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Impact of the foraging activity of Apis mellifera (Hymenoptera: Apidae) on increasing yields of Gossypium hirsutum (Malvaceae) in Djoumassi (Garoua, Cameroon)

To evaluate the impact of the foraging behaviour of Apis mellifera in increasing boll and seed yields of Gossypium hirsutum, its pollination activities were studied in Garoua for two seasons, from September through October 2018 and 2019. Observations were made on 540 flowers divided into four treatments of which two were differentiated according to the presence or absence of flower protection regarding insect visits; the third was made of protected and opened flowers, to allow Apis mellifera visits and the fourth made of opened flowers then closed without any visit. The bee’s daily rhythm of activity, their foraging and pollination activities were evaluated. Results showed that, on Gossypium hirsutum flowers, Apis mellifera intensely collected pollen and nectar. Data obtained allow the classification of this Malvaceae as a highly polliniferous and nectariferous bee plant species. Through its pollination efficiency, Apis mellifera has increased the fruiting rate by 29.66% and 13.95%, the mean number of seeds per boll by 24.53% and 14.89% and the percentage of normal seeds by 3.89% and 8.87% in 2018 and 2019 respectively. Hence, the conservation of Apis mellifera colonies close to Gossypium hirsutum fields is recommended to improve its boll and seed yields.


Introduction
Pollination is a vital ecosystem service, an estimated 87.5% of all flowering plants rely on insect pollination [1,2]. Cropinsect pollinator interactions are very important to maintain pollinator populations, ensure pollination services, increase honey yields and therefore benefit beekeepers [3]. The most important crop pollinator species is the western honeybee (Apis mellifera L.), providing roughly 50% of global crop pollination [4]. Worldwide, Honeybees are among the major pollinators of the world's cultivated food and cash crops, such as G. hirsutum (Malvaceae) [5,6].
Gossypium hirsutum is grown for its seeds rich in lipids (36%) and fiber used in textile industry [7]. Cotton leaves are trilobed and flowers are cream and produce approximately 20,000 cream pollen grains per flower and produce low volume of nectar [8,9,10]. Considered as a major bee plant species [6], cotton is primarily self-pollinating, although out-crossing can occur [11]. Before our work, the flowering insects of G. hirsutum were studied in Cameroon [12,5,6] and in Australia [13,14] where A. mellifera was the most frequent visitor on G. hirsutum flowers. Near Narrabi in New South Wales, no bee was seen on its flowers, and although small number of wasps and flies were recorded [15]. In USA [8] and in Brazil [16], Bumblebees (Bombus sp.) and Apoids are respectively the main cotton pollinators' insects. In Paris, [7] reported that laying A. mellifera colonies in cotton plantations leads to increase yields of 5 to 30%.
Entomofauna of a plant species can vary from one region to another [17], it is therefore, important to deepen the investigations on the relationship between G. hirsutum and A. mellifera in Cameroon, to supplement the existing data. The main objective of this work was to contribute to the understanding of the relationships between G. hirutum and A. mellifera, for their optimal management in Garoua. Specific objectives were to: (a) determine the place of A. mellifera in G. hirsutum flowering insects; (b) study the activity of this Apidae on cotton flowers; (c) evaluate the impact of flowering insects including A. mellifera on bolls and seeds yields; (d) estimate the pollination efficiency of A. mellifera on this plant species.

Study site, experimental plot and biological material
The studies were conducted from June to October, in 2018 and 2019, in the locality of Djoumassi (latitude of 9°23'16.512' N, a longitude of 13°23'20.627 E, an altitude 169 m.a.s.l.), Benoue Division, in the city of Garoua, North Region (Cameroon). This region belongs to the Sahel-Sudanian ecological zone (MINEPAT, 2014). It has Sudanian climate type characterized by two annual seasons: a rainy season (April to October) and a dry season (November to March). August is the wettest month of the year [18,19]. Annual rainfall varies from 750 to 1250 mm and the mean annual temperature is 27 °C [20,19].
The experimental plot was an area of 437 m 2 . The animal material was mainly represented by insects naturally present in the environment and four and two colonies of A. mellifera in 2018 and 2019 respectively, located near the experimental field. The plant material was G. hirsutum seeds obtained from the Institute of Research and Agricultural Development (IRAD) of Garoua.

