Amegilla Sp. (Hymenoptera: Apidae) Visitors to Cotton (Gossypium Hirsutum, Malvaceae) Flowers and Their Role in Crop Pollination at Djoumassi (North, Cameroon)

Journal of Experimental Agriculture International

42(4): 145-157, 2020; Article no.JEAI.56961 ISSN: 2457-0591 (Past name: American Journal of Experimental Agriculture, Past ISSN: 2231-0606)

Amegilla sp. (Hymenoptera: Apidae) Visitors to Cotton (Gossypium hirsutum, Malvaceae) Flowers and Their Role in Crop Pollination at Djoumassi (North, Cameroon)

Adamou Moïse1,2*, Mazi Sanda1, Taimanga1,3 and Yatahaï Clément Minéo1

1Laboratory of Applied Zoology, Department of Biological Sciences, Faculty of Science, University of Ngaoundéré, P.O. Box 454, Ngaoundéré, Cameroon. 2Faculty of Medicine and Biomedical Sciences of Garoua, University of Ngaoundéré, P.O. Box 454, Ngaoundéré, Cameroon. 3Institute of Fisheries and Aquatic Sciences, Department of Agronomy, University of Douala, P.O. Box 2701, Douala, Cameroon.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors AM and MS designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors YCM and Taimanga managed the analyses of the study and the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JEAI/2020/v42i430509 Editor(s): (1) Dr. Fernandez-Luqueno, Fabian, Center for Research and Advanced Studies of the National-Polytechnic Institute (Cinvestav Saltillo), Mexico. Reviewers: (1) Lia Maris Orth Ritter Antiqueira, Universidade Tecnológica Federal do Paraná, Brasil. (2) Diana Salles Sampaio, Universidade Federal de Uberlândia, Brazil. Complete Peer review History: http://www.sdiarticle4.com/review-history/56961

Received 17 March 2020 Original Research Article Accepted 22 May 2020 Published 09 June 2020

ABSTRACT

Aims: To evaluate the impact of a single flower visit of Amegilla sp. on the production of Gossypium hirsutum. Study Design: A randomized complete block design was used in a split plot scheme of 437 m2 delimited and divided into eight subplots, each measuring 8*4.5 m2. Place and Duration of Study: Locality of Djoumassi, Benoue Division, in the city of Garoua, the North Region of Cameroon from June through October 2018 and 2019.

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*Corresponding author: E-mail: [email protected];

Moise et al.; JEAI, 42(4): 145-157, 2020; Article no.JEAI.56961

Methodology: The experiments were carried out on 540 flowers labeled at bud stage and divided in four treatments: two differentiated according to the presence or absence flowers protection regarding insect visits; the third protected and uncovered when flowers were opened, to allow Amegilla sp. visits and the fourth with the flowers protected, uncovered when they were opened, then rebagged without any visit. Results: Among 21 insect species recorded on G. hirsutum flowers, Amegilla sp. ranked second and harvested nectar and pollen. Throughout the pollination efficiency of a single flower visit, Amegilla sp. provoked a significant increase of the fruiting rate, the mean number of seeds per boll and the percentage of normal seeds by 14.89%, 12.93% and 7.01% respectively. Conclusion: The conservation and installation of Amegilla sp. nests close to G. hirsutum fields is recommended to improve its boll production and seed quality.

Keywords: Amegilla sp; Djoumassi; flowers; Gossypium hirsutum; nectar; pollen; production.

1. INTRODUCTION Previous researches have shown that Apis dorsata, Apis florea, Anthophora confuse and Pollination is one of the most important plant- Elis thoracica were the main visitors of G. animal interactions for reproduction of plants [1]. hirsutum flowers in India [15]. The primary According to Klein et al. [2] and Potts et al. [3], visitors observed by Pleasants and Wendel [16] almost 80% of wild plant species and agriculture on G. Tomentosum flowers in Hawaiian Island crops and 75% of the cultivated plant species were honeybees and carpenter bees [16]. Apis rely on insect pollinators for better fruits set and mellifera revealed their efficient role in the seeds production. Among insect pollinators, pollination of cotton flowers in Arizona in USA Honeybees Apis mellifera are the most widely [17], in Australia [18] and in Cameroon [13,14, known of all the bees [4]. They are the most 19]. In Brazil, Pires et al. [20] observed efficient pollinators of cultivated crops because Bumblebees (Bombus sp.) and Apoids on cotton their body parts are especially modified to pick flowers. In South Korea, Bombus terrestris was up pollen grains, they have body hair, have the main pollinator of G. hirsutum flowers [21]. In potential for long working hours, show flower Mexico, Velázquez-López et al. [22] have constancy and adaptability to different climates considered G. hirsutum as having a mixed [5-7]. Also, Research has shown that pollination reproductive strategy throughout its whole by honeybees increases fruit set, enhances fruit complex, rather than being primarily quality and reduces fruit drop [8]. autogamous.

