Introduction

Maize is infested in the field and during storage by various pest species. During storage the maize weevil, Sitophilus zeamais Motschulsky, 1885 (Coleoptera: Curculionidae), is the most important pest causing severe quantitative and qualitative losses. Larvae and adults feed internally in the seeds, causing losses in weight and quality and increasing infection by pathogens, which are harmful for human health, e.g., aflatoxins (^{Pacheco and Paula 1995}; ^{Hell et al. 2000}; ^{Fontes et al. 2003}).

Control of this pest is based on use of synthetic insecticide fumigants, e.g., aluminum and magnesium phosphide (inorganic precursor of phosphine) and contact products, e.g., fenitrothion and pirimiphos-methyl (organophosphate), bifenthrin and deltamethrin (pyretroid) and esfenvarelate (pyretroid) + fenitrothion (organophosphate) (^{Brasil 2016}). Because of their non-specific mode of action, acting on the central nervous system and on the energetic metabolism (Brazilian Committee of Action to Prevent Resistance to Insecticides - ^{IRAC-BR 2015}), these products can be deadly harmful to non-target organisms.

Hence, the search for viable alternatives of control to deal with this pest is more than just a reality, but a necessity. Besides, the lack of insecticides with alternative modes of action to be used in rotation for mitigating pest-resistance, this species has exhibited resistance to various synthetic insecticides (^{Ribeiro et al. 2003}). Thus, some botanical insecticides are already available including neem-based formulations, e.g., AzaMax^®, which is registered in the Brazilian Agriculture, Poultry and Supply Ministry (MAPA) for the control of field pests (^{Brasil 2016}). Additionally, other neem-based formulations, although not registered, are recommended to manage storage pests as is the case of Natuneem^®.

Azadirachtins, which make up the active ingredient (a.i.) of neem-based formulations since they are the most active and abundant phytochemical in neem and show action against at least 550 insect species (^{Gahukar 2014}), are considered to be selective to mammals based on their specific mode of action. They act on physiological processes inherent to insects, related to metamorphosis and ecdysis (^{Mordue and Nisbet 2000}). Furthermore, they are of special interest for use in the prevailing condition of the storage environment (darkness), since one of its main components which has known insecticidal activity, e.g. azadirachtin-A (makes up 73 % content of the azadirachtin), is light degraded after 2.47 days while covering foliar surfaces (^{Johnson et al. 2003}). To date, there is a lack of information concerning the efficiency of these formulations on control of storage pests, such as S. zeamais.

The first step to be taken when selecting alternative products for pest control is to define the discriminating dosages or concentrations (acute toxicity) that are effective in killing the pest. This is generally made through estimation of the LC₅₀ and LD₅₀, which are, respectively, the concentration and dosage that kill 50 % of the population. These estimations, when doubled, are expected to harm approximately 100 % of the population. The estimations are made on this range since there is a higher degree of reproducibility and they are more reliable while making assumptions for a population (^{Costa et al. 2008}). Thus, this study focused on estimating the LC₅₀ and LD₅₀ values of neem-based formulations in order to define the discriminating dosages and concentrations effective to control S. zeamais and comparing them to a contact synthetic insecticide used against the pest.

Material and methods

Results and discussion

The LC₅₀ -values for AzaMax^® and Natuneem^® were around 4 µL of formulation/g of grains while the LC₅₀ -value for Decis was 0.41 µL of formulation/g of grains (Table 1). This also means that these compounds were almost 10-fold (9.67 and 10.74) less toxic than deltamethrin (Table 2).

Topical application of neem-based formulations yielded LD₅₀ values of 51.32 and 76.76 µL of formulation/g of insects for AzaMax and Natuneem, respectively (Table 1). The LD₅₀ value for Decis was 42.75 µL of formulation/g of S. zeamais (Table 1), which was the lowest value observed in this study. Hence, it takes a dosage around 20 % and 80 % higher of AzaMax and Natuneem to cause the same mortality rate in S. zeamais while exposed to Decis (Table 2).

