Pollination Efficiency of Amegilla calens (Hymenoptera: Apidae) on Gossypium hirsutum L. (Malvaceae) Variety L457 Flowers at Meskine (Maroua, Cameroon)

Animal pollination is a key of reproduction of more plants species including Gossypium hirsutum. This Malvaceae is the best cultivated oilseed plant in tropical and subtropical regions in the world. This research evaluates the in-teraction between Amegilla calens and Gossypium hirsutum from 2018 to 2019 based on the activities of the solitary bee on the flowers of this plant and their productive impact in field. The experiment was focused on four re-peated treatments during the flowering phase: the first two were characterised by the existence or absence of protection of the flowers regarding all insects and the other two made up of flowers designed for the exclusive visit of A. calens or open then protected again without a visit from an insect or any other organism. Daily rhythm of activity, behavioural ecology, the pollination efficiency, the fruiting rate, the number of seeds per capsule and the percentage of normal seeds were evaluated. Among twenty insects species recorded A. calens is the most abundant with 30.72% of 655 visits and the most efficient pollinator. Amegilla calens is active on G. hirsutum flowers from 6 a.m. to 5 p.m. with a maximum activity situated from 8 a.m. to 9 a.m. All visits of this solitary bee are able to fertilize the flowers of Malvaceae. The mean duration of a


Introduction
The importance of pollinating insects in agricultural production, mainly with regard to that of domestic and wild bees, is no longer to be proven but remains generally unknown [1]. In the natural environment and in agroecosystems, flowering insects in general and Apoidea in particular have great ecological and economical importance because they have a positive influence on food production [2] [3]. Insect pollination in many crops is essential for fruit and seed productions [4].
Gossypium hirsutum (cotton), commonly called white gold, is the second most important oil-seed crop after Glycine max (soybean) [5].
In all these investigations, the foraging behaviour and pollination activity were carried out in detail only on Apis mellifera and Macronomia vulpina. The flowering entomofauna and the impact of insects on pollination, fruit and seed yields of a plant species may vary with the species of insect, time and space [17]. Cameroon is classified among the countries with a severe level of hunger with an overall hunger index of 17.6 with 22% of the undernourished population [18]. In this country, the demand for cotton seed is high (250.000 tons/year) while its production is low (240.000 tons/year) [19]. Currently, the processing of seed cotton produces around 15 million litres of cotton seed oil per year for an estimated population of 25 million inhabitants [19]. Cotton yields can be increased in this country if its flowering insects are well known and exploited [16]. Therefore, it is important to investigate the possibilities of increasing the production of this valuable plant in Cameroon.
The general objective of this work is to contribute to the understanding of the relationships between A. calens and G. hirsutum, for their optimal management. Specific objectives are to: 1) determine the place of A. calens in the G. hirsutum floral entomofauna; 2) study the activity of this Apidae on flowers of the Malvaceae; 3) evaluate the impact of the flowering insects including A. calens on pollination, fruit and seed yields of G. hirsutum; 4) estimate the pollination efficiency of A. calens on this plant species.

Study Site, Experimental Plot and Biological Material
The experiment was carried out from 13 th September to 12 th December 2018 and from 11 th September to 1 st December 2019 at Meskine within an experimental fields (latitude: 10˚32'26"N; longitude: 014˚14'53"E; altitude: 424 m above sea level). This Region belongs to the ecological zone with three phytogeographical areas (Sudano-Sahelian, Sahelian and Sudanian altitude) periodically flooded, with unimodal rainfall [20]. The climate is characterized by two seasons: a dry season (November to May) and a rainy season (June to October); August is the wettest month of the year [21]. Annual rainfall varies from 400 to 1100 mm [21]. The annual average temperature varies between 29˚C and 38˚C; daily temperature range between 6˚C and 7˚C [21].  [14]. Weeding was performed manually as necessary to maintain plots weeds-free.

