**8. The neem cake alternative**

O

HO

O O

H3C

**Figure 6.** The main limonoids of neem.

Figure 6. The main limonoids of neem.

including products for pet care.

cake deoiled, with a residue up to 1.5% [62].

H

232 Insecticides Resistance

<sup>H</sup> OH

H

O O H3C

O OAc <sup>O</sup>

H H

H H

CH3

O O O

nimbin

CH3

CH3 O O CH3

genera, including *Aedes*, *Anopheles*, and *Culex*, also under field conditions.

O

CH3

CH3

O <sup>O</sup> <sup>O</sup>

H3C

H H

H H CH3 O O

CH3

O O H3C

salannin

O

HO

O O

H

<sup>H</sup> OH

H

azadirachtin B

Many formulations deriving from neem seeds show antifeedancy, fecundity suppression, ovicidal and larvicidal activity, insect growth regulation, and/or repellence against insect pests, even at low dosages [51–56], including ticks, house dust mites, cockroaches, raptor bugs, cat fleas, bed bugs, *Sarcoptes scabiei* mites infesting dogs, poultry mites, and beetle larvae parasitizing the plumage of poultry. The insecticidal properties, environmental safety, and public acceptability of neem and its products have been certified by the US EPA [57] and have led to its adoption into some control programs against Diptera pests [58]. Noticeably, emulsified formulations of *A. indica* oil showed an excellent larvicidal potential against

Action mechanisms include repellence, feeding and oviposition deterrence, but hormonal effects are the key of the inversion of control strategy, changing the target from the adult everywhere dispersed to the locally maintained larvae, through growth inhibition, mating disruption, chemo-sterilization, etc. In fact, hormones are necessary for to complete the process of metamorphosis as the insects pass from larva to pupa to adult. In any case, if the larva manages to enter the pupal stage, the adult emerging from the pupa is 100% malformed, absolutely sterile without any capacity for reproduction. The insect populations decline drastically as they become unable to reproduce. However, also antifeedant and deterrent activities are important to defend crops. The ideal plant-derived product, including insecticide, should be eco-friendly, sustainable, low cost, and target specific, leaving unaffected the beneficial ones. Neem products do not leave any residue on the field, being biodegradable by the action of sunlight. Azadirachtin in open space after dissipation has a half-time of about 20 h. The degradation slowly occurs also when neem products are stored under appropriate conditions [59–60]. Neem, at usual concentrations, is harmless to nontarget and beneficial organisms like pollinators, honeybees, mammals, and other vertebrates [61]. The absence of toxicity is largely evidenced by the millenary use in Indian traditional medicine, as well as by the EPA report and the large use during the last 30 years,

Neem cake is the residue that is left over when the kernel is crushed from neem kernels containing seeds and the remaining is pressed to obtain the oil (Fig. 5). In fact, although the overall marketed name is seed neem oil, not only the seeds are utilized. Neem cake looks more like flour than cake, with differences in color and size of particles. Two products are therefore in the market: neem oil cake obtained by cold pressure, with 6% of the oil still residue, and neem

Actually, it is not approved as pesticide, and mainly it is highly appreciated as organic fertilized. Neem cake acts as a

India alone has an annual potential of 80,000 tons of oil and 330,000 tons of neem cake from 14 million plants that grow naturally. To this potentiality, the high number of cultivations actually occurring in many parts of the world must be

natural fertilizer with pesticide properties, protecting crops from nematodes, soil grubs, and white ants.

different mosquito genera, including *Aedes*, *Anopheles*, and *Culex*, also under field conditions.

Action mechanisms include repellence, feeding and oviposition deterrence, but hormonal effects are the key of the inversion of control strategy, changing the target from the adult everywhere dispersed to the locally maintained larvae, through growth inhibition, mating disruption, chemo-sterilization, etc. In fact, hormones are necessary for to complete the process of metamorphosis as the insects pass from larva to pupa to adult. In any case, if the larva manages to enter the pupal stage, the adult emerging from the pupa is 100% malformed, absolutely sterile without any capacity for reproduction. The insect populations decline drastically as they become unable to reproduce. However, also antifeedant and deterrent activities are important to defend crops. The ideal plant-derived product, including insecticide, should be eco-friendly, sustainable, low cost, and target specific, leaving unaffected the beneficial ones. Neem products do not leave any residue on the field, being biodegradable by the action of sunlight. Azadirachtin in open space after dissipation has a half-time of about 20 h. The degradation slowly occurs also when neem products are stored under appropriate

Many formulations deriving from neem seeds show antifeedancy, fecundity suppression, ovicidal and larvicidal activity, insect growth regulation, and/or repellence against insect pests, even at low dosages [51–56], including ticks, house dust mites, cockroaches, raptor bugs, cat fleas, bed bugs, *Sarcoptes scabiei* mites infesting dogs, poultry mites, and beetle larvae parasit‐ izing the plumage of poultry. The insecticidal properties, environmental safety, and public acceptability of neem and its products have been certified by the US EPA [57] and have led to its adoption into some control programs against Diptera pests [58]. Noticeably, emulsified formulations of *A. indica* oil showed an excellent larvicidal potential against different mosquito

O O H3C

H

OAc

CH3

CH3 CH3

O

O O CH3 CH3

OH O O CH3

OAc

H

CH3 O CH3

O O

OH O O CH3

OAc

azadirachtin A

H

CH3 O CH3

H

Despite the evidence of efficacy, several factors limit the massive use of neem oil in control of insect vectors. Limits include high cost, photosensibility, and persistence in the soil. A network of several Italian universities and research institutions decided to investigate the larvicidal activity of the neem cake as an alternative. In fact, neem cake is a low cost by-product of neem oil production (Fig. 7). <H1>**The neem cake alternative**

Despite the evidence of efficacy, several factors limit the massive use of neem oil in control of insect vectors. Limits include high cost, photosensibility, and persistence in the soil. A network of several Italian universities and research institutions decided to investigate the larvicidal activity of the neem cake as an alternative. In fact, neem cake is a low cost by-product of neem oil production

Fig. 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

**Figure 7.** Nem cake ready for exportation.

(Fig. 7).

Fig. 7. Nem cake ready for exportation.

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‐ dirachtin A.

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‐ achtin, 1.5 ppm salannin, and 0.3 ppm of nimbin. The fraction was more toxic than a commercial formulation at the same azadirachtin concentration.

Strategy in field trials was based on the deposit of neem cake in the typical larval sites of *Culicoides*, and again the product was found to be very effective. A treatment with neem cake at dose of 100 g/m was applied in a larval breeding site of *Culicoides* located in a riverside of a pond margin of a livestock farm in Sardinia, Italy. The treatment with the neem cake resulted in a significant reduction in *Culicoides* emergence until 28 days.

Finally, activity tests are now in progress at the ENEA laboratories to measure the larvicidal toxicity against *P. spumarius*, as main vector of *X. fastidiosa*. First experiments were positive but limited to the laboratory conditions. Field experiments are urgently needed.

Although the mentioned results need confirmation and utilization in larger scale, neem cake is a promising material for the development of newer products useful in the control of vectors of insect-borne diseases at the larval stage.
