**9. Conclusions**

The first step of the network was analytical. Several neem cakes from different producers and importers were analyzed by high-performance liquid chromatography (HPLC), evidencing still the presence of neem limonoids, but with different pattern in comparison with neem oil. Percentage was low but very different in each sample and salannin was the prevalent nortri‐ terpene (3750 ppm of azadirachtin A+B, 7980 ppm of salannin, and 1850 ppm of nimbin) [63– 65]. The high-performance thin layer chromatography (HPTLC) analyses, performed in the laboratories of Environmental Biology at the Sapienza University of Rome, allowed to evidence a great complexity of the neem cake extract, showing at least more than 30 secondary metab‐ olites spread in all range of polarity (Fig. 8). On the basis of the information obtained in the chromatographic analyses, a neem cake product was selected and used for the activity tests, realized at the ENEA laboratories. Laboratory essays evidenced a significant activity of neem

cake n-hexane and ethylacetate extract against *A. albopictus* mosquito larvae [66].

**Figure 8.** HPTLC analysis of different neem products. Mobile phase: toluene, ethyl acetate (4:6 v/v). Derivatization: Anisaldehyde. Plate on the top, visualization: UV366 nm. Plate on the bottom visualization: white light. Tracks: (1) neem oil marketed in Italy extracted with ethyl acetate, (2) neem oil marketed in India extracted with ethyl acetate, (3) neem oil marketed in Italy, (4) neem oil marketed in India, (5 and 6) neem cakes extracted with ethyl acetate, (7 and 8) neem cakes of tracks, (5) defatted and concentrated (track 8 more concentrated), (9) nimbin, (10) salannin, and (11) aza‐

In the same time, another group of the network, at the University of Sassari in Sardinia, worked on Blue Tongue disease. *Culicoides* species are vectors of BTV [67]. These insects breed in mist microhabitats, like small pools, irrigation channels, beverage sites, and drainage pipes. *Culicoides imicola* is the main vector, representing about 10% of all emerged *Culicoides* adults. In the laboratory essays, larvae of *C. imicola* resulted highly sensitive to the commercial neem cake. The larval mortality in water after 7 days gave a lethal concentration value (LC50) of 0.37 g/l. In order to define the chemical nature of active constituents, a neem cake methanol extract was separated by different solvents. Fractions of increasing polarity were assayed on *Culi‐ coides* larvae. The most active resulted the ethyl acetate fraction, containing 1 ppm of azadir‐

dirachtin A.

234 Insecticides Resistance

The previous samples are related to Southern Italy, but situations in the other parts of the world are probably very similar. Chun-Xiao et al. reported the relationship between insecticide resistance and genome mutations of *Aedes aegypti* in Southern China [68]. The relationship has been detected for several insecticides, but the mechanisms of resistance are not totally understood. The kind of resistance to both pyrethroids and DDT, known as knockdown resistance, has been related to amino acid substitution in the sodium channel. In the paper, the causes of the resistance are not only attributed to the extensive recent use of pyrethroids and related to a series of different factors, first of all the climate changes, but also associated to the rapid development of tourism, transportations, and increasing urbanization that could increase *A. aegypti* breeding sites. Therefore, the development of resistance must be considered a complex multifactor phenomenon. The complex solution should consist into a multitreat‐ ment in at least three steps.

The soil must be considered not only to sustain the plant with adequate fertilizer able to give the necessary elements but also to positively change the biome living underground. Roots must be considered not only as the corm part necessary for collection of water and minerals but also as a part of the plant integrated to the underground habitant, including the living system. Insect vectors must be controlled possibly at the larval stage, when the insects are not able to move and need mist conditions to survive. Insecticide must be eco-friendly and low cost, targeted to preserve pollinators and other useful insects. Natural substances could be the starting point to develop antibiotics of new generation, based on different mechanism and useful to be used in large scale, without relevant damages to the environment. This multidis‐ ciplinary approach highlights the need of stronger cooperation among pharmacologists, chemists, parasitologists, entomologists, and behavioral ecologists.