Estimation of the Frequency of Amegilla calens Visits on Gossypium hirsutum Flowers
The frequency of A. calens visits on G. hirsutum flowers was determined based on observations of flowers of treatments 1 and 5, every day, from 13 th to 22 nd September 2018 and from 11 th September to 1 st October 2019 according to six daily time frames: 6 -7 a.m., 8 -9 a.m., 10 -11 a.m., 12 -1 p.m., 2 -3 p.m. and 4 -5 p.m. In a slow walk along all labelled flowers of treatments 1 and 5, the identity of all insects that visited G. hirsutum flowers was recorded [22]. Specimens of all insect taxa were caught using insect net on unlabelled flowers and conserved in 70% ethanol, excluding butterflies that were preserved dry [23], for subsequent taxonomic identification. All insects encountered on flowers were registered and the cumulated results expressed as the number of visits to determine the relative frequency of A. calens in the anthophilous entomofauna of G. hirsutum [22]. Data obtained were used to determine the frequency of visits (F i ) of each insect species on G. hirsutum flowers. For each study period, Vi is the number of visits of insect i on treatment with unprotected flowers and Vt the total number of insect visits of all recorded insect species on these flowers.

Floral Product Harvested
In addition to the determination of the flower visiting insect frequency, direct observation of the foraging activity of A. calens on flowers was made in the experimental field. The floral product (nectar or pollen) harvested by A. calens during each flower visit was registered based on its foraging behaviour. Nectar foragers were seen extending their proboscis between the base of the corolla and stamens, while pollen gatherers scratched the anthers using their mandibles and legs [23] [24]. During the same time the duration of A. calens visits on flowers was registered, the type of floral product harvested by this solitary bee was noted [24]. In the morning of each sampling day, the number of opened flowers was counted. Data obtained were used to determine the relationship between the number of visits of A. calens and the corresponding flowers [22].

Duration of Visits and Foraging Speed
During the same days as for the registration of the frequency of visits, the dura- foraging speed (F s ) was calculated using the following formula: where d v is the duration (in sec) given by stopwatch and F s the number of flowers visited during d v [22]. During the observation, when a forager returned to previously visited flower, counting was performed as on two different flowers [22].

Abundances per Flower and per 1000 Flowers
The abundance of foragers (highest numbers of individuals foraging simultaneously) [26] per flower and per 1000 flowers (A 1000 ) were recorded on the same dates and daily time frames as that for the registration of the duration of visits. Abundance per flower was recorded as a result of direct counting. For determining the abundance per 1000 flowers, foragers were counted on a known number of opening flowers and A 1000 was calculated using the following formula:

Foraging Ecology
The disruption of the activity of foragers by competitors or predators and the attractiveness exerted by other plant species on A. calens was assessed by direct observations. For the second parameter, the number of times that the solitary I. Kodji Issaya et al. bee left these Malvaceae flowers to another plant species and vice versa was noted through the investigation period.
During each observation date, temperature and relative humidity in the station were registered every 30 minutes using a mobile thermo-hygrometer installed in the shade [22], from 6 a.m. to 6 p.m. and destined to be uncovered then rebagged without the visit of insects or any other organism; as soon as each flower of these treatments was opened, the gauze bag was removed and this flower was observed for up to 10 minutes, while avoiding insect or any other organism visits. At the maturity, fruits were harvested and counted from each treatment. The fruiting rate, the percentage of seeds per fruit and the percentage of normal seed were then determined for each treatment [28].

Evaluation of the Impact of Flowering Insects Including
Evaluation of the effect of insects including A. calens on G. hirsutum production was based on the impact of flowering insects on pollination, the impact of pollination on G. hirsutum fruiting and the comparison of the fruiting rate, the number of seeds per fruit and the percentage of normal (that is well developed) seed [29] of treatments 1, 2, 4, 5, 6 and 8. For each year, the fruiting rate due to the foraging insects including A. calens (Fri) was calculated using the following formula [29]: where FX, FY and FZ are the fruiting rates in treatment X (flowers left in free pollination), treatment Y (flowers protected from all insect visits) and treatment Z (flowers bagged then uncovered and rebagged without insect or any other organism visit). The fruiting rate of a treatment (F) is where b is the number of fruits formed and a the number of viable flowers initially set [30]. The impact of flower visiting insects including A. calens on the percentage of seeds per fruit and the percentage of normal seeds was evaluated using the same method as mentioned above for the fruiting rate [30].

Assessment of the Pollination Efficiency of Amegilla calens on
Gossypium hirsutum The contribution of A. calens on the fruiting rate, the number of seeds per fruit and the percentage of normal seeds, was calculated using the data of treatments , where FA is the fruiting rate in treatment A (flowers visited exclusively by A. calens).
The impact of A. calens on the fruiting rate, number of seed per fruit and the percentage of normal seeds were evaluated using the same method as mentioned above for the fruiting rate.