Preparation of experimental plot, sowing and weeding
From 24 th to 29 th June 2018 and from 03 rd to 06 th July 2019, the experimental plot was cleaned and divided into eight subplots, each measuring 8*4.5 m 2 . Four seeds were sown per hole and six lines per subplots were made, each line having six holes. Holes were separated by 50 cm from each other, while lines were 75 cm. Weeding was performed manually during the flowering periods [5].

Determination of the reproduction mode of Gossypium hirsutum
On September 25 th , 2018, 240 flowers from untreated subplots at the bud stage were labeled among which 120 flowers were left unprotected (treatment 1), while 120 others were bagged using gauze bags nets (treatment 2) to prevent insects' visits ( Fig. 1). In similar subplots, on October 04 th , 2019, 240 flowers at the bud stage were labeled of which 120 were unprotected (treatment 5), while 120 were bagged (treatment 6). For each cropping year, two weeks after shedding of the last labeled flower, the number of bolls was assessed in each treatment. The fruiting index was then calculated as described by Tchuenguem et al. (2001) [21] : Pi = Fb/Fa, where Fb is the number of bolls formed and Fa the number of viable flowers initially set. The allogamy rate (TC) from which derives the autogamy rate (TA) was expressed as the difference in fruiting indexes between treatment X (unprotected flowers) and treatment Y (bagged flowers) [21]. For each year of study, Fx = [(Vx / Vi) * 100], where Vx is the number of visits of A. mellifera on flowers of free treatment and, Vi the total number of insect visits on flowers of the same treatment [21].
During our investigations, before starting with the record of visit, the number of opened flowers was counted. The same days as for the registration of frequency of visits, the floral products (nectar and/or pollen) collected by each worker bee were recorded for the same date and daily time frame. The study of this parameter indicates whether A. mellifera is strictly polliniferous and / or nectariferous on G. hirsutum flowers [22]. This can give an idea of its involvement in the pollination of this plant and honey production. The duration of the individual flower visits was recorded (using a stop watch) according to six daily time frames: 7 -8 h, 9 -10 h, 11 -12 h, 13 -14 h, 15-16 h and 17 -18 h. The foraging speed expressed as the number of flowers visited by a worker bee per minute according to Jacob -Remacle [23] was calculated using the following formula : Vb = (Fi/di) *60 where di is the time given by the stopwatch and Fi is the number of flowers visited during di.
The abundance of foragers (highest number of individuals foraging simultaneously) per flower or per 1000 flowers (A1000) were recorded on the same dates and time slots as the duration of visits. Abundance per flower was recorded as a result of direct counting. To determining the abundance per 1000 flowers, some foragers were counted on a known number of opened flowers and A1000 was calculated using the following formula: A1000 = [(Ax / Fx) *1000], where Fx and Ax are respectively the number of flowers and the number of foragers effectively counted on these flowers at time x [24]. The disruption of the activity of foragers by competitors and / or predators and the attractiveness exerted by other plant species on this insect was assessed by direct observations. For the second parameter, the number of times the worker bee went from G. hirsutum flowers to another plant species' flowers and vice versa was noted throughout the periods of investigations. During each day, temperature and relative humidity of the station were recorded after every 30 minutes using a mobile thermo-hygrometer (HT-9227) installed in the shade.

Evaluation of the effect of insects including Apis mellifera on Gossypium hirsutum yields
The evaluation of the effect of insects including A. mellifera on G. hirsutum yields was based on the impact of flowering insects on pollination, the impact of pollination on G. hirsutum fruiting, and the comparison of fruiting rate, the mean number of seed per boll and the percentage of normal seeds of treatments 1 and 2 (2018) and 5 and 6 (2019). For each year, the fruiting rate due to the foraging insects including A. mellifera (Fri) was calculated using the following formula : where FX and FY are the fruiting rates in treatment X (flower left in free pollination) and treatment Y (flower protected from all insect visits), and Eg the effect of the gauze bag net who can be calculated using the formula Eg = FY-FZ, where FZ is the fruiting rate in treatment Z (flowers protected then opened and closed without insect or any other organism visit).
The fruiting rate of a treatment (F) is where b is the number of seeds formed and the number of viable flowers initially set [25].
The impact of flower visiting insects including A. mellifera on the number of seeds per boll and the normal seeds were evaluated using the same method as mentioned above for the fruiting rate.