Up to date, no previous study has been reported Currently, dramatic losses of managed on the relationships between G. hirsutum and honeybees with suspected worldwide losses of Amegilla sp. Besides, the activity and diversity of wild pollinators are focusing research on an flowering insects of a plant vary with place and impending still poorly documented pollination period [23]. The main objective of this study was crisis. However, alternative pollinators that are to contribute to the understanding of the not directly managed by humans, like wild bees, relationships between G. hirsutum and Amegilla are important in tropical agricultural crops [9]. sp. for their optimal management. It had four Among these wild bees, Amegilla sp. is highly specific objectives: determine the place of efficient as pollinator of crops [10,11] such as Amegilla sp. in G. hirsutum floral entomofauna; Gossypium hirsutum. study of the activity of this bee on cotton flowers;

assess the impact of flowering insects including Commonly known as cotton, G. hirsutum is one Amegilla sp. on boll and seed production of this of the most important crops for humanity. It is Malvaceae; evaluate the pollination efficiency of placed among the top ten most widely grown this Apidae on G. hirsutum. crops in the world even though its main purpose is not food [12]. In addition to the appreciation for 2. MATERIALS AND METHODS its ﬁbers, cultures learned to use the whole plant for many uses, from controlling reproduction and 2.1 Materials pharmaceuticals to pigments and cattle feed [12]. 2.1.1 Study site and experimental plot Cotton flowers produce nectar and pollen that attract insect pollinators that play a crucial role in The studies were conducted from June to the pollination biology of this Malvaceae [13,14]. October, in 2018 and from July to October 2019,

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in the locality of Djoumassi (latitude of treatment. The fruiting index (Pi) was then 9°23’16.512’N, a longitude of 13°23’20.627E, an calculated as described by Tchuenguem [28]: altitude of 169 m.a.s.l.), Benoue Division, in the city of Garoua, the North Region of Cameroon. Pi = Fb/Fa, where Fa is the number of viable This region belongs to the Sahel-Sudanian flowers initially set and Fb the number of the ecological zone [24]. It has Sudanian climate formed bolls. The allogamy rate (Alr) from which type characterized by two annual seasons: a derives the autogamy rate (Atr) was expressed rainy season (April to October) and a dry season as the difference in fruiting indexes between (November to March). August is the wettest treatment X (unprotected flowers) and treatment month of the year [24,25]. Annual rainfall varies Y (bagged flowers): from 750 to 1250 mm and the mean annual temperature is 27°C [24,26]. The experimental Atr = {[(PiX - PiY)/ PiX] * 100}, where PiX and plot was an area of 437 m2. PiY are the fruiting indexes in treatments X and Y respectively; Alr= 100 - Atr. 2.1.2 Biological material 2.2.3 Determination of the place of Amegilla The animal material was mainly represented by sp. on Gossypium hirsutum insects naturally present in the environment and entomofauna three and two nests of Amegilla sp. in 2018 and 2019 respectively, located close to the Observations were conducted on flowers of th experimental field the plant material was G. treatments 1 and 3, every day, from 26 nd th hirsutum seeds supplied by the Institute of September to 02 October 2018 and from 05 to th Research and Agricultural Development (IRAD) 12 October 2019. During each observation day, of Garoua in Cameroon. before starting visit counts, the number of opened flowers in each treatment was counted. 2.2 Methods Data were taken according to six daily time frames: 6 - 7 am, 8 - 9 am, 10 - 11 am, 12 - 13 2.2.1 Preparation of experimental plot, pm, 14 - 15 pm and 16 - 17 pm. In a slow walk sowing and weeding along all labeled flowers of treatments 1 and 3, the identity of insects that visited G. hirsutum th th rd th From 24 to 29 June 2018 and from 03 to 06 flowers was recorded [29]. All insects July 2019, the experimental plot was delimited, encountered on flowers were registered and the ploughed and divided into eight subplots, each cumulated results expressed as the number of 2 th measuring 8*4.5 m . On June 30 2018 and July visits to determine the relative frequency of each th 07 sowing was done on six lines per subplot, insect species in anthophilous entomofauna of G. each of which had 16 holes per line [27]. Four hirsutum [13]. Data obtained were used to seeds were sown per hole. Holes were determine the frequency of visits (Fi) of each separated 50 cm from each other, while lines insect species on G. hirsutum flowers. For each were 75 cm apart. From germination to the study period, Fi= [(Vi / Vt) * 100], with Vi the development of the first flower, the field was number of visits of insect i on treatment with regularly weeded with hoe and was performed unprotected flowers and Vt the total number of manually as necessary to keep plots weed-free insect visits of all recorded insect species on until the maturation of bolls. A week after these flowers recorded [29]. Specimens (3 to 5) germination, the plants were thinned and only for all insect taxa, excluded Apis mellifera were two were left per hole. caught using insect net on unlabeled flowers and conserved in 70 % ethanol, excluding butterflies 2.2.2 Determination of the reproduction mode that were preserved dry recorded [30] for of Gossypium hirsutum subsequent taxonomic identification.

