Effects of Botanical Extracts on Foraging and Pollination Activity of Apis Mellifera (Hymenoptera: Apidae) on Glycine Max (Fabaceae) Flowers at Bini (Ngaoundere, Cameroon)

ABSTRACT


INTRODUCTION
Glycine max is an annual herb that can reach 110 cm in height at maturity.The alternate leaves are compound and generally formed of three leaflets (Gallais & Bannerot, 1992).The inflorescence is a cluster bearing 5 to 35 white, yellow or red flowers.The nectar and pollen produced by these flowers attract insects (Milfont et al., 2013).The seeds are widely used in human food and play a beneficial role in human and animal health (Sherif, 2013).It consists of over 33% protein and carbohydrates, and excellent amounts of dietary fiber, vitamins and minerals.It also consists of 18% oil, making it an important crop for the production of edible oil (Inoussa et al., 2020).In Cameroon, the demand for G. max seeds was quick to take off ; however, this dynamics is scientifically poorly documented apart from sparse statistics (Nzossié & Bring, 2020).The decline in productivity would be linked particularly to the physico-chemical and biological degradation of the soil (FAO, 2018) and especially to the pressure exerted by pests on crops.To deal with this situation, stakeholders need to adopt sound management of insect pests and pollinators (Adamou et al., 2022;Mohammadou et al., 2023).Unfortunately, pollinator management is ignored by most African farmers (Eardley et al., 2010); because the management of these arthropods is not generally included in the agricultural programs of several countries on this continent, despite their positive role in food security (Fontaine et al., 2006a(Fontaine et al., , 2006b;;Vamosi et al., 2006).In effect, to increase yields and meet everincreasing market demand, farmers resort to synthetic pesticides that have caused more damage than they have resolved (Bambara & Tiemtoré, 2008).In Cameroon, farmers use pesticides on crops without consideration for their effects on bees who are exposed to them through direct contact with spray residues on plants, through ingestion of contaminated pollen and nectar, or through exposure to nesting sites or contaminated materials.These pesticides affect the physiology, behavior and reproductive potential of the bee (Baskar et al., 2017), which results in sub-optimal agricultural production.Many plants through their extracts provide natural insecticides and can therefore be used as a substitute for synthetic chemical insecticides (Tembo et al., 2018;Kosini et al., 2021;Adamou et al., 2022;Mohamadou et al., 2023).In fact, insecticides of plant origin are more biodegradable (Bakkali et al., 2008;Suthisut et al., 2011a,b) and present, with a few exceptions for certain pure compounds, low toxicity for mammals (Regnault-Roger et al., 2012, El-Wakeil, 2013) and beneficial arthropods (Tembo et al., 2018).Aqueous extracts of Callistemon rigidus, Lippia multiflora and Plectranthus glandulosus have been tested effective in the field against cowpea pests, with a yield improvement varying between 120 and 260% (Kapeuhag et al., 2021).Organic extracts from the leaves https://journals.e-palli.com/home/index.php/ajasetAm.J. Agric.Sci.Eng.Technol.7(3) 37-46, 2023 of these plants have also been successfully tested against post-harvest pests (Danga et al., 2015;Dessenbe et al., 2022).Since these plants are available in rural areas, they constitute an effective, accessible and inexpensive means of combating legume pests.Although some research has been conducted on the impact of the use of pesticidal plants on non-target arthropods (Mkenda et al., 2015;Tembo et al., 2018;Adamou et al., 2022;Mohamadou et al., 2023), this remains a neglected area of research that requires further investigation to understand possible trade-offs of using more plant-based pesticide products.The present investigation made it possible to evaluate the impact of certain biopesticides (aqueous extracts of three cosmopolitan plants Callistemon rigidus, Lippia multiflora and Plectranthus glandulosus) on beneficial soybean arthropods, including Apis mellifera.

MATERIAL AND METHODS Study Site and Biological Material
The works were carried out from May to August 2019 and from May to September 2020, in Bini (Ngaoundéré) in the Adamawa Region, Cameroon.Cosmos sulphureus, Helianthus annuus, Tithonia diversifolia, Phaseolus vulgaris, Zea mays, Ipomea batatas, Arachis hypogea and Abelmoschus esculentus were the most frequently observed plants.According to Djoufack et al. (2012), the climate of this region is of the Sudano-Guinean type, mild and cool, characterized by two seasons: a rainy season (April to October) and a dry season (November to March).The average annual temperature is 22°C and the average annual humidity is 70% (Amougou et al., 2015).The experimental field was an area of 1064 m2 centered on a point of latitude: 07°24'33.9''N;longitude: 013°32'53.6''E;altitude: 1087m.The seeds of G. max (variety HOULA 1, 120 days) sown were purchased at the station of the Institute of Agricultural Research for Development (IRAD) in Garoua.The biopesticides were produced from the fresh leaves of Callistemon rigidus, Lippia multiflora and Plectranthus glandulosus, harvested in the Adamaoua region, respectively at Bini, on the cliff and at Nyanbaka.The latter were identified at the National Herbarium of Cameroon by comparison with the samples registered under the numbers 18564/ SRFCam, 9051/SRFCam, 60652/HNC, respectively for C. rigidus, L. multiflora and P. glandulosus.