Evaluation of the pollination efficiency of Apis mellifera on Gossypium hirsutum
Parallel to the setup of treatments 1, 2, 5 and 6, 600 flowers at bud stage were protected in 2018 and 2019, to form two treatments: Treatment 3 (2018) or 7 (2019): 400 flowers protected using gauze bag nets to prevent insect or any other organism visits and destined to be visited exclusively by A. mellifera. As soon as the first flowers of treatments 3 and 7, the gauze bag was gently removed and the flower observed for up to 10 minutes; the flower visited once by A. mellifera was marked and then protected again ; Treatment 4 (2018) and 8 (2019): 200 flowers protected using gauze bag nets and destined to be opened then closed without the visit of insects or any other organism. As soon as the first flowers of treatments 4 and 8 were opened, the gauze bag was gently removed and the flower was observed for up to 10 minutes, while avoid insect or any other organism to visit it.
At the maturity, bolls from each treatment were harvested and counted from. The fruiting rate, the number of seeds per boll and the percentage of normal seeds were then calculated for each treatment.

Statistical 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, Microsoft Excel 2013 software was also used.

Reproduction mode of Gossypium hirsutum
The fruiting indexes of G. hirsutum were 0.94, 0.88, 0.86 and 0.68 for treatments 1, 2, 5 and 6 respectively. Thus, in 2018, the autogamy rate was 79.07% and the allogamy rate was 20.93%. In 2019, the corresponding figures were 93.62% and 6.38%. For the two cumulative years, the autogamy rate was 86.35% and the allogamy rate was 13.66%. It appears that G. hirsutum has a mixed mating system with the predominance of autogamy.

Frequency of visits
Amongst the 288 and 342 visits of 15 and 21 insect species recorded on its flowers in 2018 and 2019 respectively, A. mellifera was the most frequent insect with 139 visits(48.26%) and 178 visits (52.05%) in 2018 and 2019 respectively ( Table 1). The difference between these two percentages is not significant (2 = 0.89; df = 1; P > 0.05).

Floral products harvested
From our observations and during the two flowering periods, A. mellifera were found to harvested intensively pollen and slightly nectar on flowers (Fig. 2).

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.  3A and B). The correlation was highly significant between the number of G. hirsutum opened flowers and the number of

Daily visits
The worker bee was active on G. hirsutum flowers throughout the day. The correlation was significant between the number of A. mellifera visits and relative humidity in 2018 (r = 0.83; df = 4; P <0.05) and was not significant in 2019 (r = 0.79; df = 4; P > 0.05) (Fig. 4A and B). The correlation was significant between the number of A. mellifera visits and the temperature in 2018 (r = 0.87; df = 4; P < 0.05) and was not significant in 2019 (r = 0.28; df = 4; P > 0.05).

Duration of a visit per flower
In For the two cumulated years‚ the mean duration visit for pollen harvest was 36.54 sec and 35.02 sec for nectar.

Abundance of Apis mellifera foragers
In 2018, the highest mean number of A. mellifera simultaneously active was one per flower (n = 54, s = 0) and 403.

Foraging speed of Apis mellifera on Gossypium hirsutum flowers
During our observations, A. mellifera visited between 0.64 and 15 flowers/min in 2018 and between 0.52 and 16.67 flowers/min in 2019. The mean foraging speed was 4.31 flowers/min (n = 92, s = 2.14) in 2018 and 3.91 flowers/min (n = 77, s = 2.42) in 2019. The difference between these means is not significant (t = 0.16; df = 167; P > 0.05). For the two cumulative years, the mean foraging speed was 4.11 flowers/min.

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: Bidens pilosa (Ne, Po), Mangifera indica (Ne), Zea mays (Po), Vigna subterranea (Ne), Vigna unguiculata (Ne, Po), Ceratotheca sesamoides (Ne, Po) and Commelina diffusa (Ne, 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.