On September 25th 2018, 240 flowers at bud 2.2.4 Study of the foraging activity of stage were labeled and divided in two Amegilla sp. on Gossypium hirsutum treatments: 120 unprotected flowers (treatment flowers 1) and 120 bagged flowers using gauze bags net to avoid all visits (treatment 2). On October 04th 2.2.4.1 Floral product harvested 2019, the same treatments were performed (treatments 3 and 4). For each cropping year, a The floral products (nectar or pollen) harvested week after shedding of the last labeled flower, by Amegilla sp. during each floral visit were the number of bolls was assessed in each recorded based on its foraging behavior. Nectar

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foragers were expected to extend their proboscis A1000= [(Ax / Fx) * 1000], where Fx and Ax are in the corolla, while pollen gatherers were respectively the number of flowers and the supposed to scratch anthers using mandibles number of foragers effectively counted on these and legs. During the same time that Amegilla sp. flowers at time x [34]. visits on flowers were registered, the type of floral product collected by this bee was noted The disruption of the activity of foragers by [28,29,31]. competitors or predators and the attractiveness exerted by other plant species on Amegilla sp. 2.2.4.2 Duration of visits and foraging speed was assessed by direct observations. For the second parameter, the number of times that this During the same days of taking data on bee left G. hirsutum flowers to other plant frequency of visits, the duration of individual species and vice versa was noted through the flower visits was recorded (using stopwatch) investigation period [28]. according to six time frames: 7 - 8 am, 9 - 10 am, 11 - 12 am, 13 - 14 pm, 15 - 16 pm and 17 - 18 During each daily period of investigation, ambient pm. The stopwatch, previously on zero was temperature and relative humidity in the station switched on as soon as an insect landed on a were registered every 30 minutes using a mobile flower. It was stopped when the insect leaves the thermo-hygrometer (HT9227) [28] installed in the flower. The related duration of visit corresponds shade. to the red value on the stop watch [32]. 2.2.5 Evaluation of the impact of the Moreover, the number of visits during which the flowering insects including Amegilla sp. bee came into contact with the stigma [32] was on Gossypium hirsutum yields registered. Concerning the foraging speed (Fs) which is the number of flowers visited by an Parallel to the constitution of treatments 1, 2, 3 individual bee per minute [33], data were and 4, 600 flowers at bud stage were protected registrated during the same dates and according in 2018 and 2019 and two treatments were to same time frames and daily period as for formed: duration of visits. The stopwatch, previously on zero was switched on as soon as an insect -Treatment 5: In 2018, 200 flowers were landed on a flower and stopped when it protected using gauze bag nets to prevent insect disappears. Concomitancy, the number of visited visits and destined to be visited exclusively by flowers was counted. The foraging speed was Amegilla sp. In 2019, the same treatment was calculated using the following formula: performed (treatment 7). As soon as the flowers were opened, each flower of treatments 5 and 7 Fs = (Nf / dv) * 60, where dv is the time (sec) were inspected. Hence, gauze bag was given by a stopwatch and Nf the number of delicately removed and this flower was observed flowers visited during dv. for up to 10 minutes; the flowers visited by Amegilla sp. were marked and then reprotected. During the observation period, when a forager returns to previously visited flower, counting is -Treatment 6: In 2018, 100 flowers were performed as two different flowers [28]. protected using gauze bag nets and destined to be uncovered then rebagged without the visit of 2.2.4.3 Abundance per flower and per 1000 insects or any other organism. In 2019, the same flowers treatment was performed (treatment 8). As soon The abundance of foragers (highest number of as each flower of treatments 6 and 8 was individuals foraging simultaneously) per flower opened, the gauze bag was removed and the flower was observed for up to 10 minutes while and per 1000 flowers (A1000) were recorded on the same dates and daily periods as the avoiding the visit by Amegilla sp. or any other registration of duration of visits. Abundance per organism. flower was recorded as a result of direct counting. For determining the abundance per At maturity, bolls were harvested and counted 1000 flowers, foragers were counted on a known from each treatment. The mean number of seeds number of opened flowers and A1000 was per boll, the percentage of normal (well calculated using the following formula: developed) seeds [29] were then evaluated.

