Apis Mellifera L.) in Yield Response of Canola Under Rainfed Conditions

POLLINATION EFFICIENCY AND ROLE OF MANAGED HONEYBEES (APIS MELLIFERA L.) IN YIELD RESPONSE OF CANOLA UNDER RAINFED CONDITIONS

TASLEEM AKHTAR

07-arid-168

Department of Entomology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi Pakistan 2019

POLLINATION EFFICIENCY AND ROLE OF MANAGED HONEYBEES (APIS MELLIFERA L.) IN YIELD RESPONSE OF CANOLA UNDER RAINFED CONDITIONS

TASLEEM AKHTAR

(07-arid-168)

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Entomology

Department of Entomology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi Pakistan 2019

ii iii iv

vii

DEDICATION

I Dedicate This Humble Effort to My Parents for Their

Encouragement and Tremendous Love for Me

viii

CONTENTS

Page

List of Tables x

List of Figures xi

Acknowledgments xix

ABSTRACT xx

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 6

2.1 POLLINATOR FAUNA OF CANOLA 7

2.2 ABUNDANCE OF APIS MELLIFERA ON CANOLA 10

2.3 FORAGING BEHAVIOR IN TERMS OF VISITATION 10

FREQUENCY OF APIS MELLIFERA

2.4 IDENTIFICATION AND QUANTIFICATION OF FLORAL 12

SOURCES OF APIS MELLIFERA

2.5 IMPACT OF WEATHER CONDITIONS ON FORAGING RATE 13

OF APIS MELLIFERA

2.6 IMPACT OF BEEHIVES DISTANCE ON FORAGING RATE OF 15

APIS MELLIFERA

2.7 EFFECT OF HONEYBEE POLLINATION ON QUALITATIVE 15

AND QUANTITATIVE PARAMETERS OF CANOLA

3. MATERIALS AND METHODS 18

3.1 STUDY AREA 18

3.2 SOWING OF CROP 18

3.3 IDENTIFICATION OF ALL POLLEN SOURCES DURING 18

BLOOMING PERIOD OF BRASSICA NAPUS FROM

FORAGERS OF APIS MELLIFERA

3.3.1 Identification of Pollen Sources Transported by Apis mellifera 18

3.3.2 Pollen QuantificationTransported by Apis mellifera 20

3.4 DETERMINATION OF COLONY LEVEL POLLINATION 20

EFFICIENCY OF APIS MELLIFERA ON BRASSICA NAPUS

CROP

3.4.1 Colony Foraging Rate 21

3.4.2 Impact of Weather Factors on Foraging Rate of Apis mellifera 21

3.4.3 Colony Condition of Apis mellifera 21

3.4.4 Data Analysis 21

3.5 CONTRIBUTION OF POLLINATION BY MANAGED APIS 22

MELLIFERA IN YIELD OF BRASSICA NAPUS

3.5.1 Visitation Frequency of Managed Apis mellifera 22

3.5.2 Pollinators Diversity 22

3.5.3 Pollination Efficacy 23

3.5.3.1 Agro morphological parameters 23

3.5.3.1.1 Number of pods plant -1 23

3.5.3.1.2 Number of seeds plant-1 23

3.5.3.1.3 Seed weight pods -100 23

3.5.3.2 Seed quality parameters 23

3.5.3.2.1 Oil contents (%) 23

3.5.3.2.2 Protein contents (%) 24

3.5.3.2.3 Fatty acid profile 24

3.5.3.2.4 Germination rate (%) 24

3.5.4 Data Analysis 24

3.6 EFFECT OF BEEHIVES DISTANCE ON COLONY LEVEL 25

POLLINATION EFFICACY OF APIS MELLIFERA ON

BRASSICA NAPUS

3.6.1 Colony Foraging Rate of Apis mellifera 25

3.6.2 Colony Condition of Apis mellifera 25

3.6.3 Impact of Weather Factors on Foraging Rate of Apis mellifera 26

3.6.4 Data Analysis 26

4. RESULTS AND DISCUSSION 27

4.1 IDENTIFICATION OF POLLENS COLLECTED FROM 27

BODIES OF APIS MELLIFERA DURING BLOOMING PERIOD

OF BRASSICA NAPUS

4.1.1 Identification of Pollen Sources 27

4.1.2 Quantification of Pollens Transported by Apis mellifera Workers 29

4.2 DETERMINATION OF COLONY LEVEL POLLINATION 38

EFFICIENCY OF APIS MELLIFERA ON BRASSICA NAPUS

CROP

4.2.1 Colony Foraging Rate 38

4.2.2 Impact of Weather Condition on Foraging Rate 41

4.2.3 Colony Condition 45

4.3 CONTRIBUTION OF POLLINATION BY MANAGED APIS 56

MELLIFERA IN YIELD OF BRASSICA NAPUS

4.3.1 Visitation Frequency of Managed Apis mellifera 56

4.3.2 Diversity and Abundance of Insect Pollinators on Brassica napus 63

4.3.3 Pollination Efficacy 77

4.3.3.1 Agro- morphological parameters 77

4.3.3.1.1 Total number of pods plant-1 77

4.3.3.1.2 Total number of seeds plant-1 77

4.3.3.1.3 Seed weight 100 pods -1 78

4.3.3.2 Seed quality parameters 84

4.3.3.2.1 Oil contents (%) 84

4.3.3.2.2 Protein contents (%) 84

4.3.3.2.3 Oleic acid (%) 84

4.3.3.2.4 Linolenic acid (%) 85

4.3.3.2.5 Germination rate (%) 85

4.4 IMPACT OF BEEHIVES DISTANCE ON COLONY LEVEL 89

POLLINATION EFFICIENCY OF APIS MELLIFERA ON

BRASSICA NAPUS

4..4.1 Colony Foraging Rate 89

4.4.2 Colony Condition with Different Beehives Distance from Brassica 98

napus

4.4.3 Correlation of Weather Factors with Foraging Rate of Apis mellifera 111

CONCLUSION 123

SUMMARY 124

LITERATURE CITED 132

APPENDICES 157

xii

List of Tables

Table No. Page

4.1.1 Different floral sources visited by Apis mellifera during Brassica 30

napus blooming period at URF Koont, Gujar Khan during 2015.

4.1.2 Total numbers of pollen sources visited by Apis mellifera recorded 36

during blooming period of Brassica napus at URF Koont, Gujar

Khan during 2015.

4.1.3 Weekwise Percentage of polleniferous flora of Apis mellifera during 42

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.1 Correlation between weather factors and colony foraging rate of Apis 58

mellifera during Brassica napus blooming period at URF Koont,

Gujar Khan during 2015.

4.3.1 Abundance (%) and foraging behavior of Brassica napus (insect 80

visitors) at URF Koont, Gujar Khan during 2015.

4.3.2 Overall foraging activity of Brassica napus (insect visitors) at 82

different time intervals at URF Koont, Gujar Khan during 2015.

4.4.1 Correlation between weather factors and Apis mellifera colony 121

foraging rate for Brassica napus L. at URF Koont, Gujar Khan during

2016.

xiii

List of Figures

Figure No. Page

4.1.1 Overall Percentage of pollens collected from different plants by 44

Apis mellifera foragers during blooming period of Brassica napus

at URF Koont, Gujar Khan during 2015.

4.2.1 Colony foraging rate (Mean± SE) of Apis mellifera at different 46

timeintervals on 06-01-2015 (1st week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.2 Colony foraging rate (Mean± SE) of Apis mellifera at different 47

time intervals on 20-01-2015 (3rd week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.3 Colony foraging rate (Mean± SE) of Apis mellifera at different 48

time intervals on 27-01-2015 (4th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.4 Colony foraging rate (Mean± SE) of Apis mellifera at different 49

time intervals on 03-02-2015 (5th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.5 Colony foraging rate (Mean± SE) of Apis mellifera at different 50

time intervals on 10-02-2015 (6th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

xiv

during 2015.

4.2.6 Colony foraging rate (Mean± SE) of Apis mellifera at different 51

time intervals on 17-02-2015 (7th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.7 Colony foraging rate (Mean± SE) of Apis mellifera at different 52

time intervals on 10-03-2015 (10th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.8 Colony foraging rate (Mean± SE) of Apis mellifera at different 53

time intervals on 17-03-2015 (11th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.9 Colony foraging rate (Mean± SE) of Apis mellifera at different 54

time intervals on 24-03-2015 (12th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.10 Average of nine weeks of colony foraging rate of Apis mellifera 55

at different time intervals during blooming period of

Brassicanapus at URF Koont, Gujar Khan during 2015.

4.2.11 Temperature (ͦC), relative humidity (%) and rainfall (mm) along 59

with colony foraging rate of Apis mellifera during blooming

period of Brassica napus at URF Koont, Gujar Khan during

2015.

4.2.12 Brood population area (cm2) per colony of Apis mellifera in 61

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

4.2.13 Stored food area (cm2) per colony of Apis mellifera in blooming 62

period of Brassica napus at URF Koont, Gujar Khan during

2015.

4.3.1 Visitation frequency (Mean ± SE) of Apis mellifera on 06-01- 66

2015 (1st weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.2 Visitation frequency (Mean ± SE) of Apis mellifera on 20-01- 67

2015 (3rd weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.3 Visitation frequency (Mean ± SE) of Apis mellifera on 27-01- 68

2015 (4th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.4 Visitation frequency (Mean ± SE) of Apis mellifera on 03-02- 69

2015 (5th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.5 Visitation frequency (Mean ± SE) of Apis mellifera on 10-02- 70

2015 (6th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.6 Visitation frequency (Mean ± SE) of Apis mellifera on 17-02- 71

2015 (7th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

xvi

4.3.7 Visitation frequency (Mean ± SE) of Apis mellifera on 10-03- 72

2015 (10th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.8 Visitation frequency (Mean ± SE) of Apis mellifera on 17-03- 73

2015 (11th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.9 Visitation frequency (Mean ± SE) of Apis mellifera on 24-03- 74

2015 (12th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

4.3.10 Overall average of visitation frequency (Mean± SE) of Apis 75

melliferaat different time intervals on Brassica napusat URF

Koont, Gujar Khan during 2015.

4.3.11 Numbers of pods per canola plant in response to different 83

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar

Khan during 2015.

4.3.12 Numbers of seeds per canola plant in response to different 86

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar

Khan during 2015.

4.3.13 Seeds weight 100 canola pods-1 in response to different 87

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar

Khan during 2015.

xvii

4.3.14 Percentage of oil contents of canola seeds in response to different 88

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar

Khan during 2015.

4.3.15 Percentage of protein contents of canola seeds in response to 91

different treatments (T1= Open plots allowing free visits of bees+

other pollinators, T2= Plots caged without bees) at URF Koont,

Gujar Khan during 2015.

4.3.16 Percentage of oleic acid of canola seeds in response to different 92

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar

Khan during 2015.

4.3.17 Percentage of linoleic acid of canola seeds in response to 93

different treatments (T1= Open plots allowing free visits of bees+

other pollinators, T2= Plots caged without bees) at URF Koont,

Gujar Khan during 2015.

4.3.18 Percentage of germination rate of canola seeds in response to 94

different treatments (T1= Open plots allowing free visits of bees+

other pollinators, T2= Plots caged without bees) at URF Koont,

Gujar Khan during 2015.

4.4.1 Effect of beehives distance from Brassica napus crop on colony 99

foraging rate of Apis mellifera (Mean± SE) per 10 minutes on 04-

02-2016 (1st weekly interval) at URF Koont, Gujar Khan during

2016.

xviii

4.4.2 Effect of beehives distance from Brassica napus crop on colony 100

foraging rate of Apis mellifera (Mean± SE) per 10 minutes on 18-

02-2016 (3rd weekly interval) at URF Koont, Gujar Khan during

2016.

4.4.3 Effect of beehives distance from Brassica napus crop on colony 101

foraging rate of Apis mellifera (Mean± SE) per 10 minutes on 25-

02-2016 (4th weekly interval) at URF Koont, Gujar Khan during

2016.

4.4.4 Effect of beehives distance from Brassica napus crop on colony 103

foraging rate of Apis mellifera (Mean± SE) per 10 minutes on 03-

03-2016 (5th weekly interval) at URF Koont, Gujar Khan during

2016.

4.4.5 Effect of beehives distance from Brassica napus crop on colony 104

foraging rate of Apis mellifera (Mean± SE) per 10 minutes on 10-

03-2016 (6th weekly interval) at URF Koont, Gujar Khan during

2016.

4.4.6 Effect of beehives distance from Brassica napus crop on colony 105

foraging rate of Apis mellifera (Mean± SE) per 10 minutes on 24-

03-2016 (8th weekly interval) at URF Koont, Gujar Khan during

2016.

4.4.7 Brood and stored food area of Apis mellifera at different hives 113

distance (Mean±SE) on 02-02-2016 (1st weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

xix

4.4.8 Brood and stored food area of Apis mellifera at different hives 114

distance (Mean±SE) on 11-02-2016 (2nd weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.9 Brood and stored food area of Apis mellifera at different hives 115

distance (Mean±SE) on 18-02-2016 (3rd weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.10 Brood and stored food area of Apis mellifera at different hives 116

distance (Mean±SE) on 25-02-2016 (4th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.11 Brood and stored food area of Apis mellifera at different hives 117

distance (Mean±SE) on 03-03-2016 (5th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.12 Brood and stored food area of Apis mellifera at different hives 118

distance (Mean±SE) on 10-03-2016 (6th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.13 Brood and stored food area of Apis mellifera at different hives 119

distance (Mean±SE) on 17-03-2016 (7th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.14 Brood and stored food area of Apis mellifera at different hives 120

distance (Mean±SE) on 25-03-2016 (8th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

4.4.15 Effect of temperature (ͦC), relative humidity (%) and rainfall 122

(mm) on Apis mellifera visits/10 minutes per canola plant at

weekly interval on Brassica napus in URF Koont, Gujar khan

during 2016.

xxi

ACKNOWLEDGEMENTS

First of all, praise is due to ALMIGHTY ALLAH with his compassion and mercifulness to allow me finalizing this Ph. D dissertation. Countless salutations the most perfect ,(صلیاہلل علیہ وسلم ) for HOLY PROPHET MUHAMMAD among and of ever born on earth, who is forever a true torch of guidance and knowledge for humanity as a whole.

I would like to express my sincerest gratitude to my supervisor Dr. Muhammad Asif Aziz, Department of Entomology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi for his guidance and help in completion of this dissertation, critical assessments, inspiring guidance and helpful suggestions in the conduct of this work and particularly in reviewing the manuscript.