Experimental Device, Sowing and Maintenance of the Culture
The experimental plots were cleared, plowed and landscaped according to the procedure of the completely randomized block device with four repetitions for each experimental unit.Each experimental plot consisted of 44 experimental units of 4 x 3.5 m spaced from each other by one meter furrows.On May 1, 2019 and May 22, 2020, sowing was done in rows, with 6 rows of 10 pockets each and three seeds per pocket.The spacings were 36.36 cm on the lines and 50 cm between the lines.From germination (May 7, 2019 andMay 29, 2020) to the appearance of the first flowers (June 27, 2019 andJuly 19, 2020), weeding was regularly carried out with a hoe, every two weeks.From flowering to pod maturation, weeding was done by hand.

Process for Obtaining Biopesticides
The middle fresh leaves of C. rigidus, L. multiflora and P. glandulosus harvested in the morning, were cleaned and dried separately in the shade.After drying, they were pounded separately using a wooden mortar, then sieved with a 0.2mm mesh sieve and stored in dry plastic bags tightly closed in a refrigerator at 4°C until use for extractions.For each of the powders derived from plants, 75, 150 and 225g were weighed using a CAMRY brand electronic scale (accuracy 0.01g) and introduced separately into a container containing 1.5 L of water to obtain the concentrations 5, 10 and 15% respectively.After stirring and maceration for 24 hours, the mixtures were sieved using a 0.2 mm mesh sieve.

Insecticide Treatment
The experimental units were treated with two types of insecticides: synthetic pyrethroid, Decis (Deltamethrin 15g/L) used as reference and three concentrations (5%, 10% and 15%) of aqueous extracts of the three species of plants (C.rigidus, L. multiflora and P. glandulosus) tested in this study.The experimental units were distributed in a completely randomized design with four repetitions: 1 control x 3 plants x 3 extracts x 3 concentrations x 1 reference insecticide (Decis) x four groups of flowers (group 1:120flower buds whose open flowers have been left free to pollinate, group 2: 120 flower buds whose open flowers have been protected from insects using gauze bags, group 3: 200 flower buds whose open flowers have been protected, then uncovered for 10 minutes for an exclusive visit by A. mellifera before being protected again, and group 4: 100 flower buds with open flowers protected, then uncovered for 10 minutes and protected again, without visit insects or any other organism).The spraying of insecticides was done using a manual sprayer at sunset, five weeks after sowing, and repeated every 7 days until the last flower faded.The extracts and the reference insecticide (recommended dilution is 3mL/15L of water) were applied at the rate of 268L/ha and 2.1L/ ha respectively.

Data collection
At the flower bud stage, the flowers were grouped as described above and labeled.The observations for the determination of the biological diversity of the anthophile insects of G. max were made on the flowers left in free pollination.Observations were made every day, during five time slots: 8 -9 a.m., 10 -11 a.m., 12 -1 p.m., 2 -3 p.m. and 4 -5 p.m.The number of morphologically identical insects was recorded.A recognition code was assigned to each visiting species.Two to five insects of each species, depending on their abundance, were captured using a sweep net and preserved in 70% ethanol, except for Lepidoptera, which were preserved in foil for later identification (Borror and White, 1991).Am.J. Agric.Sci.Eng. Technol. 7(3) 37-46, 2023 Insects not having been marked, the cumulative results of these counts were expressed by the number of visits (Tchuenguem et al., 1997).At the end of the investigations, the insects were identified using insect keys (Delvare and Arbelenc, 1989;Borror and White, 1991;Eardley et al., 2010;Gourmel, 2014).At the same time, the durations of the pollen collection visits and those of the nectar collection were recorded separately according to the different insecticide treatments, using a stopwatch.The density of foragers (Tchuenguem & Népidé, 2018) and browsing speed (Jacob Remacle, 1989) were recorded for the different treatments.The number of blooming flowers, the temperature and the ambient humidity were recorded during each time slot.The last two parameters were recorded using a portable thermo-hygrometer, installed in the shade.At maturity, the pods were harvested and counted.