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For each observed year, the fruiting rate square ( χ2) for the comparison of percentages due to the flowering insects including Amegilla using Microsoft Excel 2016 software. sp. (Pri) was calculated using the following formula: 3. RESULTS

Pri = {[(PX - PZ) / (PX + PY - PZ)] * 100} [35], 3.1 Reproduction Mode of Gossypium where PX, PY and PZ are the fruiting rates in hirsutum treatment X (flowers left in free pollination), treatment Y (flowers protected from all insect The fruiting indexes of G. hirsutum were 0.94, visits) and treatment Z (flowers bagged then 0.88,0.86 and 0.68 for treatments 1, 2, 3 and 4. uncovered and rebagged without insect or any other organism visit). Thus, in 2018, the autogamy rate was 79,07% and the allogamy rate was 20.93%. In 2019, the The fruiting rate of a treatment (Pr) is giving by corresponding figures were 93.62% and 6.38%. the following formula: Pr = [(b / a) * 100], where a For the two cumulative years, the autogamy rate is the number of viable flowers initially set and b was 86.35% and the allogamy rate was 13.66%. the number of formed bolls. It appears that G. hirsutum has a mixed The impact of flower visiting insects including reproduction mode with the predominance of Amegilla sp. on the number of seeds per boll and autogamy over allogamy. the percentage of normal seeds were evaluated using the same method as mentioned above for 3.2 Activity of Amegilla sp. on the fruiting rate. Gossypium hirsutum Flowers

2.2.6 Assessment of the pollination efficiency 3.2.1 Frequency of visits of Amegilla sp. on Gossypium hirsutum

Amongst the 288 and 342 visits of 15 and 21 The contribution of Amegilla sp. on the fruiting insect species recorded on its flowers in 2018 rate, the number of seeds per boll and the and 2019 respectively, Apis mellifera ranked first percentage of normal seeds was calculated with 139 (48.26%) and 178 (52.05%) visits in using the data of treatments 5 and 6 for 2018 2018 and 2019 respectively. Amegilla sp. ranked and those of treatments 7 and 8 for 2019. second with 96 (33.33%) and 84 (24.56%) visits

in 2018 and 2019 respectively (Table 1). The For each observed year, the contribution of difference between these two percentages is Amegilla sp. on the fruiting rate (PrX) was calculated using the following formula: PrX= significant (2 = 5.88; df= 1; P<0.05).

{[(PC - PZ) / PC] * 100}, where PC is the fruiting 3.2.2 Floral products harvested rate in treatment C (flowers visited exclusively by the Amegilla sp. individuals) and PZ is the fruiting From our observations and during the two rate in treatment Z (uncovered flowers then rebagged without the visit of insects or any other flowering periods, Amegilla sp. were found to organism) [29]. harvested intensively pollen and slightly nectar on flowers from G. hirsutum (Fig. 1).

The impact of Amegilla sp. on the number of seeds per boll and the percentage of normal seeds were evaluated using the same method as mentioned above for the fruiting rate.