I am deeply indebted to Prof. Dr Ata ul Mohsin Chairman Department of Entomology, Prof. Dr. Muhammad Naeem and Dr. Muhammad Sheraz Ahmedfor his contributions during the research work. Specialthanks are extended to Dr. Imran Bodlah, assistant professor, Department of Entomology, for the confirmation of species. I am grateful to all my teachers Dr. Humuyon Javed, Dr. Munir Ahmed, Dr. M. Farooq Nasir, Dr. Fareed Asif Shaheen, Dr. Muhammad Tariq and Dr. Asim Gulzar for their moral support and sincere help. I am also grateful to Prof. Dr. Mazhar Qayyum, Director of Quality Control, for helpful words of encouragement towards my success.

I really have no words those can express my obligations and depth of dedication to my Parents, Brothers and Sisters for their moral support and encouragement. I feel pleasure to say deep and unlimited thanks to Shagufta Bibi and Zubaria Iqbal for their moral support and help during my research work. Finally, I thank my Husband for his love, support, and for constantly believing in me.

TASLEEM AKHTAR

xxii

ABSTRACT

Canola (Brassica napus L.) is an important oil seed crop in Pakistan having the potential of bridge gap between consumption and production of edible oil in the country. Canola has high potential of yield but due to many limiting factors, farmers in Pakistan are getting three times less production than developed countries. One of these factors is the insufficient crop pollination. Pollination is an essential ecosystem service andcan be provided by managed pollinators (honeybees and other insects).

A two year study was designed to assess the possible role of Apis mellifera managed pollination to enhance the yield of Brassica napus in Gujar Khan,

Rawalpindi during 2015 and 2016. Pollen sources of A. mellifera during blooming period of B. napus comprised of 11.11% ornamentals, 33.33% weeds, 22.22% shrubs, 22.22% herbs and 11.11% crops. Quantification of pollens transported by

A. mellifera reflected maximum pollens of B. napus followed by Calendula officinalus and Sonchus asper. Studies about determination of colony level pollination efficiency of A. mellifera on B. napus crop depicted that on average maximum colony foraging rate of A. mellifera foragers with pollen loads (281.2 bees/ ten minutes) was observed at 1200 hours on 10-02-2015 (6th weekly interval).

Weather factors influenced the activity of A. mellifera colony foraging rate in the field conditions. A. mellifera foragers with pollen loads attained maximum colony foraging activity on 10-02-2015 (6th weekly interval) when the temperature was

21.5 ̊C and average R.H. was 60%. Colony condition of A. mellifera varied throughout the blooming period of B. napus. Maximum brood and stored food area of bees were observed at the end week of B. napus blooming period.

xxiii

Observations were made about insect pollinators’ visited B. napus throughout the blooming period. Out of thirty five species belonging to five orders and twenty families were recorded. A. mellifera was most frequent visitor comprised 87.66%. Managed A. mellifera had significant effect on yield of B. napus in response to two different treatments. The results about total numbers of pods plant-1, numbers of seeds plant-1 and seed weight pods-100showed significant differences between treatments and revealed that treatment T2 (Open plot free visit of bees+ other pollinators) gave maximum yield as compared to treatment T1

(Cages plants without honeybees). Seed quality parameters of B. napus crop also affected bytwo different treatments (T1=Cages plants without honeybees, T2= Open plot free visit of bees+ other pollinators). Hive distances also affected on colony level pollination efficiency in A. mellifera on B. napus. Colony foraging rate at different hive distances from the B. napus crop proved that colony foraging rate of

A.mellifera started to decrease after 200m hives distance. Maximum foraging rate of A. mellifera was at 100m hives distance from B. napus. Effect of beehives distance from B. napus on brood and food stored area of A. mellifera showed that maximum brood and stored food area was found in hives placed at 100m distance from B. napus crop.

Overall recommendation for using managed A. mellifera as an important pollinator of B. napus proved that seed yield of B. napus increased with managed pollination of A. mellifera as well as brood and stored food area of A. mellifera also increased with blooming progression of B. napus.

xxiv

Chapter 1

INTRODUCTION

Canola (Brassica napus L.) is a significant oilseed crop in the world and ranked as the second largest volume oilseed traded following soybean (Mohamed and Rateb, 2014). Its oil is of best quality with low erusic acid and glucosinolates contents. Brassica napus L. is usually grown worldwide for food and animal feed.

In Pakistan, B. napus is the major oil producing crop. After sunflower, canola is the potential crop, which can fulfill requirements of edible oil in the country. This crop is grown in irrigated and rainfed (barani) areas of Punjab, Sindh and Khyber

Pakhtunkhwa, provinces of Pakistan. The total area under its cultivation in Punjab during 2015 was 153000 ha; with total production of 148000 metric tons and an average yield of 969 kg/ha (MINFAL, 2015). Among different oil seed crops, it contributes 21% towards national oil production (Economic Survey of Pakistan,

2014).

In Pakistan, expenditures on the import of edible oil are the most important drain on the foreign exchange reserves of the country. In the past, edible oil consumption was 2.76 million tons, of which 0.857 million tons (31%) came from local resources and 1.90 million tons (69%) were imported (Anonymous,

2006). The area for B. napus production has been on increase in the past but the productivity did not increase accordingly, hence the native oil production of the country could not match the growing demand of population (Shahzad and Rashid,

2006). In spite of great attention of government to increase its productivity, canola yield is still three times less than that of advanced countries (MINFAL, 2015). This decline in production may be attributed to pests attack, diseases damage, poor soil

fertility, cultivation on marginal and sub marginal land, water stress and insufficient pollination (Free, 1999).

Pollination is an ecological service that mainly contributes in maintenance of biodiversity and food production (Allen-Wardell et al., 1998). In nature, 87.5% of reproduction occurs in angiosperms (Ollerton et al., 2011) and in agriculture,

75% of crops needs animal pollination (Klein et al., 2007). In 2005, total economic value of pollination amounted to 153 billion Euros worldwide (Abrol, 2011). It is estimated that production deficit due to the insufficient pollination in developed world ranges between 3 to 5% and up to 8% in the developing world (Aizen et al.,

2009). Stephan and Irshad (2012) reported that in Pakistan, the deficit in edible oilseed crops is $55 billion, out of which is 47% is only in toria and sarsoon which may be attributed to low and insufficient pollinator’s population per unit area

(Munawar et al., 2009).

Among different pollinating agents, insects make a considerable contribution to the crop production. Up to 90% of flowering plant species rely on insects’ pollination (Buchmann and Nabhan, 1996). In USA, about one-third of the total human diet is dependent on insect pollinated plants (McGregor, 1976;

Richards and Kevan, 2002). The flower visitation rate by insects is an important factor for reproductive success in crops (Sahli and Conner, 2007). Reproductive output of plants depends not only on the visitation rate of pollinators but also on the quantity of viable pollen shifted to the stigma, this depends on the category of insect visitors and their foraging behavior (Davila and Wardle, 2008). Insect visitors on Brassica spp. flowers play a vital role in increasing yield and quality of seed (Bhalla et al., 1983). Many insects’ species provide pollination services but

honeybees (Apis mellifera) are the most important manageable pollinators because thousands of them can be kept in each hive and transported to fields to pollinate many crop species throughout the world (Leet al., 2008).

A. mellifera has great economic importance to increase the yield and quality of commercially grown crops in the world (Hoehn et al., 2008). They are active and regular foragers due to their own nutritional needs and those of their progeny

(Kearns and Inouye, 1997) and subsequently are considered to be the best pollinators (Thapa, 2006; Greenleaf et al., 2007; Brosi et al., 2008). For its progeny development, certain behavioral characters like collection of nectar and pollens force it for flower foraging (Jams and Singer, 2008). This character is very important in honeybees because other insect just feed on pollen and nectar but do not collect them hence they may not be reliable in pollination (Free, 1993). The body of honeybees is covered with branched hairs which make them morphologically well adapted for pollen collection while their easy management in hives makes them economically suitable as mass crop pollinators (McGregor, 1976;

Du Toit, 1988). Estimated contribution of managed pollination by honeybee (A. mellifera) in increasing crop yield and quality is more as compared to honey and wax production (Shrestha, 2004). Only honeybees are responsible for 70-80% of all insect pollination (Johannsmeier and Mostert, 2001). Crops pollinated by honeybees contributed $29 billion to US farm income in 2010 (Gallai et al., 2011).

Worldwide, lot of work has been conducted on the biology and ecology of

B. napus. Most pollination biologists are in favor of insect pollination for quantitative and qualitative yields, i.e., greater seed yield, higher seed set, high seed meal lipid, more seeds per pod and more pods, earlier pod formation, earlier

cessation of flowering, faster and more uniform seed maturation and increased germination rate of seeds (Fries and Stark, 1983; Korpela, 1988; Kevan and

Eisikowitch, 1990; Sabbahi et al., 2005). An adequate pollination process involves successive bee visits to ensure the reproductive cycle of the Brassicaceae and increase their productivity indices (Abrol, 2007).

B. napus flowers have a generalized open structure (Faegri and van der Pijl,

1971) that every floral visitor can feed from them. Yellow flower with deep placement of visible nectar mostly attracts honeybees (Kunin, 1993). Among honeybees, A.mellifera is documented as the most common visitor of canola flowers (Johannsmeier and Mostert, 2001). A. mellifera accounting for from 46% to

95% of all insect pollinators of this crop (Blight et al., 1997; Koltowski, 2001;

Piere et al., 2003). Other rape pollinators, such as solitary bees can account for about 4% or sometimes 9% of all insect pollinators (Koltowaski, 2001).

Although a large number of beekeepers migrate towards the Pothwar rainfed area (Gujar Khan) of Punjab province during Brassica season, but farmers normally show reluctance to cooperate with beekeepers. The reason is that farmer’s perception that bees deprive valuable products of flowers (nectar and pollen).

Keeping in view the above situation, the research work described in this thesis aims to improve the understanding of the use of managed honeybee pollination in B. napus pollination. To achieve this, the following objectives were addressed:

1. Toidentify all pollen sources transported by A. mellifera during blooming

period of B. napus

2. To determine the colony level pollination efficacy of A. mellifera against B.

napus crop

3. To evaluate the contribution of pollination by managed honey bee towards B.

napus crop production

The findings of this work will contribute to the devise of general guidelines to maintain and improve canola (B. napus) crop pollination with managed A. mellifera.

Chapter 2

REVIEW OF LITERATURE

Pollination, the transfer of pollen grains from the anther of a plant to the stigma, is essential to produce fruits and seeds. Diversity of pollinators can increase quantity and quality of certain crops (Hoehn et al., 2008; Kremen et al., 2002).

Among these, insect pollination is very important in determining the mating opportunities of plants and increased seed set of many fruit crops, as well as the quality of seed/ fruit (Free, 1993). Hence pollination is a keystone process in natural ecosystems (De Jong et al., 2005; Collette, 2008).

Plants use different attractant cues for visiting pollinators like Brassica crop has bright yellow ray florets as visual attraction of insects while disk florets as high energy source. Presences of large number of florets per flower also increase pollination efficiency in terms of single insect visit (du Toit, 1988; Neff and

Simpson, 1990). Flower pattern and characteristics are considered important affecting the foraging behavior of insect pollinators (Thapa, 2006; Stang et al.,

2009). In pollination, insects’ contribution is over 80% and among insects, honeybees play a major role nearly 90% of the total insect pollinated crop species and therefore considered as the best pollinators (Robinson and Morse, 1989).

Crops from the Brassicaceae, depends on entomophilous pollination for seed production (Syafaruddin et al., 2006). A large number of insect pollinators visit canola flowers and play a central role to increase the quality and yield of seed

(Bhalla et al., 1983). For example, some authors found that without insect pollination Brassica could not produce high yield (Westcott and Nelson, 2001).

Managed honeybee pollination ensure maximum and high quality crop yield (Anita et al., 2012). Kevan and Eisikowitch (1990) documented that many countries used managing colonies of A. mellifera to improve their orchard and crop yields. Wild bees visit agricultural fields in less number; in that situation managed honeybee hives are the only solution for farmers to ensure crop pollination.

These studies advocate at the utilization of managed A. mellifera for increasing productivity of the canola leading to raised economy of the country.

Previous information on this topic and some related aspects have been reviewed and presented below:

2.1 POLLINATORS FAUNA OF CANOLA

Oilseed mustard, Brassica was reported to be pollinated by honey bee, Apis mellifera; solitary bee, Osmia cornifrons; native bee species, Osmia lignaria subsp. lignaria in North Central Regional Plant Introduction Station (NCRPIS), USA

(Abel et al., 2003). Likewise Saure et al. (2001) observed great numbers of bee species (Apidae), hoverflies (Syrphidae) and sawflies (Symphyta) on flowers of rape seed at Michigan State, USA. A wide range of insects were observed visiting oil seed rape (Brassica campestris) by Langridge and Goodman (1975) of which bees were main floral visitors (32.9 %) followed by hover fly (30.7 %), blowflies

(22.9 %), native bees (4.9 %) and others (8.8 %). Adegas and Nogueira (1992) reported Apis mellifera, Trigona spinipes and Dialictus spp. as the most frequent insect visitors of rape flowers (Brassica napus var. oleifera) in Brazil constituting

80.6%, 12.8% and 6.6% respectively.

The flower structure of the canola attracts large number of insect pollinators e.g. bees, flies, beetles, wasps and butterflies (Kunin, 1993). In a previous study,

Mahindru et al. (1995) documented A. dorsata, A. florae, A. mellifera and Andrena sp. on brown sarson while Chakravarty (2000) reported Eristalis, Syrphus spp., A. cerana indica, A. mellifera, Mellipona spp., Bumbus spp., Heliothis armigera,

Plusia orichalcea and Pieris brassicae as the floral visitors of B. napus at

Pantnagar, Uttarakhand. In another study, Chaudhary (2001) reported that honeybees, Megachile hera, Nomia curvipes, Chalcidoma creusa, Andrena sacrissima, Sphicodes fumipennis, Braunaspis moderata, Bombus spp., Xylocopa spp., syrphid fly, house fly and butterfly were visitors of B. campetris, B. carinata and Indian mustard whereas Ahmed (2005) documented 22 and 16 hymenopterans and 7 and 5 dipterans species from Diriyah and Derab (Saudi Arabia) respectively as mustard flower visitors. They observed honeybees as the main hymenopterans pollinators followed by other bees such as Andrena, Hexachysis, Osmia, Halictus,

Pompilus and wasps. More abundant dipterans genera on the other hand were

Agromyza, Chrysoma, Drosophila and Syrphus. Roy et al. (2014) reported 24 insect species belonging to 14 families under six orders in mustard bloom.