The differences found between the botanical and synthetic formulations were expected and agree with other findings (^{Brito et al. 2006}; ^{Dadang and Prijono 2009}; ^{Olaitan and Abiodun 2011}). They are attributed to the diversity in the nature of the products tested, i.e., neem-based products which are botanical insecticides versus deltamethrin which is a synthetic insecticide. The a.i. of synthetic insecticides such as delthametrin is more environmentally stable than any known botanical insecticide (^{Olaitan and Abiodun 2011}). Hence, the response of S. zeamais to higher concentrations of the botanical formulations can be associated with the ability of the effective concentration to withstand photo-decomposition (^{Johnson et al. 2003}), allowing for the remaining residues to show acute toxicity against the weevil. This pattern was maintained for both botanical formulations tested, strengthening this hypothesis.

Also, the differences in the mode of action may partially explain the differences found between the botanical and synthetic insecticides. Neem-based products are known to act mainly on growth and molting, also interfering on reproduction and cellular processes. These effects can increase mortality related to disruption of endocrine system controlling the described processes. Neem-based formulations also cause antifedant effects which are, however, a lot less pronounced in Coleoptera than in Lepidoptera (^{Mordue and Nisbet 2000}; Okweche et al. 2013). ^{Kavallieratos et al. (2007}) tested two azadirachtin formulations against adults of Sitophilus oryzae (Linné, 1763) (Coleoptera: Curculionidae) and found that further dosage increases and longer exposure times were needed to guarantee high rates of mortality. Deltamethrin based products, for instance, has a rapidly disabling effect on feeding insects and their death seems to be due to irreversible damage to the nervous system occurring when poisoning lasts more than a few hours (^{Rehman et al. 2014}).

The LC₅₀ of crude neem seed oil determined for S. zeamais infesting maize was found to be much lower than the values found herein, i.e., 1.46 mL of formulation/kg of seeds which is equivalent to 1.46 µL/gram of seeds (^{Nukenine et al. 2011}). However, because it was not a formulation but, instead, a crude extract or seed oil, such differences are again acceptable and confirm the potential of the tested formulations. Crude seed oil is not emulsified and hence may face an increase in individual contribution of toxic components in the final applied solution, what can act increasing toxicity. Some emulsified concentrate contains only 32 % of neem seed oil (^{Pandiyan 2011}) which is far lower than the content of crude oil (~100 %).

Concerning the differences in the LC₅₀ and LD₅₀ between the two botanical insecticides, AzaMax and Natuneem, the former was 1.11 and 1.50 more toxic than the latter, although the only significant differences found according to the 95 % CI was between the LD₅₀ values of AzaMax and Natuneem (Table 2). This result agrees with that found by ^{Brito et al. (2006)} who described that Natuneem was less toxic to adult females of Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) when topically applied than all the other tested formulations (Neemseto, Callneem and the extract of seeds powder).

Considering that AzaMax has a known amount of a.i. (azadirachtin A/B) in the commercial product while in Natuneem that is not specified, the differences in toxicity due to topical application might be caused by the differences in the concentration of the a.i. between the two formulations. In that matter, ^{Gahukar (2014)} mentions in his revision that the LC₅₀ values of five neem-based formulations tested against three major tea pests (thrips, tea mosquito bug and leaf hoppers) decreased as the azadirachtin content in the formulations increased. Also, the toxicity may vary according with the components of the formulation or used in the extraction (^{Mansour et al. 1987}) and with the part of the plant used in the formulation. For instance, ^{Mansour et al. (1993)} have tested three neem formulations against phytophagous mites and have found that the only truly acaricidal formulation among the three tested was the one having the lowest content of azadirachtin. The authors attributed such effect to the extraction of neem seed kernels with lipophilic solvents resulting in much higher oil content in the final formulation of this product. Therefore, final products possessing higher oil content act as a contact poison particularly against mites and soft bodied insects once in such formulations the variation on bioactivity can be driven from a great variation in azadirachtin content (^{Gahukar 2014}). Since both tested formulations were extracted from the seeds/kernels and then were oily, that might also account for the differences found in acute toxicity of topically applied formulations, although S. zeamais is not a soft bodied insect.

The higher values of the LD₅₀ compared to the LC₅₀ found in our study, while testing the same botanical formulations, can be explained by the increased exposition of S. zeamais in the tests performed to estimate the LC₅₀ (contact and ingestion) in comparison to the contact exposure alone (topical application). Similar differences were found by ^{Scoz et al. (2004)} in tests with fruit flies. Furthermore, the mode of action of neem-based products is diversified and incremented by ingestion (exposure of both insects and food) when compared to contact exposure alone (topical exposition) (^{Mordue and Nisbet 2000}) and this also contributes to explain the differences found concerning the LC₅₀- and LD₅₀-values. In addition, according to ^{Gahukar (2014)} azadirachtin-based products are mostly stomach poisons and when applied at higher doses, exhibit contact toxicity what also account for the differences found.

In spite of the numerical differences in the LC and LD’s values among the insecticides, the only significant differences found, according to the 95 % CI of the LCR and LDR, are those seen between the LC₅₀ values of AzaMax and Decis and Natuneem and Decis; the LD₅₀ values of Natuneem and Decis and the LD₅₀ values of AzaMax and Natuneem (Table 2). However, higher numerical values have some other implications, including economic issues.

Taking into account only the cost to treat maize and to obtain effective control of the pest, it would be much cheaper to control S. zeamais using a synthetic formulation of deltamethrin. This is the case because the effective concentration to manage S. zeamais with deltamethrin was approximately 10 times lower and the formulation is also cheaper (~US$ 25.98 per liter) compared to the doses required and prices paid for a neem-based formulation. However, other non-economical issues should be taken into consideration while selecting an insecticide and these are related to the lasting residues (^{Gahukar 2012}) and relative selectivity to some non-target organisms (^{Ziaee 2014}). This approach is especially important when seeking alternatives concerning the few options available to manage storage pests and when the known alternatives we are aware of, to manage insecticide resistance, seem to have no effect on this pest (^{Oliveira et al. 2005}). The neem-based formulations can thus be considered as alternative options. Concerning the two botanical formulations tested, it would be cheaper to manage S. zeamais with Natuneem, since despite the fact that the effective concentrations and doses to kill S. zeamais are lower for AzaMax, the latter is more expensive (~R$ 82.44 per liter) than the former (~US$ 22.56 per liter). However, AzaMax is already registered on the MAPA while Natuneem is not, which may restrict availability of this formulation on the market.

Mortality as an estimation of the acute toxicity may only be a partial measure of the deleterious effects of these products. The dynamics of botanical insecticides in the environment that they are used, mainly with respect to rapid degradation (^{Johnson et al. 2003}), also indicates sublethal effects in the physiology and behavior of pests; this is the reason why a lower concentration, even without the ability to cause any mortality, can harm a population (^{Jafarbeigi et al. 2014}) by causing deterrent effects, diminishing food intake and frass production, retarding larval and pupal development, pupal weight and adult emergence (^{Li et al. 2003}). Hence, such effects should be considered in future works for those concentrations that do not cause mortality.

Conclusions

S. zeamais can be managed with both neem-based formulations by using a doubled LC₅₀ and LD₅₀ which result in the following values: 8.02 and 8.92 µL of formulation/ g of seeds and 102.64 and 153.52 µL of formulation/g of insects for AzaMax and Natuneem, respectively. Since there were no significant differences between the LC₅₀-values of the botanical insecticides, both of them can be used to manage S. zeamais infesting stored corn. However, it would cost less to manage the pest with Natuneem. The LC_50- and LD₅₀-values of the synthetic insecticide (Decis^® 25 CE) against S. zeamais are lower than those found for neem-based formulations and in order to manage the pest with this formulation (cause mortalities rates close to 100 %) it takes 0.82 µL of formulation/g of seeds and 85.5 µL of formulation/g of insects. Such LC₅₀ values found in our study would be equivalent to use 8.02, 8.92 and 0.82 L of AzaMax, Natuneem and Decis/ton of seeds.