2.2.7 Data analysis

Data were analyzed using descriptive statistics (means, standard deviation and percentages), ANOVA (F) for the general comparison of means of more than two samples, student’s t-test for the comparison of means of two samples, Pearson correlation coefficient (r) for the study of the Fig. 1. Amegilla sp. collecting pollen in association between two variables and chi- Gossypium hirsutum flower at Djoumassi

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Table 1. Diversity of insects collecting pollen and nectar on Gossypium hirsutum flowers in Djoumassi, Cameroon during 2018 and 2019 seasons

Insects 2018 2019 Total Order Family Genus and species n1 P1 (%) n2 P2 (%) nT PT (%) Coleoptera Coccinellidae Cheilomenes lunata (Po) 6 2.08 10 2.92 16 2.5 Meloidae Coryna sp. (Po) 2 0.69 3 0.88 5 0.79 Diptera Calliphoridae (1 sp.) (Ne) 4 1.39 2 0.58 6 0.99 Muscidae Musca domestica (Ne) 5 1.74 7 2.05 12 1.9 Syrphidae Episyrphus sp. (Ne) 8 2.78 5 1.46 13 2.12 (1 sp.) (Ne) 1 0.35 3 0.88 4 0.62 Heteroptera Pentatomidae (1 sp.) (Po) 3 1.04 2 0.58 5 0.81 Hymenoptera Apidae Amegilla sp. (Ne, Po) 96 33.33 84 24.56 180 28.95 Apis mellifera (Ne, Po) 139 48.26 178 52.05 317 50.16 Formicidae Camponotus sp. (Ne) - - 5 1.46 5 0.73 Myrmicaria opaciventris (Ne) 3 1.04 1 0.29 4 0.67 Halictidae Macronomia vulpina (Ne, Po) 8 2.78 10 2.92 18 2.85 Vespidae Belonogaster juncea (Ne) 5 1.74 3 0.88 8 1.31 (1 sp. 1) (Ne) - - 2 0.58 2 0.29 (1 sp.2) (Ne) 3 1.04 4 1.17 7 1.11 Lepidoptera Nymphalidae (1 sp.) (Ne) - - 3 0.88 3 0.44 Pieridae Eurema sp. (Ne) 4 1.39 2 0.58 6 0.29 Papilio demodocus - - 11 3.22 11 1.61 (1 sp.) (Ne) - - 3 0.88 3 0.44 Orthoptera Acrididae Tettigonia viridissima (Po) - - 2 0.58 2 0.29 (1 sp.) (Po) 1 0.35 2 0.58 3 0.47 Total 288 100 342 100 630 100 15 21 21 n1: number of visits on 120 flowers in seven days; n2: number of visits on 120 flowers in eight days; pT= percentage of visits p1= (n1 / 288) *100; p2 (n2 / 342)*100; Comparison of percentages of Amegilla sp. visits (2018/2019): 2 = 5.88; df= 1; P< 0.05; po: collection of pollen; ne: collection of nectar; sp.: unidentified species

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3.2.3 Relationship between visits and between the number of Amegilla sp. visits and flowering stages the temperature in 2018 (r = 0.13; df = 4; P>0.05) and is highly significant in 2019 (r = 0.92; df = 4; The visits of Amegilla sp. were more numerous P<0.05). on treatments 1 and 3 when the number of opened flowers was high (Fig. 2A and B). The 3.2.5 Duration of a visit per flower correlation was highly significant between the number of G. hirsutum opened flowers and the In 2018, the mean visit duration by Amegilla sp. number of Amegilla sp. visits in 2018 (r = 0.94; df was 203.62 sec (n =93; s = 177.99; maxi = 1020 = 5; P< 0.01) as well as in 2019 (r = 0.95; df = 6; sec) and 324.35 sec (n =99; s = 284.62; maxi = P< 0.001). 1169 sec) for pollen and nectar collections respectively. In 2019, the corresponding figures 3.2.4 Daily visits were 231.64 sec (n =63; s = 202.64; maxi = 1063 sec) and 339.96 sec (n =73; s = 291.90; maxi = Amegilla sp. was active on G. hirsutum flowers 1201 sec) for pollen and nectar collection throughout the day with the peak situated respectively. The difference between the means between 10 and 11 am in 2018 as well as in duration of the visit for pollen harvest in 2018 and 2019 (Fig. 3). The correlation is not significant 2019 is not significant (t = 0.88, df = 154, P> between the number of this bee visits and 0.05). Also, the difference between the relative humidity in 2018 (r = 0.21; df = 4; P> means duration of visit for nectar in 2018 and 0.05) as well in 2019 (r = 0.02; df = 4; P> 0.05) 2019 is not significant (t = 0.35, df = 170, P> (Fig. 3A and B). The correlation is not significant 0.05).