Hymenopterans were found as frequent visitors (56%) followed by Coleoptera

(20%), Diptera (8%), Lepidoptera (8%), Hemiptera (6%) and Odonata (2%).

According to Bhowmik et al. (2014) total 19 insect species belonging to 13 families under 4 orders viz. Hymenoptera, Coleoptera, Diptera and Lepidoptera were recorded on B. juncea. Abdul- Rehman and Rateb (2014) reported that honeybees, carpenter bees, house flies and horse flies were the dominant visitors on canola plants. Koltowski (2007) documented that honeybees worker were the predominant group of pollinating insects of rapeseed and their contribution in the total number of insects on flowers accounted for nearly 95%. In another study,

addition to bees, canola plants were also visited by many flies, butterflies and true bugs (Williams, 1986). Atmowidi et al. (2007) reported 19 insect species on mustard crop. Goswami and Khan (2014) recorded nineteen species of insect pollinators belonging to two orders and nine families as visitors of B. juncea. Abrol

(1989) reported twenty pollinators’ species belonging to twelve families of orders

Hymenoptera and Diptera and reported A. mellifera, A. cerana, Halicid bees,

Halictus spp. and Lasioglossum spp., as the most important pollinators of canola crop. According to Ali et al. (2011) insect pollinators’ fauna of canola consisted of

35 species belonging to 3 orders and 14 families. Hymenopteran pollinators were found to be dominant and most frequent visitors during entire flowering season of canola. Eastham and Sweet (2002) reported members of Hymenoptera and Diptera as the most important pollinators of Brassica crop.

Kunjwal et al. (2014) observed different insect pollinators visiting B. juncea and reported thirty insect pollinators’ species belonging to ten families belonging to

4 orders visited Brassica flowers. Fifteen insect species were identified as important pollinators of sunflower which comprised;A. mellifera, A. dorsata, A. cerana, X. pubescence, X. fenestrata, , Musca domestica, Vespa orientalis, Polistis spp, Megachile lanata, M. femorata, M. cephalotes, Bombus spp., Andrena spp.,

Nomia melandria and Papilio spp.(Gupta, 1999). Four insect orders were found dominant pollinators including Hymenoptera, Lepidoptera, Coleoptera and Diptera with honeybees as the most frequent pollinators of sunflower with 89% abundance among all insect pollinators (Jadhav et al., 2011). According to Kumar et al.

(2005), different insect pollinators visiting sunflower were A. mellifera, A. cerana,

A. dorsata, A. florea, M. domestica and other insect pollinators contributing 7.81,

10.04, 4.13, 13.75 , 13.38, and 40.89 % abundance of all pollinators in winter as well as 14.52, 12.86, 10.37, , 15,77, 8.30 and 38.17 % in summer. Syrphid flies are also documented as efficient pollinators of oilseed rape crop (Jauker and Wolters,

2008). Work of Kamel et al. (2013) on sesame blooms, Sajjad et al. (2008) on onion, Sabir et al. (1999) on linseed, Hanh et al. (2014) on cucumber, Saeed et al.

(2012) on bitter gourd and Khan and Khan (2004) on apple also reported maximum abundance of Hymenopterans on these crops as compared to other insects.

2.2 ABUNDANCE OF APIS MELLIFERA ON CANOLA

Honeybees are reported as the most frequent visitor of canola plants (Free and Nuttall, 1968). According to Bhowmik et al. (2014) out of three honeybee species, the abundance of A. mellifera was maximum (18%) followed by A. dorsata

(16%) and A. ceranaindica (14%). Similarly Mahfouz et al. (2012) documented that among the bees, the number of A.mellifera was maximum followed by

Xylocopa spp. and Anthidium spp. In another study, Kunjwal et al. (2014) revealed the maximum abundance of A. mellifera in all the varities of B. juncea. Bommarco et al. (2012) recommended that because of high abundance, A. mellifera had the major contribution to canola pollination in their study. In another study, managed

A. mellifera was much more abundant (97%) than native bees (Witteret al, 2014).

Sabbahi et al. (2005) found the highest abundance of A. mellifera in the fields of canola and estimated this bee as the main pollinators of canola. Pordal et al. (2007) reported A. mellifera as the most abundant specie of three different cultivars of winter canola.

2.3. FORAGING BEHAVIOR IN TERMS OF VISITATION FREQUENCY

OF APIS MELLIFERA

A. mellifera has the tendency to increase cross pollination rates, because these bees can easily move between clusters of flowers on different plants, rather than settling in one cluster on a single plant, they also deliver pollen between flowers more quickly than any other pollinator species (Hayter and Cresswell,

2006). Bees entirely feed on pollen and nectar (Masierowska, 2003) and in order to satisfy the colony’s needs they visit a great number of flowers (Corbet et al., 1991).

The attractiveness of a crop to honeybees is related to its flower fragrance (Mussery and Fernandes, 2000) and abundant food resources (Williams, 1980; Mesquida et al., 1988). For an effective pollinating agent, this behavior should favour the transportation of anther to stigmas on the same plant or different plant species

(Freitas and Paxton, 1996). Honey bees are included in this perspective, whose foraging activities are favorable to the increase of crop productivity (D‘Avila and

Marchini, 2005).

The insects flower visitation rate is likely to be an important factor of reproductive success in crops (Sahli and Conner, 2007). Honey bees have long tongue and have advantage of nectar collection from complex angiosperm flowers

(Winston, 1987). During nectar collection, their body brushes with pollens and become covered with pollens (Free, 1993). Kumar and Singh (2005) reported that

A. mellifera dominated over the rest of the Apis species visiting Brassica sp. while

A. florea was least dominant. It was reported that Apis spp. was highest in the week having comparatively higher temperature and lower relative humidity. They also recorded abundance of Apis sp. at different hours of the day and noticed their peak activity at 1300 h (27.76) and minimum at 1500 h (14.91). Sarangi and Baral

(2006) observed that the population of A. cerana indica, A. mellifera and A. dorsata

on mustard reached their peaks at 1100 h.

2.4 IDENTIFICATION AND QUANTIFICATION OF ALL FLORAL

SOURCES OF APIS MELLIFERA

Co-evolution and mutualism relationship is found between honeybees and flowering plants. Therefore success of apiculture depends mainly on the availability of the food source from suitable flowering plants in different areas (Crane, 1990).

So it requires understanding the bees’ plant relationship to study food preferences of honeybees and their pollination requirement.

The identification of bee flora plants is of essential factor for beekeepers. It indicates the food sources used to collect nectar and pollen, mainly in areas of natural vegetation (Hower, 1953). Knowledge of the bee flora is also important for conservation and management programmes for bee pasture plants. Several studies have been done on plant pollen morphology worldwide (Raj, 1969; Sowunmi,

1973; Tomb et al., 1974; Nair and Kapoor, 1974; Gill and Chinnappa, 1982).

Poderoso et al. (2012) and Silva (2012) revealed that the families Arecaceae,

Asteraceae, Myrtaceae, Fabaceae, Rubiaceae, Poaceae and Urticaceae in Sergipe were identified as important food sources for A. mellifera. Novais et al. (2009) performed an experiment in an area of Caatinga, and reported that pollen grains belonged to the family Fabaceae, in high counts and decaled it as an important family supporting A. mellifera and other species. In another study, Lima (2007) observed that the M. misera has pollen type available only as a resource for bees, and is regarded as a plant with high beekeeping potential. Modro et al. (2011) studied pollen flora of the Vicosa region, State of Minas Gerais, these authors reported that these plants are the most visited by A. mellifera, mainly due to the

collection of pollen in plants of Cecropia spp. Shubharani et al. (2013) reported sixty eight species belonging to thirty nine families, useful as a source of bee forage. In another study, Zaitoun and Vorwohl (2003) revealed that the honeybees mostly visited by 44 flowering plant species belonging to 16 families. A total of forty six plant pollen types, distributed in nineteen families, were found in Bahia region of Brazil. Fabaceae has the maximum diversity of pollen types. The families

Asteraceae, Anacardiaceae, Rubiaceae, and Myrtaceae were represented by three pollen types each, and Lamiaceae, only two pollen types (Alves and Santos, 2014).

Noor et al. (2004) has done studies on the palynology of cultivated plants of

Rawalpindi, Pakistan. In her explored study, 15 cultivated ornamental plant species have been studies. The selected species are Baugainvellia glabra, Brassica campestris, Canna indica, Callendula officinalis, Chrysanthemum indicum,

Consolida ambigua, Catharanthus roseus, Hibiscus ciricus, Hibiscus rosa,

Jatrophaintegrrima, Jasminum grandiflora, Rosa indica, Rosa alba, Tagetis petala,

Tecoma stans and Tradescantia. Perveen and Qaiser (2010) conducted the study on family Moringaceae and Berberidaceae for pollen anaysis. Several plant taxonomists identify different plant species on the basis of their phenotypic character. These pollen morphological studies can provide a source for the identification of plant species.

2.5 IMPACT OF WEATHER CONDITIONS ON FORAGING RATE OF

APIS MELLIFERA

Honeybees visit various plants for nectar and pollen (Lane and David 2006;

Mattu et al., 2012). However, foraging activities of these pollinators are influenced by climate factors (Tirado et al., 2013) and unpredictable environmental conditions

in terms of timing and location of available food (Biesmeijer and Ermers, 1999;

Lane and David, 2006). Besides, their foraging behavior is regulated by temperature, relative humidity, season, topography and availability of floral resources (Hossam et al., 2012). Omoloye and Akinsola (2006) indicated negative correlation between the visitation rate of honeybees and temperature. While in another study, foraging rate of bees tended to be positively associated with air temperature (r= +0.21) while there was a negative tendency with relative humidity

(r= -0.19) (Contreras et al., 2013). While according to Peat and Goulson (2005) temperature did not significantly influence the foraging behaviour of bees. Kumar et al. (2002) showing the most intensive bee activity at 18.3 degrees centigrade and

60% humidity in Punjab, India. Miklič (1996) observed the most intensive bee activity at 20 to 28 ̊C and relative humidity at 40 to 50%. In another previous study; bees do not forage during rainy days (Tripath, 2011).

With respect to environmental factors which influence foraging activity of

A. mellifera were observed to start their foraging activity at mean temperatures of

6.57 °C (Tan et al., 2012) while in another study, their foraging rate was found low at 16 °C (Joshi and Joshi, 2010). At 20 °C, the highest activity of honeybees were recorded (Tan et al., 2012) while in another region, lowest foraging activity was found at 43 °C (Blazyte-Cereskiene et al., 2010) as well as below 10 °C (Joshi and

Joshi, 2010). Furthermore, significantly negative correlation (r = −0.09) was found between foraging activity and ambient temperature (Abou-Shaara et al., 2013).

Thus, it is reported by Cooper and Schaffer (1985) that foraging activities of honeybees are influenced by elevated temperature. Relative humidity had less of an effect on flight activity of bee (Joshi and Joshi, 2010). Martins (2004) reported that

honey bee foraging activity patterns varied with temperature throughout the day.

Sarangi and Baral (2006) observed that the population of A. cerana indica, A. mellifera and A. dorsata on mustard reached their peaks when average temperature was 25.4 ͦC as compared to the population of A. florea which attained its peak at

1300 to 1400 h at an average temperature of 27 ̊C. They also reported that the population of Apis species increased with the decrease in R.H. and attained peak at

47.1% R.H. except A. florea whichpeaked at 43.2%.

2.6 IMPACT OF BEEHIVES DISTANCE ON FORAGING RATE OF

APIS MELLIFERA

Manning and Wallis (2005) reported that honeybees foraging activities started to decline more than 200m from the apiary. The mean foraging distance for

A. mellifera was 1526.1 meter while foraging distances of pollen collecting worker bees in simple landscapes had a mean of 1743 meter and 1543.4 m in complex landscapes (Steffan-Dewenter and Kuhn, 2003). The mean foraging distances for small colonies of Apis mellifera was 670 meter and for large colonies in July it was

620 m, while in August the values were 1430 m for small bee colonies and 2850 m for large bee colonies (Beekman et al., 2004). Hagler et al. (2011) reported the foraging range of honey bees from 45 m to 5983 m.

2.7 EFFECT OF HONEYBEE POLLINATION ON QUALITATIVE AND

QUANTITATIVE PARAMETERS OF CANOLA

In Brassicaceacrops, cross pollination is done by insect pollinators’ activity

(Hayter and Cresswell, 2006). A large number of insect pollinators on Brassica flowers are thought to play an essential role in the quality and yield of seed (Bhalla et al., 1983). For example according to some authors, without an adequate cross

pollination Brassica could not produce high seed yield (Westcott and Nelson,

2001). Several hymenopterous insects have been reported to visit the crop at blooming (Kumar et al., 1998) out of which honey bees (Apis spp.) affect substantial pollination of the crop (Kumar and Singh, 2005) and augmentation of the crop yield (Sharma and Abrol, 2004; Singh et al., 2004 and Chhuneja et al.,

2007). Another study revealed that the introduction of hives of A. mellifera in controlled pollination helps to increase the production of crops such as rapeseed

(Brassica napus) (Sabbahi et al., 2006).

Honeybees and other insects are known to take an important role in seed setting of Brassica crops and their hybrid seed production (Westcott and Nelson,

2001; Singh and Singh, 2002). In Pakistan, Perveen et al. (2000) stated that foraging of honeybees on sarson (Brassica campestris L.) resulted in maximum yield in terms of 1000 grain weight and germination percentage of 1990 kg/ha, 3.81 g and 95.8%, respectively. Munawar et al. (2009) conducted an experiment on canola and concluded that flowers caged with bees yielded more number of pods, higher number of seed per pod and seed weight compared to flowers excluding insect visitation. Another study revealed that the introduction of managed A. mellifera in controlled pollination helps in increasing the production of crops such as Brassica napus (Sabbahi et al., 2006). Pollinating services performed by A. mellifera to increase canola productivity has been widely documented (Sabbahi et al. 2005; Duran et al. 2010; Ali et al. 2011; Jauker et al. 2012). Another study revealed 53% increase in yield and 50% economic benefit to farmers with Apis mellifera L. as the most regular and effective insect visitor (Nderitu et al. (2008).

Chambo et al. (2011) studied the influence of Apis mellifera pollination on seed

setting in sunflower and concluded that heads visited by honeybees produced 43% more seed yield than those which were restricted to insect pollinators. Rajasri et al.

(2012) conducted study on the consequence of honey bee pollination on sunflower and found that seed setting percentage, seed yield, seedling vigor, germination percentage, oil content and field emergence was significantly high compared to self pollinated sunflower heads. Brar et al. (2010) revealed an increase of 20 to 80 percent in seed yields of radish using A. mellifera.

Munawar et al. (2009) conducted an experiment on canola and concluded that flowers caged with bees yielded more number of pods, higher number of seed per pod and seed weight compared to flowers excluding insect visitation.

According to observation of Calmasur and Ozbek (1999), share of A. mellifera among all species of bees was 80-88 percent and wild bees contributed 12-20 percent of total visitation. Toria (Brassica campestris var. toria) is also a highly cross pollinated crop and its seed production depends on insect pollinators

(Chhuneja et al., 2007). The entomophilic pollen has large number and greater amount of amino acids (Singh and Singh, 1991) and is nutritionally superior to anemophilic pollen (Stanley and Linskens, 1974). Koutensky (1958) showed that in

Czechoslovakia three fields of B. campestris, well provided with honeybee colonies, had seed harvests which were 775, 830 and 820 kg/ha greater than control plots without colonies nearby. Free and Nuttal (1968) also reported an increase of

13 percent in the seed yield of Brassica napus plots with bees as compared to those without bees.

Chapter 3

MATERIALS AND METHODS

3.1. STUDY AREA

Purposed research was conducted to assess the role of managed Apis mellifera on pollination and yield of Brassica napus at University Research

FarmKoont, Gujar Khan, Department of Entomology , Pir Mehr Ali Shah , Arid

Agriculture University Rawalpindi located at 233̊ 06̍ N and 73̊ 00̍ E at an elevation

518.76 meter under arid conditions.

3.2. SOWING OF CROP

Brassica napus variety Chakwal sarsoon was sown for two consecutive years (2015-2016) on an area of 2 acres. Row to row and plant to plant distance was maintained as 45cm and 15cm respectively. Line sowing was done with hand drill.

3.3. IDENTIFICATION OF ALL POLLEN SOURCES DURING

BLOOMING PERIOD OF BRASSICA NAPUS FROM FORAGERS

OF APIS MELLIFERA

Collections of pollen from flower visitors can provide some evidence about the variety of species visited. Pollen identification is also important to measure the pollination success. So in order to perform this experiment, five colonies of equal bees’ strength were placed in the experimental area of 2 acres. Following parameters were assessed.

3.3.1. Identification of Pollen Sources Transported by Apis mellifera

For reference pollen collection, field survey in University Research Farm,

Koont, Gujar Khan was conducted in order to identify the local bee plants. The samples of ripe pollen grains were collected from mature flower directly from the field after the plant has been confirmed as bee plant by visual observation that bees are foraging on plant either for nectar or for pollen or both. The mature pollen grains of the identified bee plant species are collected and preserved in 70% alcohol for further investigation (Mailula and Nofemela, 2017).

For pollen identification transported by bees, ten bees with pollens attached on their body were collected from each hive on weekly interval. Acetolysis protocol was used for pollen identification (Dafni et al., 2005). For acetolysis, pollen suspension was prepared by washing bee body in 70% ethanol. Then 5ml of pollen suspension was added in 2ml glacial acetic acid for 10minutes. After that centrifuged it for 3minutes at 2400rpm and supernatant was discarded. Then 10ml of acetolysis mixture (glacial acetic acid and concentrated sulfuric acid 9:1) were added in it and heated the solution in water bath at 70 °C for 12minutes. This solution was cool down for a 5minutes and again centrifuged it at 2400rpm for 3 minutes and supernatant was discarded. After that, it was resuspended in distilled water, centrifuged and discarded the supernatant. After acetolysis, pollens were preserved for archival reference slides by the protocol proposed by Erdtman

(1969). In this protocol, a base stock of jelly was prepared by combining 10g gelatin, 30ml glycerin and 35ml distilled water. Then added a drop of the prepared jelly and a sample of pollen and stain on a clean microscope slide and the slide were gently warmed, stirred to thoroughly homogenize the mixture. Then a cover slip was added, sealed with nail polish around the edges. Then pollens were identified using Identification keys and online image databases (Kearns and Inouye,

1993).

3.3.2. Pollen Quantification Transported by Apis mellifera

From each colony, 10 bees were collected on weekly interval and pollen quantity transported by bees was determined by washing bee body in known quantity of 70% ethanol. Haemocytometer were cleaned with ethyl alcohol. Small drop of pollen suspension were taken with a pipette and placed in center of haemocytometer. Cover slip was placed properly over haemocytometer. Pollen suspension was allowed to settle at the bottom chamber for 2 minutes before counting. The chamber is 0.1 mm high and divided into 25 medium squares of 0.04 mm2 each, which are further subdivided into 16 small squares of 0.0025 mm2 each.

This means a volume of 0.1 ml in the chamber, 0.004 ml in each medium square and 0.00025 ml in each small one. For each pollen sample, the pollen grains of five medium squares at the center, left and right corners at the top and bottom of the chamber were counted under binocular microscope 100x magnifications (Human et al., 2013) which were repeated for making 10 individual observations. Pollen counting was done by using following formula.

Total number of pollen counted × Diluted factor Pollens per ml = Area of squares counted (mm2) × Chamber depth (mm)

3.4. DETERMINATION OF COLONY LEVEL POLLINATION

EFFICIENCY OF APIS MELLIFERA ON BRASSICA NAPUS CROP

For monitoring the pollination efficacy of A. mellifera, five colonies of equal bee strength were selected and foraging activity of A. mellifera was observed on B. napus during the whole blooming period. Following parameters were

assessed.

3.4.1. Colony Foraging Rate

The no. of bees foraging from a colony were estimated by counted the returning bees with pollen to the hive for 10 minutes at 1000, 1200 and 1400 hours by standing at the side of bee hive and the numbers of bees returning with pollens were counted (Baker and Jay, 1974).

3.4.2. Impact of Weather Factors on Foraging Rate of Apis mellifera

Weather condition i.e. rainfall, temperature and relative humidity effect the foraging behavior of honeybees. Meteorological data obtained from Department of

Environmental Sciences, PMAS-AAUR; were used for interpretation of meteorological conditions. Multiple measurements (at 10 am, 12pm and 2pm) of weather factors in Koont farm, Gujar Khan were recorded during the study to correlate them with the foraging activity of bees.

3.4.3. Colony Condition of Apis mellifera

Colony conditions from A. mellifera colonies were evaluated by mapping the comb areas of capped and uncapped worker brood (eggs, larvae) and stored food (honey and pollen) with the help of frame holder with 2 x 2 cm wire grid to measure the area covered by brood and food on weekly interval (Imdorf et al.,

1987). This was done for all combs (both sides) in each hive.

3.4.4. Data Analysis

SPSS programmed ver. 12 was used for analyses of colony foraging rate using analysis of variance. Means were compared by using LSD test at P = 0.05

(NorusICE, 2002). Multiple measurements (at 10 am, 12pm and 2pm) of weather

factors in Koont farm, Gujar Khan were recorded during the study to correlate them with the foraging activity of bees by Pearson product-correlation coefficient.

3.5. CONTRIBUTION OF POLLINATION BY MANAGED APIS

MELLIFERA IN YIELD OF BRASSICA NAPUS

To find out the contribution of managed A. mellifera to pollination of

Brassica napus, five colonies of bees were placed in the corner of the field. The data were taken from pre-blooming to pod harvesting of the crop. Following parameters were evaluated during the experiment.

3.5.1. Visitation Frequency of Managed Apis mellifera

Apis mellifera’s abundance was determined throughout the flowering period by scaned sampling of 15 plants in each of the four plots located in experimental site. Visitation frequency (no. of visits per plant per minute) of Apis mellifera was observed for 60 seconds on each canola plant with stop watch. Sampling was done by walking in 4 plots in the experimental area. The data was taken at 1000 hours,

1200 hours and 1400 hours on weekly basis throughout the blooming season of B. napus.

3.5.2. Pollinators Diversity

For determining diversity of pollinators, the insects were captured from 4 transects of 25 m length for 10 minutes at 1000, 1200 and 1400 hours at weekly interval throughout the blooming period of B. napus and placed in insect killing bottle containing potassium cyanide for some time to kill them. Pinning of those insect pollinators were done and carried to the Biosystematics Laboratory,

Department of Entomology, Pir Mehr Ali Shah Arid Agriculture University

Rawalpindi. Identification of these insect pollinators was made at genus level.

Voucher specimens were deposited at the Biosystematics Laboratory, Museum of

PMAS- Arid Agriculture University Rawalpindi.

3.5.3. Pollination Efficacy

This parameter was arranged with 2 treatments and 4 replications. Plot size was 4m² in each replication. In first treatment, plants remained uncovered for whole flowering period. In the second treatment, before commencement of flowering, 10 plants per replication were covered by muslin cloth to prevent insect pollinators. These plants remained covered throughout the blooming period. At the end of crop cycle, the plants were harvested for assessing the following yield parameters:

3.5.3.1 Agro morphological parameters

3.5.3.1.1 Number of pods plant -1

Total number of pods was counted from ten selected canola plants in each treatment at the time of harvest and their average was calculated.

3.5.3.1.2 Number of seeds plant-1

Number of seeds from each pods of canola plant was counted at the time of harvest and average was calculated.

3.5.3.1.3 Seed weight pods -100

Hundred pods were randomly selected from each sample of each treatment at the time of harvest. Then after threshing, recorded the seed weight of 100 pods by using electrical balance and their average was calculated.

3.5.3.2 Seed quality parameters

3.5.3.2.1 Oil contents (%)

Seed oil contents were measured by Near- Infrared Reflectance Spectro scopy system (NIRS) (Sato et al., 2008).

3.5.3.2.2 Protein contents (%)

Protein contents were determined by Near Infrared Reflectance Spectro scopysystem (NIRS) (Sato et al., 2008).

3.5.3.2.3 Fatty acid profile

Fatty acid profile (Oleic acid and linolenic acid) was determined by Near-

Infrared Reflectance Spectro scopy system (NIRS) (Sato et al., 2008).

3.5.3.2.4 Germination rate (%)

Seed germination rate was assessed by placing the two types of seeds (one obtained from caged pollinated plants and the other type from open pollinated plants) in 8 plastic petri dishes with double layers of Whatman filter paper. On the sowing date, the filter paper was saturated with distilled water and then kept moist for 2 days. On the second day the germination was scored as successful with the appearance of two cotyledons of the seedling in seeds of open pollinated plants

(Bhowmik et al., 2014).

3.5.4. Data Analysis

SPSS statistical programm ver. 12 was used to analyze the data about pollinator’s density and diversity, pods/plant, seeds/plant, seed weight/ 100 pods, oil content, fatty acid profile and protein content using analysis of variance

(ANOVA). Means were compared by using LSD test at P= 0.05 (NorusICE, 2002).

3.6. EFFECT OF BEEHIVES DISTANCE ON COLONY LEVEL

POLLINATION EFFICACY IN APIS MELLIFERA ON BRASSICA

NAPUS

To investigate the effect of beehives distance on bee pollination efficacy, three hives distances 100m, 200m and 300m from B. napus were taken as treatment. In each treatment, three bee colonies were placed as replication. The colonies were placed in the experimental area of 2 acres with the start of blooming period till crop harvesting. Following parameters were investigated.

3.6.1. Colony Foraging Rate of Apis mellifera

Three hives distances 100m, 200m and 300m from B. napus were taken as treatment. In each treatment, three bee colonies were placed as replication. The no. of bees foraging from each colony were estimated from hives distance 100m, 200m and 300m by counted the foragers returning with pollen to the hive for 10 minutes at 1000, 1200 and 1400 hours by standing at the side of bee hive and the numbers of bees foragers returning with pollens were counted (Baker and Jay, 1974).

3.6.2 Colony Condition of Apis mellifera

The colony conditions of Apis mellifera were evaluated from hives placed at different distance 100m, 200m and 300m by mapping the comb areas of capped and uncapped worker brood (eggs, larvae) and stored food (honey and pollen) with the help of frame holder with 2 x 2 cm wire grid to measure the area covered by brood and food on weekly interval (Imdorf et al., 1987). This was done for all combs (both sides) in each hive. This was done one time in a year.

3.6.3 Impact of Weather Factors on Foraging Rate of Apis mellifera

Effect of weather factors with foraging rate of bees in different hive distance were correlated with average of temperature, relative humidity and rainfall in Koont farm, Gujar Khan.

3.6.4 Data Analysis

SPSS statistical programmed var. 12 was used to analyze the data colony foraging rate at different beehives distance by using analysis of variance

(ANOVA). Means were compared by using LSD test at P= 0.05 (NorusICE, 2002).

The correlation of weather factors with foraging rate of A. mellifera were determined by correlation.

Chapter 4

RESULTS AND DISCUSSION

4.1. IDENTIFICATION OF POLLENS COLLECTED FROM BODIES OF

APIS MELLIFERA DURING BLOOMING PERIOD OF BRASSICA

NAPUS

Collection of pollens from flower visiting insects can provide reasonable evidence about the variety of plant species visited. Pollen identification is also important to measure the pollination success of the crop. So in order to perform this experiment, five colonies of equal bee strength were placed in the experimental area of 2 acres to identify and quantify all pollen sources available to A. mellifera during blooming period of B. napus at University Research Farm Koont, Gujar

Khan.

4.1.1. Identification of Pollen Sources

Pollens collected by A. mellifera foragers during blooming period of

B.napus from different floral sources were identified in the laboratory and results are presented in Table 4.1.1. According to which, 18 plant species belonging to 11 families were recorded as forage source for A. mellifera. Information of each floral source and pollen morphology is also given in Table 4.1.1. Family Asteraceae contributed 6 plant species as pollen sources followed by Brassicaceae and

Solanaceae with 2 plant species each. The remaining families had one floral source each. Among 18 identified pollen sources, 2 species belonged to crops, 6 to weeds,

4 to shrubs and herbs each, and 2 to ornamental plants, respectively (Table 4.1.2).

Pollen morphology differs among various plant species. Pollens showed variation

in Symmetry, exine, structure and sculpture. Euphorbia spp., B. napus and Eruca sativa pollens were spherical, pollens of family Malvaceae were echinate. Pollens of Petunia spp., and Tecomastans were prolate in shape. The pollen shape of

Celosia argentea, Lupinus perennis and Asphodelus tenuifolius were spheroid. The species belonging to family Asteraceae had spinolous type of pollens and those belonging to Malvaceae had those of echinate type. There was variability in the pollen type of plant species belonging to family Fabaceae. Pollen grains of plant species belonging to family Euphorbiaceae were inaperturate and reticulate (Table

4.1.1).

