Biological control uses antagonistic living organisms, predators of pests such as nematodes, arthropods, as well as parasitoids, corn borer, wasps sympiesis virudila, Macrocentrus cingulum), tachin fly Lydella thompsoni, Parasitoids of aphids, Wasps aphidin , etc. (Anonymous 2, 2014; Christine & Josée , 2009).

This fight against pests of crops and harvests is mainly done through synthetic insecticides, approved or not. Among them: organochlorines, whose active ingredients can be Dichloro-Diphenyl-trichloroethane (DDT) and other chlorinated derivatives; organophosphates, the active ingredients of which may be malathion, phosmet, pirimiphos-methyl, pirimiphos-ethyl, parathion-methyl, etc. ; carbamates having among others as active ingredients: bendiocarb, carbarl, methi ocarb, etc. insecticides from the pyrethroid family may have as active ingredients: acrinathrin, allethrin, bromethrin, etc. neonicotinoids include clothianidin, dinotefuran, imidacloprid, etc. (Anonymous 2, 2014). However, according to the World Health Organization (WHO), few years ago, synthetic pesticides are the cause of the death of 200,000 people each year around the world, and in Africa these risks are increased. The United Nations Environment Program (UNEP) also notes that in sub-Saharan Africa, the potential cost of treating diseases linked to pesticides between 2005 and 2009 was estimated at 6.3 billion; and that of 2005 and 2020 was estimated at 90 billion dollars (Aggrey, 2017; Anjarwalla et al, 2016). On August 26^th 2024, all the Cameroonians’ medias have reported on a tragedy where six persons died after eating maize crops which had been treated with synthetic pesticides.

For a long time, there was no comprehensive scientific study aimed at beetle insects such as S. Zea mays. This is due to the fact that it is in tropical countries and particularly in Africa, where research was not developed that these insects are big threats, because the climate of western and rich countries is not favorable to them. Note, however, the studies by Tamgno & Ngamo (2014) who worked on the use of Neeem ( A. indica) as alternative to synthetic insecticides for the protection of maize and sorghum seeds in the logone valley. Diabate et al. (2020), worked on the toxicity of aqueous extracts of A. indica and Jatropha curcas on Plutella Xylostella by contact. Furthermore, the Ministries of Agriculture of certain West African countries such as Benin, have recommended the informal use of A. indica extracts to farmers as a Biopesticide (Sikirou et al., 2020). In Cameroon, there is almost no usable data on this subject. Our study provides additional scientific informations to that field. This will convince skeptics and provide assurance for the effective use of A. indica as a Bioinsecticide. 

We face a double problem which is that of the invasion of insect pests of crops, and storage areas such as maize weevils, as well as damage to the health of consumers and the environment, due to synthetics pesticides or insecticides used in the fight against harmful organisms. The urgency of the development of natural insecticides or plant-based Bioinsecticides is signaled and proves to be a strategic element in the quest for large production, food security and environmental protection.

The question is to know how can we effectively control pests such as weevils which affect plants and stored foodstuffs such as maize, without polluting the environment or harming the health of consumers? And the research hypothesis we are working on is that: the toxic effects of Azadirachta indica (Neem)-based bio-insecticide extracts can effectively control corn weevils, while helping to reduce environmental pollution by replacing the synthetic insecticides used. So, our general objective is "to study the toxic effects of oil and powder extracts from the seeds of A. indica on S. Zea mays (Coleoptera: Curculionidae) of maize in storage. The specifics objectives were to evaluate the repellent capacity of Azadirachta indica seed oil extract on corn weevils in storage, its efficacy on maize weevils in storage, and the contact efficacy of seed powder extracts on maize weevils in storage

II-MATERIALS AND METHODS

 II.1. Material

II.1.1. Study site

The main site of the study is the Center for Research on Medicinal Plants and Traditional Medicine of the Institute of Medical Research and Medicinal Plants studies (CRPMT-IMPM) of Ngoa-ékelle in Yaounde. Samples of A. indica were collected at Mokolo market in Yaoundé (Cameroon) and S. Zea mays at Mfoundi market. The manipulations at the CRPMT-IMPM were carried out in particular in the laboratory of botany and traditional medicine for the repulsion and contacts assays of the A. indica extracts on S. Zea mays and the laboratory of Galenics and Pharmaceutical Technology for the pressed oil sample. machine. The experiment was done in ambient average temperature of about 25 ° C.