(A)

(B) Fig. 2. Seasonal variations of the number of Gossypium hirsutum opened flowers and the number of Amegilla sp. visits on these organs in 2018 (A) and 2019 (B) at Djoumassi

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(A)

(B)

Fig. 3. Variation of the temperature, the humidity and the number of Amegilla sp. visits on Gossypium hirsutum flowers according to the daily frames time in 2018 (A) and 2019 (B) at Djoumassi

For the two cumulated years‚ the mean 2018 and that in 2019 is not significant (t = 0.08; duration visit for pollen harvest was 217.63 P > 0.05). sec and 332.16 sec for nectar harvest. 3.2.7 Foraging speed of Amegilla sp. on Gossypium hirsutum flowers 3.2.6 Abundances of Amegilla sp. During observations, Amegilla sp. visited In 2018, the highest mean number of Amegilla between 0.22 and 14.21 flowers/min in 2018 and sp. simultaneously active was one per flower (n = between 0.4 and 12.90 flowers/min in 2019. The 57, s = 0) and 233.33 per 1000 flowers (n = 57; s mean foraging speed was 1.90 flowers/min (n = = 104.08; maxi = 500). In 2019, the 70, s= 2.34) in 2018 and 2.67 flowers/min (n = corresponding figures were 1 per flower (n = 69; 62, s = 2.63) in 2019. The difference between s = 0) and 234.78 per 1000 flowers (n = 69; s = these means is not significant (t = 1.75; df= 130; 99.74, maxi = 500). The difference between the P> 0.05). For the two cumulated years, the mean mean number of Amegilla sp. per 1000 flowers in foraging speed was 2.29 flowers/min.

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3.3 Influence of Neighboring Floral higher than that of flowers bagged during their flowering period (treatment 2). During each observation periods, flowers of • The means number of seeds per boll were many other plant species growing in the study 24.29, 18.5, 25.02, 20.15, 23, 20, 22.90 and area were visited by Amegilla sp., for nectar and 20 in treatments 1 to 8 respectively. The / or for pollen. Among these plants were: differences between these eight Abelmoschus esculentus, Hibiscus sabdariffa percentages are highly significant (F= and Vigna unguiculata. During the whole 149.65; df1= 7; df2 = 813; P< 0.001). Two - observation periods, Amegilla sp. foraging on G. to - two comparisons showed that the hirsutum were not seen flying from G. hirsutum difference observed was highly significant flowers to the neighboring plant flowers and vice between treatments 1 and 2 (t= 36.24; df= versa. 184; P< 0.001) as well as between treatments 3 and 4 (t= 6.03; df= 216; P< 3.4 Influence of Wildlife 0.001). Hence, in 2018 and 2019, the mean number of seeds per boll from exposed The foragers of Amegilla sp. were disturbed in flowers was higher than that from their foraging activity by other arthropods that flowers bagged during their flowering period. were either by competitors for nectar and/or • The percentages of normal seeds were pollen or by other biotic and abiotic factors. 91.61%, 81.34%, 92.96%, 82.31%, 88.96%, These disturbances resulted in the interruption of 83.65%, 89.40% and 82.21% in treatments 1 some visits. In 2018, for 96 visits of Amegilla sp., to 8 respectively. The differences between four (4.16%) were interrupted by wind, three these eight percentages are highly (3.13%) by Amegilla sp. individuals, five (5.21%) significant (χ2= 300.14; df= 7; P< 0.001). by Cheilomenes lunata and 13 (13.54%) by Two - to - two comparisons showed that the Belonogaster juncea. While in 2019 for 84 visits, difference observed was highly significant three (3.57%) were interrupted by wind, 11 between treatments 1 and 2 (χ2= 94.99; df= (13.10%) by A. mellifera and seven (8.33%) by 1; P< 0.001) as well as between treatments Cheilomenes lunata. For their load of nectar 3 and 4 (χ2= 124.42; df= 1; P< 0.001). and/or for pollen, some individuals of Amegilla Hence, in 2018 and 2019, the percentage of sp. who suffered such disturbances were forced normal seeds from exposed flowers was to visit more flowers and/or plants during the higher than that from flowers bagged during corresponding foraging trip. their flowering period. 3.5 Impact of Flowering Insects Including In 2018, the contributions of the anthophilous Amegilla sp. on Gossypium hirsutum insects including Amegilla sp. in the fruiting rate, Yields the mean number of seeds per boll and the percentage of normal seeds of G. hirsutum were The fruiting rate, the mean number of seeds per 34.29%, 18.82% and 8.91% respectively. In boll and the percentage of normal seeds in the 2019, the corresponding figures were 18.73%, different treatments of G. hirsutum are shown in 19.94% and 11.55% respectively. For the two Table 2. cumulate years, the contributions of flowering This table shows that: insects were 26.51%, 19.38% and 10.23% for the fruiting rate, the mean number of seeds per • The fruiting rates were 86.66%, 68.33%, boll and the percentage of normal seeds 94.17%, 87.5%, 70%, 51%, 76% and 74% in respectively. treatments 1 to 8 respectively. The differences between these eight 3.6 Pollination Efficiency of Amegilla sp. percentages are highly significant (χ2= on Gossypium hirsutum 91.63; df= 7; P< 0.001). Two - to - two comparisons showed that the difference During pollen and nectar harvests on the cotton observed is highly significant between flowers, individuals of Amegilla sp. always came treatments 1 and 2 (χ2= 11.57; df= 1; P< into contact with anthers and stigma, increasing 0.001) and not significant between the possibilities of G. hirsutum pollination. With treatments 3 and 4 (χ2= 3.20; df= 1; P > this pollen, they flew frequently from flowers to 0.05). Consequently, in 2018, the fruiting flowers inside the same or different individuals of rate of exposed flowers (treatment 1) was G. hirsutum.

