Impact of insect pollinators on yields of Glycine max L. (Fabaceae) at Yaoundé (Cameroon) Douniaa, Clautin Ningatoloumb, Chantal Doukaa, Elono Azang Pierre Stephana, Amada Brahimc, Joseph Lebel Tamessea, Fernand-Nestor Tchuenguem Fohouod

To appreciate the impact of insect pollinators on the pod, seeds, and seed weight yields of Glycine max, the pollinating activities of flowering insects were studied in Yaoundé, during the two mild, rainy seasons in 2016 and 2017 (March-June). Observations were made on 45 to 20400 flowers per treatment. The flowers were subjected to different treatments: Free flowers (Treatment 1), bagged flowers (treatment 2), castrated and free flowers (treatment 3), and castrated and bagged flowers (treatment 4). Some (8695 and 3325) flowers of Glycine max (Fabaceae) were observed in 2016 and 2017, respectively, for the diversity and Frequency of insect visits. For results, 1527 visits of 13 insect species distributed in seven orders were recorded on G. max flowers. The most dominating Hymenoptera observed was Apis mellifera, with 40.20 % of the total insect visits. The highest number of insect pollinators harvested in the flowers of this Fabaceae was between 8h-9h. The studied insects have a positive impact on the yields of this plant. This positive impact of the pollinator insects on the yields was 26.29 %, 16.13 %, 15.02 and 4.45 % in fructification rate, number of seeds pod, the weight of seeds, and percentage of normal seeds respectively. The avoidance of pesticide treatment of plants during flowering could be a good management strategy to improve on plant yield.


Introduction
Glycine max is a plant that originated from China (Hymowitz, 1970). This plant is annual, herbaceous, and can reach a height of 1.5m (Gallais and Bannerot, 1992); Soybean is grown primarily for its seeds, which have many uses in the food and industrial sectors (USDA, 2002;Tchuenguem and Dounia, 2014). It is a major vegetable source of protein for man and other animals (Tien et al., 2002;MINADER, 2012). The United State of America are the largest Producers of soybean in the world, the production in Cameroon is estimated at 12544 tons. This production is low and the demand for seeds is high in this country (MINADER, 2012). The flowers of G. max produce nectar and pollen which attracts insects Tchuenguem and Dounia, 2014;Dounia et al., 2016). The reproduction system is autogamous/allogamous (Tchuenguem and Dounia, 2014;Kengni, 2017). Therefore, it is important to investigate on the possibilities of increasing the production of this plant in Yaoundé (Cameroon). This can be possible if flowering insects of G. max in this region are known and exploited. The researches conducted in Brazil by Milfont et al. (2013), in Cameroon by Tchuenguem and Dounia (2014) and Dounia et al. (2016) in the Far-Nord Region and Kengni et al. (2015) in the Adamaoua Region revealed that Apoïdea visits G. max flowers and collect nectar and pollen. No previous research has been reported on the relations between G. max flowers and the flowering insects in Yaoundé (Centre Region, Cameroon), although, the activities of pollinator insects on the flowers can vary with Region (Tchuenguem, 2005). The main objective of this research was to gather more data on the relations between G. max and flowering insects. Specific objectives were (a) to determinate the diversity of flowering insects of G. max, (b) to evaluate the Frequency of these insects on G. max flowers, and (c) to evaluate the impact of flowering insects on pollination and yield of this plant.

Site and biological materials
The studies were conducted from March to June in 2016 and 2017 ( m i ld r ai ny s e a son ) in the fields located at the campus of Higher Teacher's Training College of University of Yaoundé I (Latitude 10° 62' N, Longitude 14° 33' E and altitude 756 m) in the Center Region of Cameroon. The animal material was represented by insect pollinators naturally present in the environment. The plant material was represented by the seeds of G. max provided by the Institute of Agricultural Research for Development in Nkolbisson (Yaoundé).

Planting and maintenance of culture
On t h e 12 th o f M a r c h 2016 and t h e 1 5 th of 2017, the experimental plot was cleaned and divided into 12 subplots, each measuring 1.5m × 1m. Seeds were sown on two lines per subplot; each line had three holes and each hole received five seeds. The spacing was 0.5m between rows and 0.5m on rows. Each hole was 5 cm depth. Two weeks after germination (March 26 th , 2016 and March 29 th , 2017), the plants were thinned and only two were left per hole. From thinning to the opening of the first flower (May 12 th , 2016 and May 21 th , 2017), weeding was performed manually as necessary to keep the plot weeds-free.

Diversity and Frequency of flowering insects on the flowers of Glycine max
On May 22 th , 2016, 12 subplots carrying 144 plants were labeled. Three subplots carrying 36 plants were left for open pollination (treatment 1), three subplots carrying the same number of plants like treatment 1 were protected with gauze mesh to prevent pollinator insects (treatment 2), 66 flowers were on three subplots contained by 36 plants like treatment 1 where some flowers were destined to be castrated (treatment 3) and 66 flowers were on three subplots with 36 plants where some flowers were destined to be castrated and then protected with gauze mesh like treatment 2 (treatment 4). For castration the stigmata were delicately remove using tongs. On May 31 st , 2017, the experiment was repeated. On June 30 th , 2016 and 2017 the pods were collected and the seeds were calculated.
The diversity of flowering insects that visited G. max flowers was appreciated; capture was done on flowers of treatment 1 and insects were conserved, described and identified using the method of Borror and White

Impact of flowering insects on the pollination of Glycine max
The evaluation of the impact of flowering insects on the pollination of G. max was done in the study and the Frequency of insect visits was calculated. It was to record the number of times that the insect's body comes in contact with the anther of flower. This can indicate the possibility of flowering insect to participation in the self-pollination and cross-pollination (Delaplane et al., 2013). To determine the different categories of pollinators, the regularity index (Id) was calculated using the formula: Id = [(P / 100) × (f / 100)], where P and f are the percentage of insect visits and the relative Frequency of insect visits.