Impact of flowering insects including Apis mellifera on Gossypium hirsutum yields
During nectar or pollen harvest on G. hirsutum, foragers always shook flowers and regularly contacted anthers and stigma, increasing self -pollination and/or cross -pollination possibilities of this plant species. Table 2 Gives the fruiting rate, the mean number of seeds per boll and the percentage of normal seeds in different treatments. It appears from this table that: -The fruiting rates were 86.66%, 68.33%, 94.17% and 87.5% in treatments 1, 2, 5 and 6 respectively. The differences between these four percentages are highly significant (χ2= 33.14 ; df= 3 ; P < 0.001) . Two -to -two comparisons showed that the difference observed is highly significant between treatments 1 and 2 (χ2 = 11.57; df = 1; P < 0.001) and not significant between treatments 5 and 6 (χ2 = 3.20 ; df = 1 ; P > 0.05). Consequently, in 2018, the fruiting rate of exposed flowers (treatment 1) was higher than that of flowers bagged during their flowering period (treatment 2).
-The means number of seeds per boll were 24.29, 18.5, 25.02 and 20.15 in treatments 1, 2, 5 and 6 respectively. The differences between these four means are highly significant (F = 285.71; df1 = 3; df2 = 400; P < 0.001). Two -to -two comparisons showed that the difference observed was highly significant between treatments 1 and 2 (t = 36.24; df = 184; P < 0.001) as well as between treatments 5 and 6 (t = 6.03; df = 216; P < 0.001). Hence, in 2018 and 2019, the mean number of seeds per boll from exposed flowers was higher than that from flowers bagged during their flowering period.
In 2018, the contribution of anthophilous insects including A. mellifera in the fruiting rate, the mean number of seeds per boll and the percentage of normal seeds were 34.29%, 18.82% and 8.91% respectively. In 2019, the corresponding figures were 18.73%, 19.94% and 11.55% respectively. For the cumulative years, the contributions of flowering insects were 26.51%, 19.38% and 10.23% for the fruiting rate, the mean number of seeds per boll and the percentage of normal seeds respectively.
The insect pollinators played a significant role in increasing bolls and seeds yields of G. hirsutum.

Pollination efficiency of Apis mellifera on Gossypium hirsutum
During pollen harvest on cotton flowers, workers of A. mellifera always contact anthers and stigma, thereby increased the pollination possibilities of G. hirsutum as they frequently flew from flowers to flowers on the same plants and / or on other flowers of the neighboring plants of cotton. Table 3 reveals that: -The fruiting rates were 72.5%, 51%, 86% and 74% in treatments 3, 4, 7 and 8 respectively. The differences between these four percentages are highly significant (χ2 = 48.28 df = 3 ; P < 0.001). The difference was highly significant between treatments 3 and 4 (χ2 = 15.71; df = 1; P < 0.001) and significant between treatments 7 and 8 (χ2 = 6.87; df = 1; P < 0.05). Hence, in 2018 and 2019, the fruiting rate of flowers protected and visited exclusively by A. mellifera was higher than those protected, opened and closed without insect or any other organism visit.
-The means number of seeds per boll were 26.5, 20, 23.5 and 20 in treatments 3, 4, 7 and 8 respectively. The differences between these four means are highly significant (F = 922.57; df1 = 3; df2 = 438; P < 0.001). The difference was highly significant between treatments 3 and 4 (t = 9.31; df = 194; P < 0.001) as well as between treatments 7 and 8 (t = 5.06; df = 244; P < 0.001). These results pointed out that flowers visited by A. mellifera have the highest number of normal seeds compare to those protected then opened and closed without the visit of insect or any other organism.
The differences between these four percentages are highly significant (χ2 = 67.83; df = 3; P < 0.001). The difference was significant between treatments 3 and 4 (χ2 = 6.59 ; df = 1 ; P < 0.05) and highly significant between treatments 7 and 8 (χ2 = 61.67 ; df = 1 ; P < 0.001). These results pointed out that flowers visited by A. mellifera have the highest number of normal seeds compare to those protected then opened and closed without the visit of insect or any other organism.
In 2018, the contribution of A. mellifera via a single flower visit were 29.66% for the fruiting rate, 24.53% for the mean number of seeds per boll and 3.89% for the percentage of normal seeds. In 2019, the corresponding figures were 13.95%, 14.89% and 8.87% respectively. For the two cumulate years, the contribution of A. mellifera on the fruiting rate the mean number of seeds per boll and the percentage of normal seeds were 21.81%, 19.71% and 6.38% respectively.