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Table 2. Fruiting rate, number of seeds per boll and percentage of normal seeds according to different treatments of Gossypium hirsutum in 2018 and 2019 at Djoumassi

Years Treatments NF NFF FrR (%) Seeds/boll TNS NS %NS m sd 2018 1 (Uf) 120 104 86.66 24.29 1.41 2478 2270 91.61 2 (Pf) 120 82 68.33 18.5 0.71 1635 1330 81.34 2019 3 (Uf) 120 113 94.17 25.02 6.17 2727 2535 92.96 4 (Pf) 120 105 87.5 20.15 5.70 1864 1526 82.31 2018 5 (FvA) 200 140 70 23.04 14.85 3225 2869 88.96 6 (Fbwv) 100 51 51 20 3.49 838 701 83.65 2019 7 (FvA) 200 152 76 22.90 5.38 3481 3112 89.40 8 (Fbwv) 100 74 74 20 4.57 1400 1151 82.21 Uf: unprotected flowers; Pf: protected flowers; FvA: flowers bagged then uncovered and exclusively visited by Amegilla sp.and rebagged; Fbwv: flowers bagged then uncovered and rebagged without insect or any other organism visit; NF: Number of flowers; NFF: Number of formed fruit; FrR: fruiting rate; TNS: Total number of seeds; NS: Normal seeds; % NS: Percentage of normal seeds; m: mean; sd: standard deviation

The comparison of the fruiting rate (Table 2) seeds due to Amegilla sp. were 27.14%, 13.19% shows that the difference observed between and 5.97% respectively. In 2019, the 2 treatments 5 and 6 is highly significant (χ = corresponding figures were 2.63%, 12.66% and 11.81; df= 1; P < 0.001) and not significant 8.04% respectively. 2 between treatments 7 and 8 (χ = 0.08; df= 1; P > 0.05). Therefore, in 2018, the fruiting rate of For the two cumulated years, the numeric flowers protected and visited exclusively by contribution of Amegilla sp. via a single flower Amegilla sp. (treatments 5 and 7) was higher visit on the fruiting rate, the mean number of than that of flowers bagged, opened and closed seeds per boll and the percentage of normal without the visit of insect or any other organism seeds were 14.89%, 12.93% and 7.01% (treatments 7 and 8). respectively.