Incidence of flowering insects on Glycine max yields
This evaluation was based on the impact of visiting flowers on pollination, the impact of pollination on fructification of G. max, and the comparison of yields [fruiting rate, mean number of seeds per pod, weight of seeds and percentage of normal (well developed) seeds] of treatments 1, 2, 3 and 4.
-The fruiting rate due to the activity of insects (Fri) was calculated as follows : Where Frx and Fry are the fruiting rates in each treatment.
-The fruiting rate (Fr) is: Where F2 is the number of pods formed and F1 the number of flowers initially set.
-The percentage of mean number of seeds per pod due to the activity of insects (Spi) was calculated using the formula: Where Spx and Spy are the percentages of seeds per pods in different treatments.
-The percentage of weight of seeds due to the activity of insects (Wsi) was calculated as follows: -The percentage of normal seeds due to the activity of insects (Nsi) was calculated as follows:

Data analysis
Data were analyzed using descriptive statistics, student's t-test for the comparison of means of the two samples, correlation coefficient (r) for the study of the association between two variables, chi-square (χ2) for the comparison of two percentages, ANOVA for the comparison of many samples. We also used SPSS statistical software and Microsoft Excel.

Diversity and Frequency of entomofauna of Glycine max
Among the 188 and 1339 visits of 8 and 12 insect species counted on G. max flower in 2016 and 2017. For the two cumulated years; seven Orders of anthophilous insects were found on G. max flowers including: Diptera, Coleoptera, Hemiptera, Hynemoptera, Lepidoptera, Orthoptera and Nevroptera (Table 1). Thirteen (13) flowering insects were represented on G. max flowers : constant species that include (Apis mellifera, Dysdercus voelkeri, Halictus sp., Lipotriches collaris, Musca domestica and Synagris cornuta) and accidental species (Acrea acerata, Calliphiridae, Catopsilia flerella, Coleoptera, Delta sp., Orthoptera and Nevroptera) ( Table 2). Flowering insects have been active on the flowers of G. max from 8 am to 17 pm, with a peak of visits between 8 and 9 am in 2016 and 2017 (Table 3).   Accidental species

Impact of flowering insects on pollination of Glycine max
Three categories of pollinators were observed on flowers of G. max in 2016 and 2017 (Table 4) : -Major pollinators (Id > 0,05 and/or p > 50 %) Apis mellifera and Halictus sp.

Impact of anthophilous flowering insects on yield of Glycine max
During foraging behaviour on flower of G. max, flowering insects always shook flowers and are regularly in contact with the anthers and stigma (p = 76.38 %), increasing cross pollination possibility of G. max fruiting rate, number of seeds per pod, weight of seeds and percentage of normal seeds in different treatments (Table  4).
a. The comparison of the fruiting rate showed that the difference was very highly significant between treatments in 2016 (F = 9.02, df = 3, P < 0.001) and in 2017 (χ2 = 6.23, df = 2, P < 0.001). The difference observed was highly significant between fruiting rate of free opened flowers (treatment 1) and that b. The comparison of the mean number of seeds per pod showed that the difference was highly significant between treatments in 2016 (F = 6.44, df = 3, P < 0.001) and in 2017 (F = 5.83, df = 2, P < 0.001). The difference observed was highly significant between mean number of seeds per pod in treatment 1 and treatment 2 (t = 13.38, df = 58, p < 0.001), the same observation was fund in treatment 1 and treatment 3 (t = 6.75, df = 37, p < 0.001) and the difference observed was significant between mean number of seeds per pod in treatment 1 and treatment 4 (t = 2.21, df = 29, p < 0.02) in the first year. In the second year the difference was significant between mean number of seeds per pod in treatment 1 and treatment 3 (t = 2.41, df = 35, p < 0.02). The mean number of seeds per pod in treatment 1 was higher than treatments 2, 3 and 4 in 2016 as well as in 2017. The mean number of seeds per pod due to the action of insects was 28.61 in 2016 and 3.65 % in 2017. For the two cumulated years, the mean number of seeds per pod due to the influence of insects was 16.13 %.
c. The comparison of the mean weight of seeds showed that the difference was significant between treatments in 2016 (F = 4.98, df = 3, P < 0.001) and not significant in 2017 (F = 1.09, df = 2, P > 0.05).
The difference was significant between weights of seeds in treatment 1 and in treatment 2 (t = 1.37, df = 198, p < 0.02) in 2016. The weight of seeds due to the action of insects was 15.02 % in 2016.
The difference observed was highly significant between the percentage of normal seeds of in treatment 1 and treatment 2 (χ2 = 91.43, df = 1, p < 0.001), the same observation was fund in treatment 1 and treatment 3 (χ2 = 19.87, df = 1, p < 0.001) in the first year. In the second year the results were χ2 = 829.81, df = 1, p < 0.001 in treatment 1 and treatment 2 and χ2 = 554.10, df = 1, p < 0.001 in treatment 1 and treatment 3. The percentage of normal seeds of treatment 1 was higher than treatments 2 and 3 in 2016 as well as in 2017. The mean percentage of seeds due to the action of insects was 5.31 % in 2016 and 3.60 % in 2017. For the two cumulated years, the mean number of seeds per pod due to the influence of insects was 4.45 %.