II.1.2. Biological material.

The animal material used was S. Zea mays, and the plant material, included A. indica s and Zea mays seeds (figure 1). Samples of S. zea mays which are of the genus Sitophilus, of the order of Coleoptera and of the family of Curculionidae were collected at the Mfoundi market in Yaoundé, in untreated maize waste (stalks, bran), infested and sorted before the sale.

The shelled kernels and seeds of A. indica A. juss, were purchased at the Mokolo market in Yaoundé, from the northern part of Cameroon.

The North Cameroon white maize seeds used were purchased at the Mfoundi market in Yaoundé.

Figure 1: Biological Material A1&A2: Seeds and Kernels of Azadirachta indica; B: Sitophilus Zeamais (Scale: 5); C: Maize in storage.

II.1.3. Equipment and others.

Equipment used:

The manual crushing machine, the manual winepress machine, 1-liter glass jars, venti-line brand oven, syringes, filter paper, petri dishes, glass jars, gangs, sampling tubes, sensitive balance, camera, cotton, acetone, etc.

Figure 2. Equipment. A: sensitive equilibrium; B: Oven; C et D: Sitophilus zeamais rearing device; E: syringes; F: Neem oil; G: petri dishes; H: glass jar; I: oil press

 

II.2. Methods

                This study is an experimental study, carried out in the laboratories mention above.

II.2.1. Collection and breeding of corn weevils.

 The samples of S. zeamais were collected from a maize seller's store at the Mfoundi market in Yaoundé. They were collected with cob debris and maize bran containing a few handfuls of seeds, in a bag (Nyetam, 2022). Subsequently, they were transposed into a bucket covered with a transparent fabric, then into glass jars, as they approached use. Breeding was therefore done in mass.

II.2.2. Choice and packaging of the maize variety.

The variety chosen is white maize from North Cameroon (large and small grains) found in our markets, for breeding and experimentation. Before the experiment, we stored our seeds in clean glass jars. They were put in the refrigerator three days before the experiment in order to kill the germs, then removed and dried (Boeke et al., 2001).

II.2.3. Preparation of Azadirachta indica seed extracts.

The almonds previously dried at ambient temperature are put in an oven at 60 °C, during twelve hours. A quantity of 843 g of A. indica seed almond powder is obtained after molding 860 g of dried seed almonds using a manual mill, shortly before the extraction process and shortly before the various necessary tests requiring the powder (Nyetam, 2022).

800 g of crushed A. indica seed powder are introduced into a makeshift cylindrical press with a grid and cotton at its base, then pressed so as to drop its oil into a container placed below.

Efficiency (% ) =(Mass of extract (g) Mass of plant material (g) X100

II.2.4. Effectiveness of A. indica seed extracts on weevils.

The repulsion test or extract avoidance test is used to determine whether weevils, given the choice, are able to detect the extract from A. indica seeds and sidestep it.

The repellency assay is used to calculate the percent repellency of seed oil on corn weevils by the preferential zone method described by Mc Donald et al. (1970). Indeed, the discs of whatman filter paper n° 2 of 9 cm in diameter were divided into two equal parts of 31.79 cm² of surface (Fig. 3). Four concentrations were prepared by dilution in 0.5 ml acetone, of the following seed oil volumes: 100, 200, 300 and 400 µl. The solutions thus obtained were homogenized by manual stirring and produced on one half of the disc (using an insulin syringe). The other half, treated only with 0.5 ml of acetone, served as a control. Ten minutes were then allowed to elapse, the time for the solvent to evaporate completely. The following oil concentrations were thus obtained (volume/area): 3.14 µl/cm², 6.29 µl /cm², 9.43 µl /cm² and 12.58 µl /cm². The filter paper discs were reconstituted depending on the treated halves and the control halves using clear adhesive tape and placed in petri dishes (fig.3). Fifteen adult weevils, just taken from their rearing medium, were placed in the center of each paper, and the petri dishes covered. Four replicates were performed for each oil concentration. The counting of weevils on each half of the disc was carried out after 1 hour, 2 hours, 3 hours and 4 hours, of treatment under laboratory conditions. The percentage repellency is calculated according to the formula used by Nerio et al. (2009) as follows:

PR(%) =Nac- Nh(Nac+Nh)X100

PR= Percentage of repulsion

Nac= number of weevils present on the half-disc treated with acetone.

Nh= number of weevils present on the half-disc treated with the dose of oil.

Figure 3 : Repellency test.

Average repellency percentage is calculated and assigned to the different repellent classes according to the classification of Mc Donald et al (1970), represented by the following Table I:

Table I. Repellent classes according to the classification of Mc Donald et al (1970).

Repellent classes	Repulsion intervals (%)	Properties
Class 0	< 0,1	Not repellent
Class I	0,1-20	Very weak repellent
Class II	20,1-40	Weakly repellent
Class III	40,1-60	Moderately repellent
Class IV	60,1-80	Repellent
Class V	80,1-100	Very repellent

Mc Donald et al., 1970.

Volumes of 100, 200, 300, 400 µl of A. indica oil are prepared by dilution with 1ml of acetone and mixed with 30 g of corn contained in 1000 ml glass jars. The whole was homogenized by manual stirring and left for 5 to 10 minutes for complete evaporation of the solvent. The respective concentrations of 3.33; 6.66; 9.99 and 13.33 µl/g were obtained. Then, 15 adult insects were introduced into each jar, and the latter was covered with porous fabric held in place with elastic ties. The control dishes were prepared under the same conditions and treated only with acetone (1ml). Four tests of 15, 15, 10, 10, insects, are carried out per dose and for the control, the results expressed for an average of 25 insects. The jars are stored under ambient conditions in the laboratory.

Fifteen adult maize weevils are introduced into transparent polystyrene jars containing 30 g of white maize seeds. These are treated with different doses of A. indica seed powder.

Four doses of A. indica seed powder (1, 5, 10, 15 g) are used for 30 g of maize seeds; i.e. 3.33 respectively; 16.67; 33.33 and 50 % of the weight of the seeds, control batches (0%) are carried out in parallel with untreated seeds. Four repetitions are carried out for all the doses and for the control. The jars are stored under ambient conditions in the laboratory.

Dead insects were counted every 24 hours during a period of the experiment. The mortality rate was assessed through the ratio of the number of dead insects to the number of insects initially introduced according to the following formula.

MR(%)=NCMNCIX100

With, MR (%): Mortality rate; NCM: number of dead weevils; NCI: number of weevils introduced.

Mortality was calculated and corrected according to Abbott's formula taking into account natural mortality (Mt) observed on the control (Abott, 1925, cit. Ambela minkeng, 2018).

Mc=Mo-Mt100-Mtx100

Mc: corrected mortality; M0: mortality of the sample tested; Mt: mortality in the untreated control.

And the LD₅₀ or CL₅₀ of Neem oil and powder were estimated according to the time of exposure of the insects to the different doses, using the smallest dose whose mortality rate is closer to 50 % with the rule of three or by plotting a standard curve giving the variations in mortality as a function of the increasing concentrations of the products.

 

 

II.2.5. Statistical analysis of data

Data were measured or calculated for each response variable at the test level (percentages of mortality, repellency, etc.). The raw data were visually explored to check for consistency, entered into MS-Excel, and then statistically analyzed according to the Mc Donald et al.  (1970) classification. For these statistical analysis, computer software called graph pad Prism and Excel were used.

III. RESULTS

III.1. Extraction Yield.

With 860 g of dried almonds, we obtained 843 g of powder, i.e. a yield of 98.02 % and by introducing 800 g to the press, we had 56.5 g of oil, with a concentration of 1.3 g/ ml, i.e. a yield of 7.06 %, and a quantity of approximately 44 ml of A. indica oil. This yield is lower than what would have been obtained by solvent extraction, but the raw and natural character of the extract obtained could have advantages in activity and is easily assimilated to the oil extracts used by the farmers (Table II).

Table  II. Extraction Yield

Excerpt name	Masses of plant material	Mass of the extract obtained	Yield
Powder	860g (almonds)	843 g	98,02 %
Pressed oil	800g (Powder)	56,5 g	7,06 %

 

III.2. Effectiveness of A. indica seed extracts on weevils.

-Depending on doses

At the dose of 200 µl (6.29 µl/cm2), the difference in the rate of repulsion is significant with the other doses; Neem oil is very repellent at 200 µl, while it is moderately repellent at doses of 100 µl (3.14 µl/cm²), 300 µl (9.43 µl/cm²), 400 µl (12.58 µl/cm²). Because, we note that at 300 µl and 400 µl, its effect is no longer only repellent, but also lethal or paralyzing. the insects which venture into the part of the disc treated with A. indica oil, are paralyzed, unable to flee and thus find death. The overall repellency average is 62.25 %(Tables III).

 

 

Table III. Repellent effect of oil extracts from A. indica seeds on weevils depending on amounts

The amount of oil	Number of individuals		Average percent repellency	Effect	repellency class
	In the untreated part (Nac)	in the treated part(Nh)
100µl (3,14µl/cm2)	80	20	60 %	Moderately repulsive	III
200µl (6,29µl/cm2)	94	6	88 %	very repulsive	V
300µl (9,43µl/cm2)	77	23	54 %	Moderately repulsive (but kills)	III
400µl (12,58µl/cm2)	74	26	48 %	Moderately repulsive(but kills°	III

 

-Depending on duration

No matter the concentration, the Neem oil is repellent toward S. zeamais after 2 hours, and Moderately repulsive during the first two hours (Table III). A. indica oil is Repulsive of class IV (Table IV).

 

Table IV. Repellent effect of oil extracts from A. indica seeds on weevils depending on durations.

	Duration	Average percent repellency	Repellency class	Effect
Azadirachta indica oil	1h	56	III	Moderately repulsive
	2h	56	III	Moderately repulsive
	3h	64	IV	Repulsive
	4h	74	IV	Repulsive
	Mean	62,5±8,54	IV	Repulsive

 

The contact of maize treated with different doses of A. indica oil, with S. Zea mays  [Coleoptera: Curculionidae], shows that the mortality rate increases with the duration of contact and reaches its maximum on the 4th day for the 100 µl dose. From the 1st day, mortality reaches 100% at the dose of 400 μl and approaches this maximum for the doses of 200 μl and 300 μl, because there is no significant difference between their mortality rates, from the 1st to the 4th. day. The effectiveness of the 300 µl dose does not increase on the 2nd and 3rd day, compared to the 200 µl dose which proves to be slightly more effective during this period and compared to the 400µl dose, the effectiveness of which is maximum from the first 24 hours. The DL₅₀ or CL₅₀ decreases when the exposure time increases, and varies from 94.62 µl during o1 day to 55.11 µl when 4 days are needed to kill fifty percent of Sitophilus zeamais (Table V&VI).