The comparison of the mean number of seeds 4. DISCUSSION per boll (Table 2) shows that the difference observed between treatments 5 and 6 is Results obtained from these experiments significant (t = 2.25; df= 189; P <0.05) and highly indicated that G. hirsutum has a mixed significant between treatments 7 and 8 (t = 4.20; reproduction mode that is allogamous- df= 224; P <0.001). These results pointed out autogamous with the predominance of autogamy that flowers visited by Amegilla sp. have the over allogamy. The same result has been highest number of seeds per boll (treatments 5 obtained by Basga et al. [13] at Djamboutou in and 7) than that of flowers bagged, opened and Cameroon, indicating that autogamy rebagged without visit of insect or any other predominates over allogamy to G. hirsutum. At organism (treatments 6 and 8). Dang [14] and Maroua [19] in Cameroon according to Mazi et al. [14] and Dounia et al. The comparison of the percentage of normal [19] allogamy predominates over autogamy to seeds (Table 2) shows that the difference this plant species. observed between treatments 5 and 6 is highly 2 significant (χ = 18.25; df= 1; P < 0.001) and Amegilla sp. was the second insect visitor of 2 treatments 7 and 8 (χ = 48.38; df= 1; P < 0.001). cotton flowers in 2018 as well in 2019 and Hence, in 2018 and 2019, the percentage of harvested pollen and nectar. The significant normal seeds of flowers protected and difference between the two percentages of visited exclusively by Amegilla sp. was higher Amegilla sp. visit for the two study years could be than that of flowers bagged, opened and the consequence of climatic factors and seasonal rebagged without visit of insect or any other variations in flower resources availability. It can organism. also be attributed to the variation of the number of Amegilla sp. nests in the study site from one In 2018, the fruiting rate, the mean number of year to another (three nests in 2018 and two in seeds per boll and the percentage of normal 2019).

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Other observations have revealed that A. During the collection of nectar and/or pollen on mellifera was the most frequent insect visitors on each flower, Amegilla sp. individuals regularly flowers of G. hirsutum after Lasioglossum sp. come into contact with the stigma and anthers. [14] and Macronomia vulpina [19] at Dang and at They were also able to carry pollen with their Maroua in Cameroon respectively. This shows hairs, legs and mouth accessories from a flower the attractiveness of A. mellifera workers with of one plant to stigma of another flower of the respect to the floral products of G. hirsutum. same plant (geitonogamy), to the same flower (autogamy) or to that of another plant The peak of activity of this bee was situated (xenogamy). The foragers can thus influence between 10-11 h, which should be the daily self-pollination and cross-pollination [39] This has period of highest availability of nectar and pollen also been observed by others studies such as on on this Malvaceae flowers. The activity of this A. mellifera at Dang [14], Maroua [19] and bee was conditioned by the temperature and Djamboutou [13] in Cameroon. humidity of the study area. In Cameroon, particularly at Dang [14] and Maroua [19], the 5. CONCLUSION same observations have been made on A. mellifera. The results obtained from this study reveal that Gossypium hirsutum is a plant that highly The high abundance of foragers per 1000 benefits from the pollination by insects. Amegilla flowers, and the positive and significant sp. is the efficient insect of cotton and harvests correlation between the number of G. hirsutum nectar and pollen. The comparison of boll and flowers and the number of the visits of this bee, seed sets of flowers visited exclusively by underscores the attractiveness of G. hirsutum Amegilla sp. with those of flowers bagged then nectar and pollen for Amegilla sp. This opened and rebagged without visit of this bee or attractiveness could be partially explained by its any other organism underscores the value of high production and the accessibility of these Amegilla sp. in increasing the fruiting rate, the products [13,14,19]. mean number of seeds per boll and the The significant difference observed between the percentage of normal seeds of G. hirsutum. duration of pollen harvest and that of nectar Thus, the conservation and installation of collection could be explained by the accessibility Amegilla sp. nests close to G. hirsutum is or availability of each of these floral products or recommended to improve its boll production as the variation of diversity of flowering insects in well as seed quality and to stabilize the 2018 and in 2019. Pollen is produced by the population of this bee in the Garoua region. anthers, which are on the top of the stamen and are thus easily accessible to Amegilla sp. CONSENT whereas nectar is between the base of style and stamens and is thus less accessible [36]. The All authors declare that ‘’written informed consent weight of Amegilla sp. played a positive role: was obtained from the patient (or other approved when collecting nectar and/or pollen, by shaking parties) for publication of this case report and flowers. This movement could facilitate the accompanying images. A copy of the written liberation of pollen by anthers, for the optimal consent is available for review by the Editorial occupation of the stigma. This phenomenon was office/Chief Editor/Editorial Board members of also reported by Dounia and Tchuenguem. [37] this journal." for Macronomia vulpina on flowers of the same plant species at Maroua. COMPETING INTERESTS

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