In present study, an attempt was made to explore all available beeflora during Brassica blooming period, so that the beekeepers can get useful information regarding main crop (B. napus) and alternate foraging sources. Analysis of the data showed that the bee flora of University Research Farm Koont, Gujar Khan consisted of two crops. Among which B. napus was the main crop but bees also collected pollens from E. sativa. Besides these, bees also collected pollens from different weeds, shrubs, herbs and ornamental plants. The flowering plants of an area to be used as bee pasture are of great value necessary to maintain bee colonies in good conditions (Baptist and Punchihewa, 1980). It has been observed that foraging activities of bees were slow in the start of blooming period. However when most of the plants came into full bloom at the end of February till March, the foraging activities also increases. At the same time, the colonies constructed new combs, built up their population very rapidly and intensified brood rearing activity.

During this period, B. napus was recorded as the most dominant bee forage source both as pollen and nectar source. These results are in agreement with Noor et al.

(2009) who identified Brassica spp. as a major bee forage plant for bees. In present study, 6 weeds i.e. P.hysterophorus, H. autumnale, S. nigrum, A. arvensis, C. arvensis, A. tenuifolius were reported as supporting bee flora. Weeds are natural vegetation of any area which can fulfilled pollen requirements of A. mellifera. In dearth period when agro horticultural crops are not in blooming condition, weeds and wild flowering plants can serve as alternate food sources for honeybees. Weed species constituted the bulk of the honey bee diet between the mass flowering crop periods (up to 33.33%) and are therefore suspected to play a critical role at this time period. Requier (2015) also reported weeds as pollen resources for honeybees.

Present study also demonstrated different ornamental flowers (C.officinalis, C. indicum), herbs (S. asper, A. rosea, L. perennis, T. officinale) and shrubs

(E.uphorbia spp., Petunia spp, T. stans, C. argentea) during the B. napus flowering season. Their number per unit area was less or lesser quantity of pollen. These were an important source of pollen for Apis mellifera when major flora is absent. These findings are in line with Dalio (2012) who stated that minor sources are utilized by bees during the time of scarcity of major bee flora.

4.1.2. Quantification of Pollens Transported by Apis mellifera Workers

The pollens collected by A. mellifera foragers from different floral sources at URF, Koont during blooming period of Brassica napus were separated from their bodies and quantified (Table 4.1.3). According to results pollen grains observed from bee foragers belonged to families Brassicaceae, Asteraceae,

Malvaceae, Solanaceae, Primulaceae, Convolvulaceae, Euphorbiaceae, Fabaceae,

Bignoniaceae, Xanthorrhoeaceae and Amavanthaceaea. Among these observed families, maximum numbers of B. napus pollens from Brassicaceae were observed

Table 4.1.1. Different floral sources visited by Apis mellifera during Brassica napus blooming period at URF Koont, Gujar Khan during 2015.

Sr. No Pollen type Plant name and Family name Common name Morphology Flowering Forage period source 1. Monoporate, Spherical Brassica napus Sarsoon Jan- Mar Crop shape, thick layer covering

(Brassicaceae) the structure

2. Monoporate, pointed at its Eruca sativa Taramira Jan- Mar Crop one end and shows bilateral

(Brassicaceae) symmetry

3. Pantoporate, pores 32, Calendula officinalis Marigold Feb- Mar Ornamental echinate, radial symmetry, plant (Asteraceae) reticulate exine

Parthenium hysterophorus Carrot weed Porate, spinolous, spheroid Feb- May Weed

shape. radial symmetry (Asteraceae)

Porate, spinolous, spheroid Helenium autumnale Sneeze weed Dec- Jan Weed shape. radial symmetry (Asteraceae)

Porate, spinolous, spheroid Sonchus asper Sow thistle Aug- Feb Herb shape. radial symmetry (Asteraceae)

Pantoporae, spheroid, Alcea rosea Holly hock Sep- Jan Herb radial symmetry (Malvaceae)

8. Colporate, prolate, Solanum nigrum Black night Jun- Jan Weed oblatespheroid, radially (Solanaceae) shad symmetrical

9. radial symmetry, exin Anagallis arvensis Red chick weed Feb- Aug Weed epsilate (Primulaceae)

10. Tri-periporate, echinate Convolvulus arvensis Field bindweed Sep- Jan Weed pollen, bilateral symmetry (Convolvulaceae)

11. Thicker outer layer but Euphorbia sp. Spurge Nov- Jan Shrub smaller in size than that of (Euphorbiaceae) Brassicaceae.

12. Prolate, sub-spheroid, Petunia sp. Petunia Feb- May Shrub exinepsilate and thin, (Solanaceae) bilateral symmetry

13. Triangular aperture, Tecoma stans Yellow bells Mar- Apr Shrub prolate-spheroid,clavate (Bignoniaceae) surface,bilateral symmetry

14.

Asphodelus tenuifolius Spheroid pollens, bilateral symmetry, thick layer on (Xanthorrhoeaceae) Onion weed Dec- Feb Weed either side of the structures.

15. Spheroid shaped pollens, Lupinus perennis Sundial lupine Mar- Jun Herb bilateral symmetry, (Fabaceae) prolate

16. Exine reticulate, porate, Taraxacum officinale Dandelion Mar- Sep Herb spheroid, spinolous, radial (Asteraceae) symmetry

17. Spheroid shape, radial Celosia argentea Silver Jan- Jun Shrub symmetry cockscomb (Amaranthaceaea)

18. Exine reticulate, porate, Chrysanthemun indicum Daisy Jan- Dec Ornamental spheroid, spinolous, (Asteraceae) plant radial symmetry

Table 4.1.2. Total numbers of pollen sources visited by Apis mellifera recorded during blooming period of Brassica napus at URF Koont, Gujar Khan during 2015.

Plant species No. of species Percentage

Crops 2 11.11

Shrubs 4 22.22

Herbs 4 22.22

Ornamental plants 2 11.11

Weeds 6 33.34

Total 18 100

from pollen samples of 5th (03-02-2015) (78.10%), 6th (10-02-2015) (77.43%) and

7th (17-2-2015) (76.81%) week of study and minimum numbers of B. napus pollens were found on 10th (10-03-2015) (24.86%), 11th (17-03-2015) (25.32%) and 12th

(24-03-2015) (5.53%) week of the study. After Brassicaceae, Asteraceae proved an important source of pollens for A. mellifera. From Asteraceae, the pollens collected from Sonchus asper forager bees start increasing (25.5%) on 10th week of study

(10-03-2015) and reached to maximum (30.1%) on 12th week (24-03-2015).

Calendula officinalis was also an important botanical pollen source followed by B. napus and S. asper. Forager bees collected pollens from C. officinalis in the same pattern as followed in the case of S. asper i.e. start increasing on 10th week of study

(24.3%) and reached to maximum on 12thweek (29.1%). A. mellifera foragers also used Taraxacum officinale as a pollen source. Pollens of T. officinale were reached on first week of study (06-01-2015) and foragers continued their collection until last week (24-03-2015) of observation. In our study area (URF Koont, Gujar Khan) pollens of other plant species (P. hysterophorus, H. autumnale,, C. indicum, A. rosea, A. esculentus, S. nigrum, Petunia spp.,A. arvensis, C. arvensis, Euphorbia spp., T. stans, A. tenuifolius, L. perennis and Celosia spp.) were found less in numbers from A. mellifera body during B. napus blooming period (Table 4.1.3).

Fluctuation in pollen collection from different plants visited by A. mellifera foragers during different weeks of the studyshowed that from B. napus abundant pollens were available for A. mellifera foragers from 03-02-2015 to 17-02-2015 which comprised 5th, 6th and 7th weeks of study. This was the period of maximum blooming of flowers. In the 2nd (13-01-2015), 8th (24-02-2015) and 9th (03-03-2015) weeks of the study due to unfavorable environmental conditions (Rainfall) the

numbers of the forager bees did not visit flowers. On the last three weeks (10th, 11th and 12th) due to ending of B. napus flowering period, pollen loads were comprised of higher percentages of S. asper, C. officinalis and T. officinale because A. mellifera are renowned for their floral constancy to visit one type of flower until resources are depleted. B. napus from Brassicaceae represented highest pollen percentage (45%) during study period followed by S. asper (21%), C. officinalis

(18%) and T. officinale (10%). Eruca sativa is also an oilseed crop but numbers of

E. sativa pollens were found less from pollen loads of forager bees. S. asper is widely spread prickly weed in URF Koont area which belongs to dandlion tribe.

This weed proved important plant taxa among polleniferous bee flora of study area

(Fig. 4.1.2).

It is evident from pollen quantification from pollen loads of A. mellifera; A. mellifera foragers obtained maximum pollens from B. napus flowers. After B. napus, percentage of pollens of S. asper and C. officinalis started to increase from first week and reached to peak on last three weeks of the study. At that time, S. asper and C. officinalis were the first spring foods for A. mellifera. Our results are coinciding with Dimou and Thrasyvoulou (2007) and Bauma et al. (2011) who stated that the bees frequently collect a wide variety of pollen types, but they generally concentrate on a few available plant species due to flower constancy behavior of bees, focusing their attention on one type of flower for a period of time.

4.2. DETERMINATION OF COLONY LEVEL POLLINATION

EFFICIENCY OF APIS MELLIFERA ON BRASSICA NAPUS CROP

4.2.1 Colony Foraging Rate

Analysis of variance of the data regarding colony foraging rate of A.

mellifera on 06-01-2015 (1st weekly interval) on Brassica napus var. Chakwal sarsoon (Appendix 1) showed significant differences (F(2,8) = 6.40, P<0.02). Means comparison illustrated maximum colony foraging rate at 1000 hours (57.6) which was significantly different from other observation time intervals. At 1200 hours maximum foraging (107.0) was observed which was at par with 1400hours (92.8)

(Fig. 4.2.1). No foraging activity was observed on13-01-2015 (2nd weekly interval) because of rain on that particular day. Analysis of variance of data regarding colony foraging rate on 20-01-2015 (3rd weekly interval) demonstrated highly significant difference (Appendix 2) between observations with two hour interval

(F(2,8) = 39.21, P<0.00). It is evident from Fig. 4.2.2 that maximum colony foraging rate was observed at 1200 hours(134.8) followed by that on 1400 hours (68.2) and

1000 hours (35.0). Highly Significant difference were observed from the analysis of variance regarding (Appendix 3) colony foraging rate on 27-01-2015 (4th weekly interval) (F(2,8) = 39.51, P< 0.00). Means comparison showed (Fig. 4.2.3) maximum foraging rate of A. mellifera at 1200 hours (182.4) followed by that at 1400 hours

(162.2) and at 1000 hours (138.0). Analysis of variance regarding foraging rate of

A.mellifera on B. napus for different time intervals showed highly significant

th differences on 03-02-2015 (5 weekly interval) (F (2,8) = 28.37, P<0.00) (Appendix

4). Maximum foraging rate of A. mellifera was observed at 1200 hours (222.0) which was statistically at par with 1400 hours (201.0) while minimum foraging rate was found at 1000 hours (55.0) (Fig. 4.2.4). Data regarding colony foraging rate on

th 10-02-2015 (6 week of observation) again showed significant differences (F(2,8) =

8.79, P<0.00) (Appendix 5). Maximum number of A. mellifera foragers were observed at 1200 hours (281.2) followed by those at 1000 (210.0) and minimum at

1400 (184.4) hours (Fig. 4.2.5). The analysis of variance revealed highly significant

th difference in colony foraging rate on 17-02-2015 (7 weekly interval) (F (2,8) =

13.36, P< 0.00) (Appendix 6). Maximum number of pollen foragers bees with pollen at 1200 hours (240) followed by 1400 hours (148.0) which was statistically at par with that 1000 hours (130.0)( Fig. 4.2.6). No foraging activity was observed on 24-02-2015 (8th weekly interval) and 03-03-2015(9th weekly interval) because of rain in these particular days. Results of colony foraging rate of A. mellifera on 10-

03-2015 (10th weekly interval) (Appendix 7) illustrated highly significant differences during various time intervals (F (2,8) = 78.61, P< 0.00). Mean colony foraging rate of A. mellifera has been presented in Fig. 4.2.7, according to which maximum pollen foragers were recorded at 1200 hours (156.0) which was statistically significant different from those at 1400 hours (105.8) and at 1000 hours

(100.6). Results obtained on 17-03-2015 (11th weekly interval) demonstrated significant differences in colony foraging rate of A. mellifera during various time intervals (F (2,8) =5.41, P<0.03) (Appendix 8). Means comparison revealed (Fig.

4.2.8) maximum number of A. mellifera with pollens at 1200 hours (137.2) followed by those at 1400 hours (127.8) which were significantly different from pollen foragers recorded at 1000 hours (117.4). The analysis of variance of the data regarding colony foraging rate of A. mellifera on B. napus on 24-03-2015 (12th weekly date of observation) showed highly significant differences between different time intervals (F (2,8) = 20.73, P<0.00) (Appendix 9). Maximum A. mellifera foragers were observed at 1200 hours (169.0) which were statistically different from those recorded on other time intervals. Foraging rate of A. mellifera at 1000 hour (80.4) was statistically similar with that observed at 1400 hours (92.4)

(Fig. 4.2.9).

Based on average of foraging rate of A. mellifera at different time intervals, maximum foraging activity was observed at 1200 hours (181.06) during the whole experiment from 6th January to 24th March, 2015(Fig. 4.2.10). Although in most cases, foraging rate at 1200 hours was maximum and significantly different from others, however there were some weekly intervals when foraging rate at 1000 hours was statistically similar with that of at 1400 hours interval i.e., 3rd, 4th and 5th weeks of observation, significant differences were observed among 1000 hours and 1400 hours in the rest weeks of observation.

4.2.2 Impact of Weather Condition on Foraging Rate

Climatic factors had clear influenceon the activity of A. mellifera colony foraging rate during B. napus blooming period. Among climatic factors, temperature had significantly significant positive and strong correlation with A. mellifera (r= 0.744**) whereas relative humidity and rainfall had significantly negative correlation (r= -0.7287**, r= -0.7094**) with foraging activity of bees on

B. napus (Table 4.2.1). It is evident from Fig. 4.2.11 that on 2nd, 8th and 9th weekly intervals due to rain colony foraging activity of A. mellifera was zero. A. mellifera foragers attained maximum activity on10-02-2015 (6th weekly interval) whenaveragetemperature was 21.5 ̊C and average R.H. was 60%, this can be because of the favorable environmental conditions and peak blooming period of B. napus. Present results are in line with Tan et al. (2012) who reported highest activity of A. mellifera at 20 ̊C. Our results are also in accordance with Kasper et al.