Impact of the Insect's Pollination Including Apis Mellifera and the Single Visit of Apis Mellifera on the Podding Rate of Glycine Max
The evaluation of the effect of insects including A. mellifera on the podding rate of G. max was based on the impact of flowering insects on pollination and the impact of pollination on G. max fruiting of treatments 1 and 2 (2019) and 3 and 4 (2020).For each year, the podding index (Pi) was then calculated according to the formula of Tchuenguem et al. (2001): Pi = Fb/Fa, Where Fa is the number of viable flowers initially set and Fb the number of pods formed.
For each year of investigation, the podding rate due to flower insects (Pi) is calculated using the formula of Diguir et al. (2020) : Pi = {[(FX -FZ)/(FX + FY -FZ)] * 100} Where FX, FY and FZ are respectively the podding rates in treatments X (flowers left free to pollinate), Y (flowers protected from insects) and Z (flowers protected, uncovered then protected again, without visiting insects).For one treatment, the podding rate (P) is : Pr = (Fb/Fa)*100, (Tchuenguem et al., 2001).The podding rate due to A. mellifera (Pr) was calculated using the formula of Diguir et al.

Data Analysis
Data were analysed using descriptive statistics, student's t-test for the comparison of means of the two samples, Pearson correlation coefficient (r) for the study of the association between two variables and chi-square (χ2) for the comparison of percentages.The transformed data was subjected to the ANOVA procedure of SPSS 16.0.Tukey's range test (DSH) was applied for the separation of means at the 5% probability threshold.Microsoft Excel software, version 2021 was also used.

Floral Products Harvested
During each flowering period, A. mellifera was seen harvesting intensively and exclusively nectar on G. max flowers.
Figure 1: Apis mellifera harvesting nectar on Glycine max flower

Relationship between Visits and Flowering Stages
The visits of A. mellifera were more numerous on treatment 1 and 3 when the number of opened flowers was high (Fig. 2A and B).The correlation was highly significant between the number of G.

Daily Visits
The honey bee were registered on G. max flowers between 8 a.m. and 3 p.m. with a peak in visits between 10 a.m. and 11 a.m., except for the sub-plots with aqueous extracts of C. Rigidus (15%), L. multiflora (15%) and P. glandulosus (15%) whose peak visits were between 12 p.m. and 1 p.m..The bee activity period coincided with the opening flowers of this Fabaceae.

Influence of Neighboring Floral
During each observation periods, flowers of many other plant species surrounding the study area were visited by A. mellifera, for nectar and / or for pollen.Among these plants were : Zea mays (Po), Vigna unguiculata (Ne), Phaseolus vulgaris (Ne), Ipomea batatas (Ne, Po), Solanum nigrum (Ne, Po), Arachis hypogea (Ne) and Richardia brasiliensis (Po).

Influence of Wildlife
The foragers of A. mellifera were disturbed in their foraging activity by biotic factors such as other arthropods that were either by competitors for nectar and/or pollen and abiotic factors like wind, rain and temperature.These disturbances resulted in the interruption of some visits.In 2019, for 1084 visits, 21 (63.64%) were interrupted by A. mellifera, 9 (27.27%) by Eurema sp. and 3 (9.09%)by Formicidae (1 sp.).While in 2020 for 721 visits, 19 (70.37%) were interrupted by A. mellifera, 7 (25.93%)by Xylocopa