Data Analysis
The data collected and aggregates were subjected to the analysis of variance (ANOVA) considering the different treatments: effect of visits of all insects, influence of or without visit, effect of an exclusive visit of A. calens and protection effect and opening and reprotection without visit, based on a fixed linear model and to the joint analysis within each year (2018 and 2019). These analyses were carried out using R commander, version i386 3.2.0. The treatment means were compared two by two by student's t-test at p < 0.05, the relationship study between two variables by Pearson correlation coefficient (r) at p < 0.05 and the comparison of percentages by chi-square (χ 2 ) at p < 0.05.

Place of Amegilla calens in Gossypium hirsutum Floral Entomofauna
Among  Table 1). The difference between the percentages of A. calens visit for two years is highly significant (χ 2 = 57.48; df = 1; P < 0.001). For the two cumulated years A. calens ranked first with 204 visits (30.72%).

Duration of Visit per Flower
The mean duration of A. calens visit per G. hirsutum flower varied according to floral product harvested. In 2018, the mean duration of a flower visit for nectar harvest was 16.29 ± 1.29 sec (n = 34; s = 7.42) and that for pollen collection was 25.80 ± 1.49 sec (n = 72; s = 12.55); the difference between these means is highly significant (t = 4.82; df = 104; P < 0.001). In 2019, the corresponding figures were 11.08 ± 0.77 sec (n = 37; s = 4.60) for nectar harvest and 21.33 ± 1.25 sec (n = 76; s = 10.82) for pollen collection. The difference between these two means is highly significant (t = 6.99; df = 111; P < 0.001). For the two cumulated years‚ the mean duration of a flower visit was 13.69 ± 0.72 sec (n = 71; s = 6.01) for nectar harvest and 23.57 ± 0.96 sec (n = 148; s = 11.69) for pollen collection. The difference between these two later means is highly significant (t = 8.22; df = 217; P < 0.001).

Floral Products Harvested
Individuals of A. calens were seen collecting nectar (Figure 1(A)) and pollen (Figure 1(B  deploys its proboscis to collect nectar. For the pollen collection the bee comes into contact with the flower from above, the thorax being in contact with the stigma; subsequently, it uses the hind legs, mandibles and abdominal hair to collect pollen which is stored at the level of their hind legs.

Daily Rhythm of Visits
Amegilla calens was active on G. hirsutum flowers from 6 a.m to 5 p.m in 2018 and in 2019. The peak of activity was situated between 8 and 9 a.m in 2018 as well as in 2019 ( Figure 2).
In 2018, the correlation was not significant between the number of A. calens visits and the temperature (r = 0.11; df = 4; P > 0.05) and between the number of visits and relative humidity (r = 0.34; df = 4; P > 0.05). In 2019, the correlation was equally not significant between the number of A. calens visits and the temperature (r = −0.12; df = 4; P > 0.05) and between the number of these visits and relative humidity (r = 0.73; df = 4; P > 0.05) (Figure 3).
In 2018 as well as in 2019 A. calens visits were apparently more numerous on G. hirsutum individual plant when their number of opened flowers was highest.
But, the correlation between the number of opened flowers and the number of visit was not significant in 2018 (r = 0.62; df = 8; P > 0.05) as well as in 2019 (r = 0.14; df = 7; P > 0.05) (Figure 4).

Foraging Speed of Amegilla calens on Gossypium hirsutum Flowers
In the experimental field, A. calens visited between 1 and 6 flowers per minute in 2018 and between 2 and 13 flowers per minute in 2019 ( Table 2). The mean foraging speed was 3.07 ± 0.33 flowers per minute (n = 69; s = 2.74) in 2018 and 2.69 ± 0.33 flowers per minute (n = 60; s = 2.52) in 2019. There is no difference between these two means (t = 0.81, df = 127; P > 0.05). For the two cumulated years‚ the mean foraging speed was 2.88 ± 0.23 flowers per minute (n = 129; s = 2.64).

Abundance of Amegilla calens
In 2018, the highest mean number of A. calens individuals simultaneously in ac-

A B
A B    we observed no passage of A. calens from G. hirsutum flowers to flowers of another plant species and vice versa. Hence during foraging trips on G. hirsutum, individuals of A. calens were faithful to this Malvaceae.

Impact of Anthophilous Insects Including Amegilla calens on Gossypium hirsutum Yields
The fruiting rate, the mean number of seeds per fruit and the percentage of normal seeds in the different treatments of G. hirsutum are shown in Table 3.
Consequently, in 2018 and 2019, the fruiting rate of unprotected flowers (treatments 1 and 5) was higher than that of protected flowers (treatments 2 and 6).
In 2018, the numeric contribution of anthophilous insects in the fruiting rate, the mean number of seeds per fruit and the percentage of normal seeds of G. hirsutum were 6.76%, 10.61% and 7.72% respectively. In 2019, the corresponding figures were 26.47%, 23% and 5.46%. For the two cumulated years, the numeric contribution of anthophilous insects including A. calens was 16.62%, 16.81% and 6.59% for the fruiting rate, the mean number of seeds per fruit and the percentage of normal seeds of G. hirsutum respectively.

Pollination efficiency of Amegilla calens on Gossypium hirsutum
During a single flower visit of A. calens for nectar or pollen harvest on G. hirsutum flowers, this bee regularly came into contact with anthers and stigma (100%), increasing the possibility of the Malvaceae pollination.
The fruiting rates ( The significant difference observed between the mean duration of a pollen harvest visit and that of nectar harvest visit could be explained by the importance and accessibility of each of these floral products. Pollen is produced by the anthers, which are on the top of the stamens, whereas nectar is between the base of the style and stamens [26]. Amegilla calens do not make honey. It harvests more pollen and less nectar to make bread which is the protein source of young larva [31]. The high abundance of individual bees per 1000 flowers and the attractiveness of G. hirsutum nectar and pollen for A. calens could be partially explained by the higher availability of these substances, their accessibility and the needs of A. calens during the flowering period of the Malvaceae. The absence of the passage of A. calens from G. hirsutum flowers to flowers of another plant species and vice versa could be explained by the fidelity of this solitary bee to the flowers of this plant during foraging bouts. This phenomenon is called floral constancy [32]. It is explained by the fact that the solitary bees in general are able to memorize the form, the color and the smell of the flowers visited during previous foraging trips [33]. These same observations have been made for honey bees on the flowers of cotton in Mayel-Ibe [3] (Maroua-Cameroon).
The disruption of visits by other insects in 2018 by Lasioglossum sp. 1 (3.77%) and Vespidae (sp. 1) (1.88%), then in 2019 by Vespidae (sp. 1) (2.65%) and Mylabris sp. (0.88%) reduced the duration of some A. calens visits. This obliged some individuals of this bee to visit more flowers during a foraging trip to maximize their pollen or nectar loads. Similar observations have been made on Apis mellifera workers foraging on the flowers of this Malvaceae in Maroua [3] and in Garoua [16].

Impact of Anthophilous Insects Including Amegilla calens on Gossypium hirsutum Yields
The numeric contribution of anthophilious insects to the fruiting rate, the number of seeds per fruit and the percentage of normal seeds of G. hirsutum was positive. During nectar and/or pollen harvest on G. hirsutum, foraging insect in general always shook flowers and regularly contacted anthers and stigma, increasing self-pollination and/or cross-pollination possibilities of this Malvaceae. Our results agreed with those obtained in: Mayel-Ibbe (Maroua) [3], Dang (Ngaoundere) [14] and Djamboutou (Garoua) [16] on this Malvaceae.

Pollination Efficiency of Amegilla calens on Gossypium hirsutum
During a single flower visit of A. calens for the collection of nectar and/or pollen on each flower, individuals of A. calens always come into contact with the stigma and anthers (100%) and thus increasing the possibilities of G. hirsutum pollination. They could thus enhance self-pollination by applying pollen of one flower on its own stigma. Amegilla calens could provide allogamous pollination through carrying of pollen with their hairs, legs, mouthparts, thorax and abdomen, which is then deposited on flowers belonging to a different plant of the same species (geitogamy) [30]. The intervention of A. calens on the pollination of G. hirsutum is especially probable since their abundance per 1000 flowers and their foraging speed were high. The positive and significant contribution of A. calens in the fruiting rate and the percentage of normal seeds of G. hirsutum is justified by the action of this bee on the pollination of visited flowers. This significant contribution of A. calens on the fruiting rate and the percentage of normal seeds of G. hirsutum is in agreement with similar findings for Apis mellifera [3] [14] [16] and M. vulpina [15] on the same Malvaceae.