Discussion
Apis mellifera was the most frequent insect visitor on G. hirsutum flowers during the observation periods. This result confirms those reported by Mazi et al. (2013) [5] at Dang and Dounia and Tchuenguem [12] at Maroua in Cameroon. According to Pires et al. (2014) [16] in Brazil, Apidae are the important flower visitor of cotton. The peak activity of A. mellifera was observed in the morning, between 8:00 and 9:00 am. This peak could be linked to the period of the highest availability of nectar and pollen in the flower of this Malvaceae [12].
The high abundance of A. mellifera per 1000 flowers and the positive and significant correlation between the number of G. hirsutum opened flowers and the number of honey bee visits underscore the attractiveness of G. hirsutum nectar and pollen to A. mellifera. The high density of workers per 1000 flowers is due to the natural faculty of honeybees to recruit a high number of workers to exploit an interesting resource of food [26]. Honeybees can smell or detect pollen or nectar odors using sensory receptors located on the flagellum of their antennae [27]. Worker communicate the location of an important resource of food to the rest of hivemates through a special dance inside the hive. The dance informs the distance and the direction from the hive to the food source [28]. The abundance per 1000 flowers was higher (429 workers for the two cumulated cropping years) than that recorded at Dang (313.9 workers for the two cumulated cropping years) by Mazi et al. [5]. This difference could be explained by the high availability of honeybee colonies on the study site in Djoumassi compare to that in Dang.
Significant differences observed between the duration of pollen harvest visit and that of nectar harvest could be explained by the accessibility of each of these floral products.
As a highly nectariferous and polliniferous bee plant with the flowering period located in the rainy season, G. hirsutum could be cultivated and protected to strengthen A. mellifera colonies. The significant difference observed between the duration of visits in 2018 and 2019 could be explained by the biotic (flower robbers and competitors for nectar and/or pollen of this Malvaceae) and abiotic (wind, rain and temperature) factors of the study area. Interruptions of bee visits took place when there was a heavy wind, collisions between visitors, visitor capture attempts by a predator or approach of flower already occupied by a first visitor. Similar observations have been made by Dounia and Tchuenguem [12] and by Mazi et al. [5] in Dang and in Maroua respectively.
The present study revealed that during one foraging trip, an individual bee foraging on a given plant species scarcely visited another plant species. This result indicates that A. mellifera showed flower constancy [29] of G. hirsutum.
During the collection of nectar and/or pollen on each flower, A. mellifera workers regularly come into contact with the stigma and anthers. They could thus enhance self-pollination, which has been demonstrated in the past [12,5]. Apis mellifera could provoke cross-pollination through carrying of pollen with their furs, legs and mouth accessories, and put them onto another flowers belonging to different plant of the same species.
The significant contribution of anthophilous insects including A. mellifera in the number of seeds per boll of G. hirsutum is similar to the findings of Dounia and Tchuenguem [12] and Basga et al. [6] at Maroua and Djamboutou respectively which showed that cotton produce less seeds per boll in the absence of efficient pollinators.
The positive and significant contribution of A. mellifera alone in boll and seed yields of this Malvaceae could be justified by its pollination possibilities. The workers have facilitated the liberation of pollen from anthers for optimal occupation of the stigma, thus increasing pollination possibilities. The flowers that were exposed to pollinators provided more seeds than protected flowers. Our findings agree those of Dounia and Tchuenguem [12], Mazi et al. [5] and Basga et al. [6] respectively at Dang, Maroua and Djamboutou on the same plant species.

Conclusion
From our observations, G. hirsutum is a plant species that highly benefits from pollination by insects among which A. mellifera is one of the most important and harvested pollen and nectar. Data on the foraging activity of A. mellifera enable the classification of G. hirsutum among the highly polliniferous and nectariferous stingless bee plant. Thus, this plant could be grown or protected to increase pollen and nectar production as bee hive product and stabilize A. mellifera colonies.