DISCUSSION
Among insect species recorded on soybean flowers, Apis mellifera was the majority with a cumulative frequency of visits of 94.65% for the two years of investigation.Thus, the main anthophilic insects of G. max in Ngaoundéré belong to the order Hymenoptera, with the dominance of the family Apidae.This result corroborates that obtained by Milfont et al. (2013) that A. mellifera is the most abundant anthophilous insect of G. max.However, the diversity and abundance of anthophilous insects of this Fabaceae may vary with regions.Because, contrary to our results and those obtained by other researchers, the Halictidae family was more abundant on the flowers of G. max in the Far North of Cameroon (Tchuenguem & Dounia, 2014) and in the Littoral Cameroon (Taïmanga & Tchuenguem, 2018).
From our observations in the field, during each flowering period and in all sub-plots treated with aqueous plant extracts or not, A. mellifera visited the flowers of G. max and intensely and exclusively collected nectar.The number of visits from A. mellifera for this harvest was proportional to the number of blooming flowers.This result highlights the good attractiveness of the floral nectar of G. max vis-à-vis A. mellifera, independently of the treatment.
Attractiveness is also linked to the abundance of floral products, which can vary depending on the number of blooming flowers.The various insecticide treatments were not repellent against A. mellifera, including the reference insecticide Decis which acts by contact and ingestion.
Indeed, the plants tested in this study are aromatic whose essential oils can be used as an alternative to acaricides and synthetic antimicrobials for an overall improvement in the state of health of honey bee colonies (Eguaras et al., 2005;Al-Ghamdi et al., 2021).The peak in A. mellifera activity was likely related to daily periods of greater nectar availability due to optimal temperature for flower development and nectar production.According to the work of Manrique & Thimann (2002), an increase in temperature stimulates the production of nectar, the availability of which is characterized by the yellow color of the flowers (Carrión-Tacuri et al., 2012).A. mellifera would therefore have associated the color (yellow) with the availability of the reward, which would have allowed it to maximize its energy gain.This illustrates the theory of optimal foraging which indicates that foragers tend to move towards abundant food resources to reduce energy losses (Baude et al., 2011).Other factors such as the spraying of insecticides had an impact on the foraging activity of the honey bee.This impact was concentration dependent, with the highest concentration (15%) being more attractive to A. mellifera.Callistemon rigidus was more active.In a similar study, Adamou et al. (2022) found that botanical extracts have an attractive effect against A. mellifera.Further studies are needed to identify the active compounds in these extracts.
According to previous studies, foragers take longer to obtain their maximum nectar load on flowers where this resource is available in large quantities.Botanical extracts would therefore increase nectar production in G. max or prevent insect competitors of A. mellifera from harvesting this floral product.The extracts therefore had no significant impact on the accessibility of the nectar collected by the foragers.
During the flowering period, and in all the sub-plots treated with aqueous plant extracts, including the positive and negative controls, the foraging activity of A. mellifera was interrupted by individuals of the same species or by other insects that were competitors for nectar collection.
Strong competition for nectar harvesting was observed in A. mellifera during the two years of observation.Eurema sp. and Xylocopa olivacea were major competitors of A. mellifera in 2019 and 2020, respectively.These interruptions occurred as a result of collisions between visitors.One of the consequences of these disturbances was the reduction in the duration of certain visits; this forced some individuals of A. mellifera to visit more flowers on a foraging trip to obtain their optimal nectar load, as reported by Djakbé et al.Similar results were found in Vigna unguiculata (Adamou et al., 2020).The impact of extracts with biopesticide effects would be linked on the one hand to their attractive property vis-à-vis this pollinating insect and on the other hand to their pesticidal property against the pests of G. max.The aqueous extracts of C. rigidus, L. multiflora and P. glandulosus were therefore more selective for A. mellifera than the synthetic insecticide Decis as reported by Pereira et al. (2020).

CONCLUSION
Botanical extracts increased the foraging ability of pollinators.In this locality of Bini, the number of insect species visiting this Fabaceae varies from year to year.It is highly visited by A. mellifera for the exclusive collection of nectar.This study demonstrated that the aqueous extracts of C. rigidus, L. multiflora and P. glandulosus with the highest concentration (15%) are attractive to this honeybee and contribute synergistically to a significant improvement in the index and fruiting rate of G. max.This means that the botanical extracts with the highest concentration (15%) were selective for A. mellifera and could be used as alternative to synthetic insecticides.The preservation of honey bee hives near soybean plantations is necessary to improve the fruiting rate.
(2017)  on Physalis minima, thenTchuenguem et al. (2018)  on Ceratotheca sesamoides.In 2019, unlike the control treatments and the reference insecticide Décis, the various botanical extracts generally significantly improved the fruiting rate of flowers left open to pollination (FL) and that of flowers pollinated exclusively by A. mellifera ( FPD).In 2020, the fruiting rate of the sub-plots left in free pollination and those visited exclusively by A. mellifera and treated with synthetic insecticide (Decis) was significantly low compared to the other treatments including the control.Fruiting rates from protected flowers were significantly low for both years.These results show a potentiation of the pollinating action of A. mellifera and botanical extracts for improving the fruiting rate of G. max.Indeed, during the nectar harvest, the workers of Apis mellifera were frequently in contact with the anthers and the stigma.They could therefore intervene directly in self-pollination, by putting the pollen of a flower on its stigma.They could also intervene in cross-pollination, by putting the pollen of one flower on the stigma of another flower of the same plant or by putting the pollen of one plant on the stigma of a flower of another plant.Therefore, A. mellifera strongly increased the pollination possibilities of G. max.

Table 1 :
Number and percentage of insects recorded on Glycine max flowers in 2019 and 2020

Table 2 :
Interruptions of Apis mellifera visits on Glycine max flowers by competing insects olivacea and 1 (3.70%) by Formicidae (1 sp.).For their load of floral products, some individuals of A. mellifera who suffered such disturbances were forced to visit more flowers during the corresponding foraging trip.Impact of the Insect's Pollination Including Apis Mellifera and the Single Visit of Apis Mellifera on the Podding Rate of Glycine MaxDuring nectar harvest on soybean flowers, honey bees and the other flowering insects always contacted anthers and stigma, thereby increased the pollination possibilities of Glycine max as they frequently flew from flowers to flowers on the same plants and / or on other flowers of the neighboring plants of soybean.Table3 reveals that: