Mortality assessment and oviposition inhibition

Total mortality of adults was obtained with concentrations 0.6 mg mL^-1 with Neem Oil Spray and PHC Neeem, with Biosave Neem was obtained with 1 mg mL^-1; with a concentration of Neemix 4.5 of 1mg mL^-1, mortality was 68.5%. CL₅₀ values denote that there is no overlap between fiducial limits, indicating different toxicity categories in the formulations. Other nim products have also shown dissimilar effectiveness as for mortality refers, as Neemazal-T/S and Neem Rose Defense EC at respective concentrations of 0.5 and 0.7 mg mL^-1 that caused 10 and 48.1% mortality in adults of T. vaporariorum (^{Von Elling et al., 2002}; ^{Chiasson et al., 2004}), lower activity to the products evaluated in this research.

In contrast, the aqueous extracts made from Nim 25 at 0.06 mg mL^-1 and ground seed (Nim 20) at 0.07 mg mL^-1, caused 14-16.9 17-20.9% mortality in adult whitefly B. tabaci, respectively (^{Gomez et al., 1997}, ^{Fernandez et al., 2013}), resulting with similar effectiveness to PHC Neeem from this work. Furthermore, ethanol extract of neem seed at 0.1 mg mL^-1 caused 49% mortality in adults of T. vaporariorum, ethanolic extract has been reported as effective in other pests (^{Simmonds et al., 2002}; ^{Coria et al., 2008}). The mortality obtained is intermediate between PHC Neeem and Neem Oil spray. Azatin EC at 0.02 and 0.1 mg ml^-1 caused 37.8 and 65% mortality in adults of B. tabaci and T. vaporariorum respectively (^{Abou-Fakhr Hammad et al., 2000}; ^{Simmonds et al., 2002}), resulting with similar activity to Neem Oil spray, unlike Margosan-O, Nim 80 and Nim Oil formulations that were more effective (^{Castillo-Sanchez et al., 2010}) than the products evaluated in this study; Margosan-O at 0.01 mg mL^-1 caused 15.5% mortality in adults of B. tabaci (^{Abou-Fakhr Hammad et al., 2000}), and Nim 80 at 0.01 and Nim Oil at 0.03 mg mL^-1 caused mortalities of 62 -65 and 33.8-80% in adults of B. tabaci, respectively (^{Gomez et al., 1997}; ^{Cubillo et al., 1999}).

Neem Oil Spray totally inhibited oviposition at 1 mg mL^-1, which, along with the concentration 0.6 mg mL^-1, produced total mortality of adult, not observing correlation between mortality and oviposition, because in the first concentration females were removed before oviposition and in the second at least one female oviposited before dying. This product caused 50% inhibition of oviposition (CIO₅₀) with 0.12 mg mL^-1. On the other hand, Biosave Neem and PHC Neeem, caused total mortality of adults at concentrations of 1 and 0.6 mg mL^-1, decreasing by 91.0 and 89.8% oviposition, respectively, denoting that both products eliminated adults after some of them oviposited. Both formulations inhibited by 50% oviposition (CIO₅₀) with 0.61 and 0.13 mg mL^-1; regarding Neemix 4.5, affecting less oviposition, with CIO₅₀ of 1 mg mL^-1. In contrast to the results obtained in this work, in other researches Neemazal-T/S at 0.5 mg mL^-1, reduced by 35% oviposition in adults of T. vaporariorum (^{Von Elling et al., 2002}), denoting similar effectiveness to Biosave Neem.

The aqueous extract of neem at 0.2 mg mL^-1 inhibited by 78% oviposition in adults of B. tabaci resulting with the same level of toxicity than Neem Oil Spray and PHC Neeem; conversely the aqueous extract of cake (Nim 25) and Nim 80 at respective concentrations of 0.06 and 0.01 mg mL^-1 inhibited in 34.6-54 and 31.7-68 % oviposition in adults of B. tabaci, respectively (^{Gómez et al., 1997}; ^{Cubillo et al., 1999}), which resulted better than the products evaluated in this investigation.

Neem Biosave and Neemix 4.5 at concentrations of 0.1-0.2 and 0.1-0.15 and 0.3-0.4 mg mL^-1 stimulated oviposition 12.8-35.9 and 41.7-52.8% and 2.8-22.2% respectively, probably in response to stress causing in female to sublethal concentrations, this can result in increased survival through enhanced oviposition rate, leading to the phenomenon of hormoligosis (^{Luckey, 1968}), in Nim Oil 0.03 mg mL^-1, caused increased oviposition of B. tabaci by 13% (^{Gomez et al., 1997}).

Repellency and inhibition of oviposition: repellency manifested itself in three logarithmic ranges from 0.01 to 1.5 mg mL^-1, with values of 0.8 to 90% from 24 to 72 h after application (Table 1). CR₅₀ values were from 0.17 to 0.85 mg mL^-1, expressing the highest effectiveness at 48 h, but does not differ in toxicity compared to 24 h in Biosave Neem, Neem Oil Spray and PHC Neeem. In contrast, Neemix 4.5 had different categories of toxicity, being less efficient at 24 h, moderately effective at 48 h and very biodegradable at 72 h, Biosave Neem was better at 24 and 48 h, with high biodegradation at 72 h; in both products, the 3rd day of evaluation CL₅₀ was ≥ 1.0 mg mL^-1, while Neem Oil Spray and PHCs Neeem showed overlap in their respective fiducial limits of CL₅₀ in the three observation times, indicating similar effectiveness and strong persistence.

Table 1. Repellence percentage of whitefly adults T. vaporariorum at 24, 48 and 72 h with four nim products. 

There are neem formulations than in similar experiments showed intermediate repellency between Biosave Neem and Neem Oil Spray, as Nim 20 and Nim 25 which prepared respectively at 0.07 and 0.06 mg mL^-1 caused 17-18 and 9-13.5% repellency in adults of B. tabaci at 48 h, respectively (^{Gómez et al., 1997}; ^{Cubillo et al., 1999}). While Margosan-O at 0.01 mg mL^-1 was similar to Biosave Neem because it caused 18.4% repellency in adults of B. tabaci at 24 h (^{Abou-Fakhr et al., 2000}).

In contrast, other products are better than those evaluated here; Neemazal-T/S at 0.01, 0.03, 0.05, 0.07 and 0.1 mg mL^-1 caused 31.7, 52.4, 69.8, 80.3 and 86.7% repellency in adults of B. tabaci at 24 h, respectively (^{Kumar and Poehling, 2006}); while Azatin EC at 0.02 mg mL^-1 caused 40% repellency of B. tabaci at 24 h (^{Abou-Fakhr et al., 2000}), as Nim 80 and Oil Nim that at concentrations of 0.01 and 0.03 mg mL^-1 resulted in 30.6-61 and 16.9-76% repellency in adults of B. tabaci at 48 h, respectively (^{Gomez et al., 1997}; ^{Cubillo et al., 1999}), while Neemazal at concentrations of 0.4 and 0.8 mg mL^-1 caused 67.83 and 70.13% repellency respectively (^{Silva et al., 2003}).

Neem Biosave and Neem Oil Spray products at concentration of 1 mg mL^-1, totally inhibited oviposition at 48 h and 48-72 h, but it did not show full repellency. Neemix 4.5 and Neem Oil Spray formulations with 1 mg mL^-1 inhibited from 98.0 to 98.5% oviposition at 24 and 48 h, respectively. PHC Neeem at 1.5 mg mL^-1 also inhibited 98% at 48 h (Table 2). In comparison the fiducial limits of CIO₅₀ at the three oviposition times, three levels of toxicity were determined, highlighting by its efficiency Biosave Neem and Neem Oil Spray, that at respective concentrations of 0.09 - 0.27 and 0.02 - 0.21 mg mL^-1 inhibited oviposition by 50% from 24 to 72 h, while Neemix 4.5 and PHC Neeem showed less activity, which required of 0.21-> 1 and from 0.04 - 0.73 mg mL^-1 to cause the same inhibition.

Table 2. Inhibition (%) of oviposition of whitefly T. vaporariorum at 24, 48 and 72 h with four nim products. 

Females treated with Biosave Neem at 0.01 (24 and 48 h) and 0.02 mg mL^-1 (from 24 to 72 h), Neemix 4.5 at concentrations of 0.03-0.1, 0.01-0.2 and 0.03-0.2 mg mL^-1 at 24, 48 and 72 h, respectively and PHC Neeem at 0.035 mg mL^-1 at 72 h, stimulate oviposition in a similar way to that observed with Nim 80 at 0.5 mg mL^-1 in adults of B. tabaci (^{Cubillo et al., 1999}).

The results of this research showed similarities with other studies; NeemAzal-T/S applied at 0.5 mg mL^-1 inhibited in 29.4% oviposition in adults of T. vaporariorum at 72h (^{Von Elling et al., 2002}), and Margosan-O, that at 48 h after application of 0.5 mg mL^-1 inhibited oviposition in 83.59%, showing similar results to those obtained with Neemix 4.5.

The combined activity of the products showed that PHC Neeem caused total mortality at 0.6 mg mL_-1 and even when it had a regular performance in repellency, it demonstrated persistence with toxic power from 24 to 72 h, in addition to inhibiting oviposition efficiently with values close to 100%; thus the commercial concentration in field (100 mL L^-1), would cause from 90 to 95% mortality, 50% repellency and 80% inhibition of oviposition at 24, 48 and 48 h, respectively, so it is suggested that although causes hormoligosis at concentration of 0.035 mg mL^-1, did not show phytotoxicity. Neem Oil Spray caused the highest mortality, repellency, oviposition inhibition and higher persistent, besides not causing hormoligosis; however, it was more phytotoxic at concentrations from 0.6 to 1 mg mL^-1.

