong spots at high and central Rf values if compared to NM (i.e. unsaturated fatty acids and triglycerides, respectively). However, the NM2 and NA2 showed abundance of the oily components, presented as red spot on the plate in contrast to the fluorescent spots. In both commercial samples, neem cake methanol and ethyl acetate extracts showed promising ovicidal (Table S1), larvicidal (Table S2) and adulticidal activity (Table S3) against A. culicifacies. All the tested neem cake extracts showed toxicity against A. culicifacies eggs. A significant effect of the tested neem cake sample (p < 0.05), the extraction solvent (p < 0.01), the tested dose (p < 0.001) and their interaction (p < 0.05) was found. NA1 was more toxic to mosquito eggs, when compared to the other three extracts. We hypothesise that the higher ovicidal effectiveness of NA1 was due to the differences in its composition, as shown by HPTLC analyses (see also Benelli, Conti, et al. 2014;. However, all the tested neem cake extracts led to a 50% reduction in egg hatchability, post-treatment with 150 ppm of neem cake (Table S1). As regard to larvicidal activity, LC 50 values were extremely low against fourth instar larvae, ranging from 1.321 (NM1) to 1.818 ppm (NA2) ( Table S2). Adulticidal activity was high against A. culicifacies, with LC 50 values ranging from 3.015 (NM1) to 3.637 ppm (NM2). Thus, the highest larvicidal and adulticidal activity were observed for NM1 extract, evidencing consistent variations according to the tested raw by-product. The slightly higher toxicity of methanol extracts from the two tested products may be linked with higher amounts of the limonoids azadirachtin A and salannin (Figure 1), two limonoids with high mosquitocidal activity already reported for a number of important mosquito vectors (see also Nicoletti et al. , 2016Benelli, Bedini, et al. 2015). In addition, our findings suggest that constituents other than limonoids could influence the insecticidal activity, as previously showed for mosquitocidal and ovideterrent assays conducted on Asian tiger mosquito, Aedes albopictus (Nicoletti et al. 2010;Benelli, Conti, et al. 2014;.  focused on A. albopictus testing six commercial neem cake samples. Three of them did not show mosquitocidal activity on newly hatched larvae, while two of them were not toxic towards late instar larvae. This highlights the key importance of comparative approaches in bioactivity surveys testing raw products from different production processes against arthropods of medical importance . Overall, our results add information about the variability in mosquitocidal activity of two neem cake products extracted with different solvents. In previous research, we evidenced that neem cake can be very different in chemical composition, and its mosquitocidal effectiveness depends on several factors, including the raw material, the used machinery and further refinery steps Benelli et al. 2016b;Chandramohan et al. 2016a;Nicoletti et al. 2016). Also from the same producer or importer, it is possible to have significant variations depending on the production year. For this reason, here we tested two commercial samples of neem cakes present in the Italian market and obtained from two different importers, highlighting our previous data on variability of composition in neem by-products and the related bioactivity of arthropod pests Chandramohan et al. 2016a). uV 366 nm, neem cake extracts. NM1 = neem cake methanol extract, commercial sample 1. Na1 = neem cake ethyl acetate extract, commercial sample 1; NM2 = neem cake methanol extract, commercial sample 2; Na2 = neem cake ethyl acetate extract, commercial sample 2.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Deanship of Scientific Research at King Saud University [Grant number N. RGP-1435-057].