(2008) who stated that temperature positively influenced the insect pollinators’ activity on foraged flowers. Similarly, rainfall has been documented as an

Table 4.1.3. Weekwise percentage of polleniferous flora of Apis mellifera during blooming period of Brassica napus at URF Koont, Gujar

Khan during 2015.

Plant species Family Percentage of pollens counted from the body hairs of Apis melliferaforagers 1st week 3rd week 4th week 5th week 6th week 7th week 10th week 11th week 12th week 6/1/15 20/1/15 27/1/15 3/2/15 10/2/15 17/2/15 10/3/15 17/3/15 24/3/15 Brassica napus 35.81 36.84 37.90 78.10 77.43 76.81 24.86 25.32 5.53 Eruca sativa Brassicaceae 4.75 4.64 5.02 3.21 2.22 2.03 3.21 3.33 3.34 Calendula officinalis 12.1 12.6 13.5 14.6 12.5 13.6 24.3 27.8 29.1

Parthinium hysterophorus 0.26 0.26 0.24 0.08 0.08 0.08 0.26 0.27 0.34

Helenium autumnale 0.30 0.27 0.27 0.09 0.09 0.09 0.26 0.28 0.32

Sonchus asper 12.8 13.6 14.6 20.2 22.8 22.8 25.5 26.4 30.1 Chrysanthemun indicum Asteraceae 0.26 0.29 0.28 0.10 0.09 0.09 0.27 0.27 0.34 Taraxacum officinale 3.26 3.25 4.23 5.08 9.08 10.08 15.25 17.26 18.12 Alcea rosea 0.26 0.26 0.23 0.08 0.08 0.08 0.25 0.26 0.32 Malvaceae Solanum nigrum 0.25 0.25 0.22 0.08 0.07 0.07 0.25 0.26 0.31 Solanaceae Petunia sp. 0.25 0.25 0.23 0.08 0.08 0.08 0.25 0.26 0.31 Anagallis arvensis 0.27 0.27 0.24 0.08 0.08 0.08 0.25 0.27 0.32 Primulaceae

Convolvulus arvensis 0.25 0.25 0.22 0.08 0.07 0.08 0.25 0.26 0.32 Convolvulaceae Euphorbia sp. 0.26 0.25 0.22 0.08 0.07 0.75 0.25 0.26 0.32 Euphorbiaceae Tecoma stans 0.26 0.26 0.24 0.08 0.08 0.08 0.27 0.26 0.32 Bignoniaceae Asphodelus tenuifolius 0.26 0.26 0.23 0.08 0.08 0.08 0.26 0.27 0.32 Xanthorrhoeaceae Lupinus perennis 0.25 0.25 0.23 0.08 0.08 0.08 0.25 0.26 0.32 Fabaceae Celosia spp. 0.25 0.25 0.23 0.08 0.08 0.08 0.25 0.26 0.31 Amavanthaceaea

Brassica napus Sonchus asper Calendula officinalis Eruca sativa Other (15) species Taraxacum officinale

10% 2% 4%

45%

18%

21%

Fig.4.1.1. Overall percentage of pollens collected from different plants by Apis

mellifera foragers during blooming period of Brassica napus at URF

Koont, Gujar Khan during 2015.

environmental factor that can disrupt the floral insect activity (McGregor, 1976). In another study, Omoloye and Akinsola (2006) also declared that A. mellifera activity was found to be positively correlated with the temperature and negatively with therelative humidity on different cultivars of oilseed crops.

4.2.3 Colony Condition

Results regarding colony condition in terms of population of A. mellifera during blooming period of B. napus are presented in Fig. 4.2.12. These results depicted minimum area of eggs (1767±175.3 cm2) on 08-01-2015 (first weekly interval) which increased with blooming progression of B. napus up to

(6414±129.5 cm2) cells per colony on 26-03-2015 (last weekly interval). Similarly, open brood was also gradually increased with evident and found more (7365±61.91 cm2) on 26-03-2015 (last weekly interval) while minimum open brood

(2978.3±196.8 cm2) was found on 08-01-2015 (first weekly interval). The area of capped brood was found significantly high (7800.8±112.2 cm2) on 26-03-2015 (last weekly interval) while lowest (2957±125.5 cm2) was observed on 08-01-2015 (first weekly interval). Fig. 4.2.12 depicted increasing trend in all of three parameters

(eggs, open brood, capped brood) during blooming period of B. napus. Fig. 4.2.13 depicted the results about colony condition in terms of food stores collected by A. mellifera during blooming period of B. napus. Results illustrated significant differences in area of stored honey from first weekly interval (7619.6±301.2 cm2) up to last weekly interval (10830±97.5 cm2). Similarly, increasing trend in area of stored pollen was also observed. More stored pollen was observed on last weekly interval (1940.6±32.1 cm2) while lowest was observed on first weekly interval

(557.8±96.5 cm2). Fig. 4.2.13 depicted increasing trend of honey and pollen with

300 280 260 240 220 200 180 160 140 120 100 80 60

Colony foraging Colony rate ten per minute 40 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.1. Colony foraging rate (Mean± SE) of Apis mellifera at different time

intervals on 06-01-2015 (1st week of study) throughout blooming

periodof Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig 4.2.2. Colony foraging rate (Mean± SE) of Apis melliferaat different day

times on 20-01-2015 (3rd week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.3. Colony foraging rate (Mean± SE) of Apis mellifera at different day

time on 27-01-2015 (4th week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig.4.2.4. Colony foraging rate (Mean± SE) of Apis melliferaat differentday

time on 03-02-2015 (5th week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.5. Colony foraging rate (Mean± SE) of Apis melliferaat different day

time on 10-02-2015 (6th week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during

2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.6. Colony foraging rate (Mean± SE) of Apis mellifera at different

day time on 17-02-2015 (7th week of study) throughout

blooming period of Brassica napus at URF Koont, Gujar Khan

during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.7. Colony foraging rate (Mean± SE) of Apis melliferaat different

daytime on 10-03-2015 (10th week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.8. Colony foraging rate (Mean± SE) of Apis mellifera at different time

intervals on 17-03-2015 (11th week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.9. Colony foraging rate (Mean± SE) of Apis mellifera at different time

intervals on 24-03-2015 (12th week of study) throughout blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

300 280 260 240 220 200 180 160 140 120 100 80 60

40 Colony foraging Colony rate ten per minutes 20 0

1000 h 1200 h 1400 h Observations time

Fig. 4.2.10. Average of nine weeks of colony foraging rate of Apis mellifera at

different day intervals during blooming period of Brassica napus at

URF Koont, Gujar Khan, 2015.

blooming progression of B. napus. Results of present study depicted that brood area showed increasing trend in a colony due to continuous availability of nectar and pollen from B. napus. As pollen is the protein diet for bees,theyrequire carbohydrates, proteins, fats, minerals, vitamins, and water to be able to rear brood for growth and development of the colony (Manning, 2008). These elements are sourced mainly from pollen and nectar. Due to continuous availability of pollen, queen was encouraged to lay eggs and fulfilled the food requirements of new brood population. Present results also depicted increasing trend of honey and pollen in the colony. Normally beekeepers want to increase the bee population size during this period instead of harvesting honey and making splits for new colonies so B. napus could be the best crop for beekeepers to increase the colony population due to abundant availability of pollen and honey.

4.3. CONTRIBUTION OF APIS MELLIFERA MANAGED POLLINATION IN YIELD OF BRASSICA NAPUS

4.3.1 Visitation Frequency of Managed Apis mellifera

Data analysis about visitation frequency of A. mellifera on 06-01-2015(1st weekly interval) (Appendix 10) revealed highly significant differences during various time intervals (F (2, 9) = 19.29, P< 0.00). Maximum numbers of A. mellifera visits were observed at 1200 hours (3.45 visits/plant/ minute) (Fig 4.3.1).

Significant differences were also observed between visitation frequencies at 1400 hours with 1000 hours. On 20-01-2015 (3rd weekly interval) highly significant differences of visitation frequency was observed (F (2, 9) = 23.13, P< 0.00)

(Appendix 11). Visitation frequency at different three time intervals of the day on

B. napus on 3rd week of study is showed in Fig.4.3.2 which indicates maximum

numbers of A. mellifera visits at 1200 hours (3.96 visits/ plant/ minute). However, visitation frequency at 1000 hours was found statistically non significant with 1400 hours. The data about number of visits per plant per minute on 27-01-2015 (4th weekly interval) demonstrated highly significant differences (F(2, 9) = 77.42, P<

0.000) (Appendix 12). A. mellifera was found most active in terms of visiting flowers at 1200 hours (4.78 visits) as compared to other two time intervals; 2.58 visits/ plant/ minute at 1000 hours and 2.96 visits/ plant/ minute at 1400 hours (Fig.

4.3.3). Visitation frequency on 03-02-2015 (5th weekly interval) indicated significant difference between treatments (F(2, 9) = 17.006, P<0.0009) (Appendix

13). Maximum visits/ plant/ minute were recorded during 1200 hours (2.8) however, during other two times, 1000 hours (1.86) and 1400 hours (2.11) visitation frequency was statistically similar (Fig. 4.3.4). Results about visitation frequency of A. mellifera on 10-02-2015 (6th weekly interval) (Appendix 14) showed significant differences between different time intervals (F(2, 9) = 56.25, P<

0.00). According to Fig. 4.3.5, maximum visitation frequency was recorded at 1200 hours (5.0) and minimum at 1000 hours (2.56). Analysis of variance data about visitation frequency of A. mellifera on 17-02-2015 (7th weekly interval) revealed significant differences between treatment groups (F(2, 9)= 8.88, P< 0.0074)

(Appendix 15). Fig. 4.3.6 indicated highest activity of A. mellifera at 1200 hours

(3.53 visits per plant per minute) followed by 1400 hours (3.1 visits per plant per minute) were significantly different with 1000 hours (2.43). Results about visitation frequency of A. mellifera on B. napus on 10-03-2015 (10th weekly interval)

(Appendix 16) revealed highly significant difference among three time intervals

(F(,9)= 15.439, P<0.001). Means comparison of the data indicated that at 1200 hours

Table 4.2.1. Correlation between weather factors and colony foraging rate of Apis

mellifera ligustica during Brassica napus blooming period at URF Koont,

Gujar Khan during 2015.

Weather Factors r P

Avg. Temperature (̊C) 0.7446 0.0055**

Avg. Relative Humidity (%) -0.7287 0.0072**

Rainfall (mm) -0.7094 0.0098**

** = Highly significant n= 12

avg of bees/ day Temprature (°C) Relative humidity(%) Rainfall (mm) 250 100 90 200 80 70 150 60 50 100 40 30 50 20 Weatherfactors 10

Average foragingAveragerate per day 0 0

Dates of observations

Fig. 4.2.11. Temperature (̊C), relative humidity (%) and rainfall (mm) along with

colony foraging rate of Apis mellifera during blooming period of

Brassica napus at URF Koont, Gujar Khan during 2015.

hours, visitation frequency was maximum (3.00) followed by that at 1400 hour

(2.21) and at 1000 hours (1.78) (Fig. 4.3.7). Analysis of variance obtained on

11thweeklyinterval (17-03-2015) indicated significant differences in visitation frequency of A. mellifera during different times interval (F(2,9) = 7.34, P<0.0128)

(Appendix 17). Maximum visits per plant per minute were recorded at 1200 hours

(2.58) followed by 1400 hours (2.06) and at 1000 hours (1.71) (Fig. 4.3.8).

Analysis of variance of dataobtained on24-03-2015 (12th weekly interval) showed in (Appendix 18) demonstrated highly significant differences in visitation frequency for three observation intervals (F (2,9) = 19.12,P<0.00). Higher mean value of visitation frequency was found at 1200 hours (2.41 visits per plant per minute) followed by that at 1400 hours (1.95 visits/ plant/ minute) and at 1000 hours (1.48 visits/ plant/ minute) (Fig. 4.3.9).

Fig. 4.3.10 shows average visitation frequency of A. mellifera on B. napus flowers at 1200 hours (3.50± 0.30) due to peak blooming period of B.napus however means comparison of visitation frequency of A. mellifera at 1000 hours was (2.13± 0.14). Similarly data of visitation frequency of A. mellifera at 1400 hours was (2.58± 0.18). Foraging activity of A. mellifera was observed throughout flowering period of B. napus. Individuals of A. mellifera generally started their foraging activity in the morning hours, which attained its peak in the noon hours of the day.

Highest visitation frequency (visits/ plant/ minute) of A. mellifera was observed at 1200 hours followed by that at 1400 hours of the day throughout the flowering period lasting from 06-01-2015 to 24-03-2015 because of high flow of

E. OB CB

12000 ) 2 10000

8000

6000

4000

2000 Areapopulation brood of colony per (cm 0

08-01-1515-01-1522-01-1529-01-1505-02-1512-02-1519-02-1526-02-1505-03-1512-03-1519-03-1526-03-15 Weeks of observation, 2015

Fig. 4.2.12. Brood population area (cm2) per colony of Apis mellifera in blooming period of Brassica napus at URF Koont, Gujar Khan during 2015.

E= Eggs , OB= Open Brood, CB= Capped Brood

SH SP

12000 )

2 10000

8000

6000

4000

2000 Honey and pollen and Honey cells colony per (cm

08-01-1515-01-1522-01-1529-01-1505-02-1512-02-1519-02-1526-02-1505-03-1512-03-1519-03-1526-03-15 Weeks of observation, 2015

Fig. 4.2.13. Stored food area (cm2) per colony of Apis mellifera in blooming

period of Brassica napus at URF Koont, Gujar Khan during 2015.

SH= Stored Honey, SP= Stored Pollen

nectar at noon which is similar to the study of Ali et al. (2011) who observed highest foraging activity of A.mellifera at 1200 hours. Present results also coincide with the findings of Goswami and Khan (2014) who observed that peek activity time of A. mellifera was at 12 hours. Results of present study are also in line with findings of Woyke et al. (2003) who reported maximum activities of A. mellifera at

1200 hours. Perveen et al. (2000) in Pakistan also found maximum foraging activity at 1200 hours during January and February. Similarly, Semida and Elbanna

(2006) documented that the abundance of pollinators differed across the time of the day and increased gradually up to maximum around the mid day (1200 hours).

Rana et al. (1997) also observed higher visitation activity of A. mellifera at 1200 hours and lowest at 0900 hours. Some other researchers reported differences regarding peak activity of A. mellifera between 0900 and 1300 hours (Nascimento and Nascimento, 2012), at 1400 hours (Kunjwal et al., 2014) and at 1500 hours

(Williams,1985). This fluctuation in visitation might be due to difference in geographical and environmental conditions because in our research area heavy fog incidence was observed in January. Thus hours of the day seemed to play an important role in foraging rate of A. mellifera because floral rewards of B. napus and foraging activity of insect visitors are directly linked with them.