This may be partly due to the damage caused on the leaf, specifically at deterrent level in the selection of oviposition site (^{Rodriguez, 2000}); while commercial dose (1.25 mL^-1), would cause between 75 and 80% mortality, 92% repellency at 48 h and from 75 to 80% inhibition of oviposition. Biosave Neem, a mixture of extracts from two plants, was the third product in efficiency; however, it caused phytotoxicity. Full mortality occurred at 1 mg mL^-1, which decreased significantly from 0.6 mg mL^-1, was the best of the four products in repellency at 24 h, the second best at 48 h, and showed strong biodegradation at 72 h, and nevertheless strongly inhibited oviposition with 1 - 0.6 mg mL^-1, stimulating it at concentrations of 0.2 - 0.01 mg mL^-1, which is important in a mixture with high degradation like this, because in field high concentrations can be sublethal at 3 d.

As for the recommended commercial concentration (1.25 mg mL^-1), this would cause 94-95% mortality, 80% repellency and 100% inhibition of oviposition at 24, 48 and 48 h. In contrast, the less efficient product was Neemix 4.5 which at 1 mg mL^-1caused moderate mortality, repellency and inhibition of oviposition at 24, 48 and 48 h respectively; however, stimulated oviposition at concentrations of 0.4 to 0.01 mg mL^-1, and even when the recommended commercial concentration could cause 95% of mortality, 90% repellency and 95% inhibition of oviposition at 24, 48 and 48 h, this product proved to be phytotoxic at such concentrations.

In general, two toxicological categories within concentrations evaluated in all products were observed, the first is between 0.4 and 1.5 mg mL^-1, range in which causes more than 50% effectiveness in mortality, repellency and inhibition of oviposition, and the second from 0.01 to 0.3 mg mL^-1 showing from 0.8 to 40% effect on mortality, repellency and inhibition of oviposition, and it is in this second category in which Biosave Neem and Neemix 4.5 show hormoligosis.

The persistent effect of formulations with oil can be considered an advantage due to lower concentrations are required to achieve the same effect than non-persistent; but the same implies greater permanence of the product in the environment and accelerates selection pressure, develops resistance and shortens product life, besides being phytotoxic. Authors note that products enriched with oil are more efficient, by the presence of insecticidal compounds and the persistence of these in the plant and the physical effect on the insect (^{Gómez et al., 1997}; ^{Cubillo et al., 1999}). ^{Stark and Walter (1995)}, mentioned that there is 62% more efficiency in four products with 5% neem oil, compared to three non-oily products, identified in the first six limonoids in addition to azadirachtin.

Taking into account the above, oily formulations in the form of emulsions (common in commercial products) should be considered the most appropriate schemes for inclusion in integrated pest management. Neem oil is an essential component in a commercial formulated product, since the addition of oil changes the polarity of the same, and in this context the formulations with lower proportion of oil and more emulsifiable can be absorbed more easily and have systemic effects up to 14 d, further demonstrating that the highest effectiveness of the products, after the first hours of application, is due to this effect (^{Silva et al., 2003}), so it could behave as non-phytotoxic insecticides, with regular persistence . This is because azadirachtin and other limonoids decompose rapidly under the effect of light, UV rays, temperature, rain, HR, pH and microbial activity, within approximately 4 d (^{Mulla and Su, 1999}). While applying crude extracts of oil turns efficient for at least 5 d, being able to increase up to 10 d, gradually decreasing, this depending on the solvent used in extraction (Abdel-Salam et al., 2005). This is because neem oil increases the toxicity of the formulations, either because it promotes penetration into insect cuticle or contains other limonoids that increase their insecticidal activity, in addition that just oil confers greater persistence to the mixture (^{Stark and Walter, 1995}).

One more reason for the differential behavior of formulations, especially those like Biosave Neem, which are extract mixtures of two plants, is that azadirachtin concentration could cause loss of synergistic effect with other compounds, in addition that at low concentrations is possible to observe repellency and feeding inhibition, but these effects won’t be appreciated at high concentrations (^{Kumar et al., 2003}).

Regarding stimulation behavior of oviposition of some nim products, another likely explanation (besides that it could show hormoligosis), is that these have a similar effect to Fenvalerate, which also induced hormoligosis in B. tabaci in cotton plants at concentrations of 25, 38 and 52 g ai ha^-1. In the evaluation of various insecticides, it was found that this product does not cause reduction of total sugars in leaves after application, so that the insect does not consider the host as unsuitable for the development of their offspring (^{Abdullah et al., 2006}).

However, studies in which the effect on fertility are determined and subsequent monitoring in the cycle of the insect are required. Considering other researches, in general, Neem Oil Spray and PHCs Neeem are more efficient than Neemazal-T/S and Neem Rose Defense EC (^{Von Elling et al., 2002}; ^{Chiasson et al., 2004}); while Azatin EC and Margosan-O, exhibit similar activity to the first. On the other hand, it is suggested that Nim 80 and Nim Oil (^{Gomez et al., 1997}; ^{Cubillo et al., 1999}), require lower concentrations than those evaluated in this research to produce similar effects.

Even though these products alone are not able to abate these populations, having a synergistic effect with some products of microbial origin such as entomopathogenic fungi, which can be used together, although its use should be subject to compatibility tests with natural enemies such as parasitoids, predators and pollinators (^{Morgan et al., 2009}; ^{Luna et al., 2011}; ^{Scudeler Santos, 2013}).