 

Table V. Contact toxic effects of Azadirachta indica seed oil on maize weevils

Duration	Corrected number of deaths and cumulative mortality rate as a function of dose and time
	0 µl	100 µl		200 µl		300 µl		400 µl		1g Sinograin
		Deaths	Rate	Deaths	Rate	Deaths	Rate	Deaths	Rate	Deaths	Rate
24h	2	13,21	52,84%	21,42	85,68%	21,42	85,68%	23,46	93,84%	23,46	93,84%
48h	2	20,40	81,6%	22,44	89,76%	21,42	85,68%	23,46	93,84%	23,46	93,84%
72h	2	21,42	85,68%	22,44	89,76%	21,42	85,68%	23,46	93,84%	23,46	93,84%
96h	3	22,68	90,72%	22,68	90,72%	22,68	90,72%	22,68	90,72%	22,68	90,72%
MOY	2,25 ±0,5	19,62 ±4,46	77,71% ±0,16	22,24 ±0,56	88,98% ±0,02	21,73 ±0,63	86,94% ±0,02	23,52 ±0,12	93,06% ±0,01	23,52 ±0,12	93,06% ±0,01

 

Figure 3: Corrected number of deaths as a function of dose and time

Table VI. LC₅₀ of Neem Oil as function of time

	24 h	48 h	72 h	96 h
DL50 ou CL50	3,15 µl/g	2,05 µl/g	1,9 µl/g	1,83 µl/g

Figure 4: LC₅₀ of Neem oil as function of time

 

The contact of maize treated with different doses of A. indica powder, with S. Zea maïs [Coleoptera: Curculionidae], shows that the mortality rate increases with the duration of contact and reaches its maximum (100 %) on the 3rd day for the dose of 5 g, 10 g and 15 g. however, the 5g dose proves to be the most effective on day 1 (93.33 % versus 46.66 %, 80 % and 86.66 %, respectively for 1 g, 10 g and 15 g). the LC₅₀ varies from 1.07 g (35,66 g/kg) for 1 day to 0.53 g(17,66 g/kg) for 2, 3 or 4 days. The 5 g (166,66 g/kg) dose is the LC₁₀₀.

Table VII. Contact toxic effects of Azadirachta indica seed powder on maize weevils

Duration	Corrected number of deaths and cumulative mortality rate as a function of dose and time
	0g	1g		5g		10g		15g		1g Sinograin
	M	1M	MR	M	MR	M	MR	M	MR	M	MR
24h	0	7	46,66%	14	93,33%	12	80%	13	86,66%	15	100%
48h	0	14	93,33%	14	93,33%	12	80%	13	86,66%	15	100%
72h	0	14	93,33%	15	100%	15	100%	15	100%	15	100%
96h	0	14	93,33%	15	100%	15	100%	15	100%	15	100%

 

 

Table VIII. LC₅₀ of Neem powder as function of time.

	24 h	48 h	72 h	96 h
DL50 ou CL50	35,66 g/kg	17,66 g/kg	17,66 g/kg	17,66 g/kg

 

 

 

 

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LIST OF ABREVIATIONS

 

LC50	:	Lethal mean Concentration killing 50 % of the population.
CRPMT	:	Center for Research on Medicinal Plants and Traditional Medicine.
DDT	:	Dichloro-Diphényl-Trichloréthane.
LD50	:	Lethal mean Dose killing  50 % of the population.
FAO	:	Food and Agricultural Organisation of the United Nations.
IMPM	:	Institute of Medical Research and Medicinal Plants studies
IRAD	:	Institut de Recherches Agricoles pour le Développement (Institute of Agricultural research for development).
IRAM	:	Institut de Recherches et d’Applications des Méthodes de Développement (Institute for research and application of development methods).
IRDA	:	Institut de recherche et de développement en agroenvironnement(Institute for reseach and development in Agro-Environment).
MINADER	:	Ministry of Agriculture and rural development.
MINEPAT		Ministère de l’économie, de la planification et de l’aménagement du territoire
Mc	:	Corrected mortality.
MR(%)		Mortality rate
OECD		Organisation for economic cooperation and development.
SDG	:	Sustainable development goals.
PR	:	Percentage of repulsion.
SND30	:	National development Strategy 2020-2030.
UNEP		United Nations Environment Program.

 

 

 

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