4.3.2. Diversity and Abundance of Insect Pollinators on Brassica napus

Canola crop was found to be visited by 35 insect species belonging to five orders on B. napus during flowering season. Out of which twenty-seven species were frequent visitors of canola flowers. These foragers comprised six bees, two wasps, twelve flies, seven butterflies, two moths, three beetles and three bugs. Bees were among the most abundant floral visitors with total abundance of 89.79%,

followed by Diptera (5.12%) and butterflies (3.24%). Moths and wasps were the rarest floral visitors with 18 and 2 individuals, respectively (Table 4.3.1). The

Hymenopteran visitors belonged to four families; five species from Apidae, (A. mellifera, A. florea, A. dorsata, Amegilla cingulata and Xylocopa spp.), one species each from Halictidae (Halictus spp.), Ichneumonidae (Ichneumon spp.) and

Sphecidae (Sphex spp.) were found during blooming period of B. napus. Among dipterans, six species were reported from Syrphidae (Eristalis tenax, Eupeodes corollae, Melanostoma spp., Ischniodon scutellaris, Episyrphus balteatus, Eristalis smilis), two from Calliphoridae (Stomorohina discolor, Chrysomya megacephala) one species each from Sarcophagidae (Sarcophaga spp.), Muscidae (Musca domestica), Tabanidae (Tabanus suleifrons) and Tachnidae (Prosena siberita).

From Lepidoptera six species (Pieris brassicae, Anaphaeis aurota, Eurema nicippe, Eurema smilax, Catopsilia pomonaand Pieris canidia) from Pieridae and one species each from Nymphalidae (Vanessa cardui), Erebidae (Callimorpha spp.) and Sphingidae (Macroglossum nycteris) were found to visit B. napus flowers. The remaining species belonged to the orders Coleoptera and Hemiptera and were found as casual visitors of the flowers and are not reported to participate in nectar or in pollen collection (Bhowmik et al., 2014 and Roy et al., 2014) (Table 4.3.1).

Pollinator’s abundance and composition may vary with geographical area, latitude and time (Ollerton and Louise, 2002). In India, Roy et al. (2014) documented 24 insect species belonging to 13 families under six orders

(Hymenoptera, Lepidoptera, Coleoptera, Diptera, Odonata and Hemiptera) on B. juncea crop. From India, Kunjwal (2014) observed 30 species visiting B. juncea flowers under three orders, 23 from Hymenoptera, 5 from Diptera and one from

Lepidoptera. In the same year, Goswami and Khan (2014) reported 19 insect visitors belonging to two orders, 15 from Hymenoptera and 4 from Diptera during mustard blossom period. Atmowidi et al. (2007) found 19 species of insect visitors on mustard crop. From Kingdom of Saudia Arabia, Ahmed (2005) reported 22

Hymenopterans and 16 Dipterans species as visitors of mustard flowers in Diriyah region and 7 Hymenopterans and 5 Dipterans species in Derab. In the present investigations, eight species belonging to Hymenoptera (A. mellifera, A. dorsata, A. florea, Amegilla cingulata, Xylocopa spp., Halictus spp., Ichneumon spp. and

Sphex spp.) were observed. Among Hymenopterans species, five species (A. mellifera, A. dorsata, A. florea, Xylocopa spp., Halictus spp.) were found as both pollen and nectar foragers and Amegilla cingulata as only nectar forager, while

Ichneumon sppand Sphex spp. were casual visitors. Shakeel et al. (2015) recorded five species of Hymenopterans on B. napus, among which A. mellifera was the major pollinator. Mahindru et al. (1995) found that A. mellifera, A. florea, A. dorsata and Andrena spp. were the dominant visitors of brown sarsoon at

Ludhiana, India. Chakravarty (2000) reported A. mellifera, A. ceranaindica,

Eristalis, Syrphus spp., A. dorsata, Bombus spp., Mellipona spp., Haliothis armigera, Pieris brassicacae and Plusiaorichalceaon B. napusat Pantnagar,

Uttarakhand, India. From present studies, it is evident that A. mellifera were the most dominant pollinator of canola crop with the highest abundance (87.66%). This shows that A. mellifera is an efficient pollinator of this crop and farmers can getmaximum benefit from their pollination services by allowing beekeepers to keep

A. mellifera colonies near their fields. In present studies, all recorded families of order Diptera are reported as nectar feeders except Syrphidae; in Syrphidae out of

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.1. Visitation frequency (Mean ± SE) of Apis mellifera on 06-01-2015

(1st weekly interval) on Brassica napus at URF Koont, Gujar Khan

during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.2. Visitation frequency (Mean ± SE) of Apis mellifera on 20-01-2015

(3rd weekly interval) on Brassica napus at URF Koont, Gujar Khan

during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.3. Visitation frequency (Mean ± SE) of Apis mellifera during 4th week

(27-01-2015) of study on Brassica napus at URF, Koont during

2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.4. Visitation frequency (Mean ± SE) of Apis mellifera on 03-02-

2015 (5th weekly interval) on Brassica napus at URF Koont,

Gujar Khan during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.5. Visitation frequency (Mean ± SE) of Apis mellifera on 10-02-2015

(6th weekly interval) on Brassica napus at URF Koont, Gujar Khan

during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.6. Visitation frequency (Mean ± SE) of Apis mellifera on 17-02-2015

(7th weekly interval) on Brassica napus at URF Koont, Gujar Khan

during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.7. Visitation frequency (Mean ± SE) of Apis mellifera on 10-03-2015

(10th weekly interval) on Brassica napus at URF Koont, Gujar

Khan during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.8. Visitation frequency (Mean ± SE) of Apis mellifera on 17-03-2015

(11th weekly interval) on Brassica napus at URF Koont, Gujar Khan

during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.9. Visitation frequency (Mean ± SE) of Apis mellifera on 24-03-2015

(12th weekly interval) on Brassica napus at URF Koont, Gujar

Khan during 2015.

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0 No. of visits per visits of No. plant per minute

0.5

0.0

1000 Hours 1200 Hours 1400 Hours Observation Times

Fig. 4.3.10. Overall average of visitation frequency (Mean ± SE) of Apis

mellifera at different time intervals on Brassica napus at URF

Koont, Gujar Khan during 2015.

six recorded species, three (Eupeodes corolla, Melanostoma spp., Ischniodons cutellaris) were found as both pollen and nectar foragers (Ali et al., 2011).

Lepidopterans were reported as nectar feeders only; they visit flowers to satisfy their nectar needs and may transfer pollens accidently, so may help in the process of pollination of canola (Jauker and Wolters, 2008; Jauker et al., 2012). Therefore, nine species of Lepidoptera recorded in present study may be regarded as secondary pollinators of this crop. Among all families of Hymenoptera, Apidea was the most abundant (89.71%) family (Table 4.3.1). Goswami and Khan (2014) recorded the maximum abundance of Apis bees (57.55%) followed by the non Apis bees (21.06%) on B. juncea in experiments without taking support from managed

A. mellifera pollination. In Hymenopterans, worker honeybees are the predominant group of pollinating insects of rapeseed and mustard, their total numbers on flowers can account up to 95% (Koltowski, 2007). In another study, Bhowmik et al. (2014) observed maximum abundance of A. mellifera (18%) followed by A. dorsata (16%) and A. ceranaindica (14%) on B. juncea along with no record of A. florea in their experiments. In contrary, A. florae ranked 2nd (1.11%) followed by A. dorsata

(0.98%) in our study (Table 4.3.2). The difference regarding abundance of A. mellifera (87.66%) may be due to the fact that we used managed pollination as compared to the non- managed pollination of A. mellifera used by Bhowmik et al.

(2014). Abundance of A. florae more than A. dorsata reflects that URF Koont,

Gujar Khan has also better potential to support a large population of A. florea.

Ali et al. (2011) and Roy et al. (2014) reported A. dorsata and A. cerana more abundant as compared to A. mellifera and A. florea; this may be due to the difference in distribution of A. dorsata and A. cerana and experimental conditions

in that area A. cerana is commonly found in hilly areas of Pakistan, while the area under study is in rainfed region. A. mellifera was the most efficient pollinator in our study, because of higher foraging rate and highest abundance. Present results are in line to those of Kumar and Singh (2005) and Bommarco et al. (2012) who declared A. mellifera as the most dominant species followed by other insect visitors on canola crop.

4.3.3. Pollination Efficacy

4.3.3.1 Agro- morphological parameters

4.3.3.1.1 Total number of pods plant-1

The number of pods per plant is a major yield determining component that contributes towards yield. The data about total number of pods plant-1 (Appendix

19) indicated that there was a significant difference of B. napus var. Chakwal sarsoon between different pollination treatments i.e., canola plants exposed to bees and other foral visitors and canola plants kept under cage (F(1,3) = 10.79, P<0.004).

-1 There was non significant effect of blocks on number of pods plant (F(3, 3) = 0.51,

P< 0.69). Means comparison of pods plant-1 expressed significant differences between plants exposed to bees and other pollinators (350.5) and those kept under under cage (166.25) without insect pollinators (Fig. 4.3.11).

4.3.3.1.2 Total number of seed plant-1

The number of seeds per plant is the most desirable character that contributes directly towards seed yield. Results about total number of seeds of different pollination treatments (Appendix 20) illustrated significant differences

(F(1, 3) =29.44, P<0.0123). There was non significant effect of blocks on number of

-1 pods plant (F(3, 3) = 0.71, P<0.60). The performance of plants exposed byA.mellifera and other pollinators were better and they produced more seeds per plant as compared to those kept in net without pollination of insects (Fig. 4.3.12).

4.3.3.1.3 Seed weight per 100 pods

Seed weight per pod is an important yield determining factor. Data regardingseed weight per 100 pods (Appendix 21) showed significant differences between plants facilitated by managed pollination of A. mellifera and plants covered with net (F (1, 3) =258.96, P<0.0005). Means comparison indicated that seed weight per 100 pods obtained from A. mellifera pollinated plants (5.41 g) was significantly different from plants pollinated under net (3.75 g) (Fig. 4.3.13).

Present results illustrated marked difference on yield parameters of canola plants having access to A. mellifera and other insect pollinators as compared to those kept under cage (only self and wind pollination). Open pollinated

(A.mellifera and other insect pollinators) canola plants had significantly higher yield than caged canola plants. Morandin and Winston (2005) reported that numbers of seeds in B. napus increased with higher population density of bees.

Same results was revealed by Dhakal (2003) who stated that seed yield in B. campestris var. toria significantly increased due to bee pollination in comparison with caged plants and those receiving other sources of pollination. These results corroborate the findings of Manning and Wallis (2005) and Sabbahi et al. (2006) that higher numbers of canola seeds were recorded in open plots than in covered plots, which show the impact of pollination on this crop. Tara and Sharma (2010) compared the qualitative and quantitative effects of pollination on controlled

(covered) and open pollinated plants of B. campestris var. sarsoon. They found that

fruit set was higher in open pollinated (88.1%) compared to controlled (80.0%) plots. They also found differences in number of seeds per pod (open, 11.2; controlled, 10.2) and mean weight of 100 seeds (open, 0.42 g; controlled 0.17 g).

Likewise Singh et al. (2004) noticed that insect pollination leads to the formation of well- shaped and large seeds than self pollinated plants. Shakeel and Inayatullah

(2013) found that average yields of canola were 189.3±1.7 pods/plant in the bee pollinated plots where as 142.2± 2.4 pods/ plant in the covered plots. They also observed an average of 15.0±0.9 seeds/pod in bee pollinated plots and 11.0±0.8 seeds/pod in covered plots. The weight of 100 seeds was 0.55±0.02g in bee pollinated plots and 0.37±0.01g in covered plots. Our results also coincide with findings of Munawar et al. (2009) who documented significant increased in the entire B. napus yield parameters caged with bees as compared to the plants without bees. Present results are also in accordance with the findings of Abdul- Rahman and Rateb (2014) who revealed significant differences between open pollinated and covered canola for three yield parameters (i.e., number of seeds per pod, weight of

1000 seeds and total yield of seeds). Williams et al. (1987) also demonstrated that honeybees clearly increased the yield of rape crop. In another study, Bhowmik et al. (2014) reported significant difference on qualitative and quantative effects of pollination on the percent fruit set; number of seeds per siliqua and mean weight of

100 seeds were compared in controlled and open pollinated B. juncea. Rosa et al.

(2011) documented that pollination induction increased seed productivity from

28.4% (autogamy) to 50.4% with bee visitation. Goswami and Khan (2014) revealed that open pollination increased the number of pods (142.83) and percent pod set (83.42) as compared to number of pods (96.64) and percent set (62.80) in

Table 4.3.1. Abundance (%) and foraging behaviour of Brassica napus (insect visitors) at URF Koont, Gujar Khan during 2015.

Foraging behavior S. N Name of the species Order Family Abundance (%) PF NF CV 1. Apis mellifera Apidae 4447 87.66 PF NF 2. Apis dorsata Apidae 43 0.85 PF NF 3. Apis florae Apidae 56 1.10 PF NF 4. Amegilla cingulata Apidae 2 0.04 NF 5. Xylocopa spp. Hymenoptera Apidae 3 0.06 PF NF 6. Halictus spp. Halictidae 4 0.08 PF NF 7. Ichneumon spp. Ichneumonidae 1 0.02 CV 8. Sphex spp. Sphecidae 1 0.02 CV 9. Eristalis tenax Syrphidae 24 0.47 NF 10. Eupeodes corolla Syrphidae 32 0.63 PF NF 11. Melanostoma spp. Syrphidae 31 0.61 PF NF 12. Ischniodon scutellaris Syrphidae 39 0.77 PF NF 13. Episyrphus balteatus Syrphidae 25 0.49 NF 14. Eristalis smilis Diptera Syrphidae 32 0.63 NF 15. Chrysomya megacephala Calliphoridae 8 0.16 NF 16. Stomorhina discolor Calliphoridae 9 0.17 NF 17. Sarcophaga spp. Sarcophagidae 19 0.37 NF

18. Musca domestica Muscidae 25 0.49 PF NF 19. Tabanus suleifrons Tabanidae 4 0.08 NF 20. Prosena siberita Tachnidae 12 0.24 NF 21. Pieris brassicae Pieridae 44 0.87 NF 22. Anaphaeis aurota Pieridae 23 0.45 NF 23. Eurema nicippe Pieridae 8 0.16 NF 24. Eurema smilax Pieridae 22 0.43 NF 25. Catopsilia pomona Lepidoptera Pieridae 13 0.26 NF 26. Pieris canidia Pieridae 16 0.31 NF 27. Vanessa cardui Nymphalidae 22 0.43 NF 28. Callimorpha spp. Erebidae 10 0.02 NF 29. Macroglossum nycteris Sphingidae 8 0.16 NF 30. Entomoscelis americana Tenebrionidae 36 0.71 CV 31. Lytta spp. Coleoptera Meloidae 42 0.83 CV 32. Aulacophora foveicollis Chrysomelidae 9 0.17 CV 33. Oncopeltus fasciatus Lygaeidae 1 0.02 CV 34. Sehirus luctuosus Hemiptera Cydnidae 1 0.02 CV 35. Bagrada hilaris Pentatominae 1 0.02 CV

Table 4.3.2. Overall foraging activity of Brassica napus (insect visitors) at different time intervals at URF Koont, Gujar Khan during 2015.

Foral visitors/15 canola plants (Average and percentage) Time Interval (Hours) Apis mellifera Apis florae Apis dorsata Beetles Diptrous flies Lepidoptrans Total floral visitors 10 00 32.08 (86.63) 0.42 (1.13) 0.33 (0.89) 0.92 (2.48) 1.78 (4.81) 1.5 (4.06) 37.03

1200 52.58 (88.79) 0.75 (1.26) 0.56 (0.95) 0.69 (1.16) 2.97(5.02) 1.67 (2.82) 59.22

1400 38.86 (87.38) 0.39 (0.88) 0.5 (0.69) 0.81 (1.82) 2.47 (5.55) 1.44 (3.25) 44.47

Mean± SE 41.17±6.03 0.52±0.12 0.46±0.068 0.81±0.07 2.41±0.34 1.54±0.07 46.91 a b b b b b Percentage 87.76 1.11 0.98 1.73 5.14 3.28 100

Means in rows sharing same letters are non significant at 0.05 level.

* Values in brackets indicate percentage of floral visitors

400 a

350

300

250 (Mean±SE)

-1 b 200

150

100 No. of Pods plant Pods of No. 50

0 T1 T2

Modes of Pollination

Fig. 4.3.11. Number of pods per canola plant in response to different

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar

Khan during 2015.

caged mustard. Similarly Thakur and Karnatak (2005) reported that highest number of pods per plant (495) in A.mellifera pollinated plants, (438) in A. cerana and

(417) in open pollinated plants whereas caged plants without pollinators produced

290 pods per plant. The contribution of A. mellifera to yield parameters of canola has been also documented by Duran et al. (2010) and Jauker et al. (2012).

4.3.3.2 Seed quality parameters

4.3.3.2.1 Oil content (%)

Increase in oil contents is the ultimate desire of grower. Results about oil content (%) of canola plants exposed to A. mellifera along with other insects verses caged canola plants (Appendix 22) illustrated significant differences (F (1, 3) =25.86,

P<0.05). The data pertaining to oil contents of two different pollination methods expressed significant differences between canola plants exposed to A.mellifera and other floral visitors (49.5± 1.6) and covered canola plants with cage (44.6± 1.43)

(Fig. 4.3.14).

4.3.3.2.2 Protein content (%)

The data regarding protein contents in canola plants supported with

A.mellifera managed pollination andcanola plants covered in cage is shown in

(Appendix 23). Analysis of variance of data revealed significant differences for protein contents (F(1, 3) =19.50, P<0.021). Means comparison of data (Fig. 4.3.15) indicated significantly higher protein contents in plants exposed to A. mellifera and other floral visitors (24.6± 0.13) as compared to caged canola plants (21.4± 0.72).

4.3.3.2.3 Oleic acid (%)

Analysis of variance of data regarding oleic acid in canola plants exposed to

A.mellifera and other floral insects and caged canola plants is presented in

(Appendix 24). Oleic acid of canola (B. napus) plants revealed non significant differences (F(1, 3) =3.66, P> 0.05) between caged plants and A.mellifera pollinated plants (Fig. 4.3.16).

4.3.3.2.4 Linolenic acid (%)

Analysis of variance of data regarding linolenic acid (%) of canola plants exposed to A. mellifera along with other insects and canola plants covered with cage (Appendix 25) illustrated non significant differences (F(1, 3) =1.15, P> 0.05).

Fig. 4.3.17 demonstrated that linoleic acid in seeds from canola plants exposed to

A.mellifera and other floral insects (10.3±0.8) was statistically similar with those in covered with cage (8.8±0.6).

4.3.3.2.5 Germination rate (%)

Results regarding seed germination rate of two different pollination treatments (Appendix 26) illustrated highly significant differences (F(1,3) = 1234.11,

P< 0.00). The performance of two different pollination methods in term of seed germination rate expressed significant differences between canola plants exposed to

A. mellifera and other insects (94.5%) and caged plants of canola (61.3%) (Fig.

4.3.18).

Results of present study depicted significant effect of A. mellifera managed pollination on oil contents. Oil contents were recorded higher on A.mellifera and other insect visitors’ pollinated plants (49.5%) and lower on canola plants under caged (44.6%). Similar result was reported by Mahmood and Furgula (1983), as higher seed oil contents were obtained when pollinated by honey bees. Similarly according to Rajasri et al. (2012), the higher oil content was recorded with honey bee (39.6%) and lower on control plot without honey bees (31.6%). In another

4000 a 3500

3000

2500 (Mean±SE)

-1 2000 b

1500

1000 No. of seeds plant seeds of No. 500

0 T1 T2 Modes of Pollination

Fig. 4.3.12. Number of seeds per canola plant in response to different

treatments (T1= Plots caged without bees, T2= Open plots allowing

free visits of bees+ other pollinators) at URF Koont, Gujar Khan

during 2015.

6 a

4 b

(Mean±SE) -1 3

1 Seed Weight Seed pods 100

0 T1 T2 Modes of Pollination

Fig. 4.3.13.Seeds weight 100 canola pods-1in response to different treatments

(T1= Plots caged without bees, T2= Open plots allowing free

visits of bees+ other pollinators) at URF Koont, Gujar Khan

during 2015.

study, Kumar et al. (2002) reported the higher oil contents from the canola crop under bee and other insect pollination and the lower oil content was obtained in control canola plants. Rajagopal (2000) found high oil contents and high seed weight due to bee pollination, as compared to hand pollination and without bee pollination in sunflower crop. Present studies have disagreement with the findings of Oz et al. (2008) who noted that insect pollination had non significant influence on protein content. This may be due to protein levels in the crop vary with seasonal conditions (Cheema et al., 2001). In the present study, mean percent seed germination of B. napus was highest in open pollinated plots allowing free visits of managed A.mellifera and other insect visitors (95.4%), followed by plots caged without A. mellifera colony (61.3%). The above results are strongly supported by the findings of Bhowmik et al. (2014) who observed that bee pollination increases the germinability of the resulting seeds of B. juncea to 36±3 per cent as germination percentage in open pollinated seeds was 96% where as in control only

60% seeds germinated. Kumaret al. (2013) also reported highest germination percentage in open bee pollinated plots (90.8%) as compared to crop from pollinators’ exclusion (80.0%) in B. juncea (cv. RLC-1). The present findings are also corroborated by the findings of Mahindru et al. (1998) who reported that intensive pollination of B. juncea by A. mellifera increased seed germination by 7.2 percent over natural pollination, which decreased by 0.23 percent upon pollinators’ exclusion over natural pollination. Eisikowitch and Kevan (1988) suggested that insect pollinators are important in seed production in B. napus, especially for obtaining high germination rate (i.e. >90% germinability) for planting. Darwin

(1888) also observed that presence of honey bees on canola increases the

germination rate of resulting seeds from 83 to 96%.

4.4 EFFECT OF BEEHIVE DISTANCE ON COLONY LEVEL

POLLINATION EFFICIENCY OF APIS MELLIFERA ON BRASSICA

NAPUS

4.4.1 Colony Foraging Rate

Results about the effect of hive distance from B. napus on colony foraging rate of A. mellifera foragers returned with pollen on 04-02-2016 (1st weekly interval) revealed highly significant differences (F(2,16)= 149.51, P<0.00) (Appendix

27). Fig.4.4.1. indicated that at hive distance 100m from B. napus crop, activity of

A.mellifera returned with pollen loads was found maximum (185.00 pollen foragers/ 10 minutes) at 1200 hours followed by that at 1400 hours (148.33 pollen foragers/ 10 minutes) and at 1000 hours (105.67 pollen foragers/ 10 minutes).

However at hive distance 200m, colony foraging rate of A. mellifera was less than

100m beehive distance at 1200 hours (177.33 pollen foragers/ 10 minutes) followed by that at 1400 hours (123.00 pollen foragers/ 10 minutes) and at 1000 hours (91.33 pollen foragers/ 10 minutes). Similarly at hive distance 300m from B. napus maximum foraging rate of A. mellifera with significant differences was observed at1200 hours (116.67 pollen foragers/ 10 minutes) followed by that at 1400 hours

(101.00 pollen foragers/ 10 minutes) and at 1000 hours (74.00 pollen foragers/ 10 minutes). Interaction of time interval and hive distances from B. napus crop

st revealed highly significant differences (F(4, 16) =12.99, P<0.00) during 1 weekly interval (Appendix 27). The data regarding foraging rate of A. mellifera at different hives distance from B. napus crop was not possible to record on 11-02-2016 (2nd weekly interval) because of rain. Different hives distances from B. napus

20 Oil contents Oil (%)

0 T1 T2

Fig. 4.3.14. Percentage of oil contents of canola seeds in response to different

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar Khan during 2015.

25 a b 20

10 ProteinContent (%)

0 T1 T2 Modes of Pollination

Fig. 4.3.15. Percentage of protein contents of canola seeds in response to

different treatments (T1= Open plots allowing free visits of bees+

other pollinators, T2= Plots caged without bees) at URF Koont, Gujar Khan during 2015.

50 a b

OleicAcid (%) 20

0 T1 T2 Modes of Pollination

Fig. 4.3.16. Percentage of oleic acid of canola seeds in response to different

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar Khan during 2015.

12 a

10 b

4 Lenolenic(%) Acid

0 T1 T2 Modes of Pollination

Fig. 4.3.17. Percentage of linoleic acid of canola seeds in response to different

treatments (T1= Open plots allowing free visits of bees+ other

pollinators, T2= Plots caged without bees) at URF Koont, Gujar Khan during 2015.

100 a

80 b 60

40 Germinationrate (%) 20

0 T1 T2 Modes of Pollination

Fig. 4.3.18. Percentage of germination rate of canola seeds in response to

different treatments (T1= Open plots allowing free visits of

bees+ other pollinators, T2= Plots caged without bees) at URF Koont, Gujar Khan during 2015.

significantly affected the colony foraging rate of A. mellifera returned with pollen

rd loads (F (2, 16) =2699.39, P<0.00) on 18-02-2016 (3 weekly interval) (Appendix

28). Fig. 4.4.2 showed maximum A. mellifera returned with pollen loads at hive distances 100m at 1200 hours (431.00 pollen foragers/ 10 minutes) followed by that at 1400 hours (381.67 pollen foragers/ 10 minutes) and at 1000 hours (342.33 pollen foragers/ 10 minutes). At hive distance 200m, maximum numbers of pollen foragers were observed at 1200 hours (356.00 pollen foragers/ 10 minutes) followed by that at 1400 hours (302.67 pollen foragers/ 10 minutes) and at 1000 hours (248.33 pollen foragers/ 10 minutes) where as significant difference was found at hive distance 300m from B. napus at 1200 hours (264.00 pollen foragers/

10 minutes) followed by that at 1400 hours (220.67 pollen foragers/ 10 minutes) and at 1000 hours (193.3 pollen foragers/ 10 minutes). Interaction of hives distance

rd and time interval during 3 week of study also showed significant differences (F(4,

16) =13.05, P<0.00) (Appendix 28). Results showed that colony foraging rate of

A.mellifera returned with pollen loadson 25-02-2016 (4th weekly interval) significantly different at hives distance (F(2, 16) =2787.06, P<0.00) (Appendix 29).

Fig. 4.4.3 clearly indicated significant difference of hives distance 100m at 1200 hours (366.67 pollen forager/10minutes) compared to other two observation times i.e. 1000 and 1400 hours ranging from 308.33 to 325.00(pollen forager/ 10 minutes). At hive distance 200m, maximum numbers of A. mellifera with pollen loads were observed at 1200 hours (237.33 pollen forager/10minutes) followed by that at 1400 hours (218.00 pollen forager/10minutes) and at 1000 hours (206.67 pollen forager/10minutes) whereas hive distance 300m revealed maximum pollen foraged bees at 1200 hours (202.00 pollen forager/10minutes) followed by that at

1400 hours (189.67 pollen forager/10minutes) and at 1000 hours (175.3 pollen forager/10minutes). Performance of colony foraging rate of A. mellifera at 100m hive distance on 03-03-2016 (5thweekly interval) revealed maximum returned bees with pollen at 1200 hours (247.33 pollen forager/10minutes) followed by that at

1400 hours (217.00 pollen forager/10minutes) and at 1000 hours (136.00 pollen forager/10minutes) whereas at 200m hive distance from the B. napus, maximum number of pollen foragers were observed at 1200 hours (198.00 pollenforager/10minutes) followed by that at 1400 hours (186.00 pollen forager/10minutes) and at 1000 hours (105.67 pollen forager/10minutes). In the same week, at hive distance 300m from B. napus, more bees returned with pollen loads were observed at 1200 hours (169.67 pollen forager/10minutes) followed by that at 1400 hours (113.33 pollen forager/10minutes) and at 1000 hours (91.6 pollen forager/10minutes) Fig. 4.4.4. Interaction of hive distance and time interval in the 5th week of study showed significant differences for colony foraging rate of

A. mellifera returned with pollen loads at different hives distance from the B. napus

(F(4, 16) =65.95, P<0.00) (Appendix 30). Results regarding colony foraging rate of

A. mellifera returned with pollen laods at different hives distance from B. napus

th crop on 10-02-2016 (6 weekly interval) showed significant differences (F(2, 16)

=999.84, P<0.00) (Appendix 31). At 100m hives distance per 10 minutes at 1200 hours, 238.33 pollen forager/10minutes followed by that at 1400 hours (211.3 pollen forager/10minutes) and at 1000 hours (78.67 pollen forager/10minutes) were observed (Fig. 4.4.5). Hive distance 200m showed maximum colony foraging rate of A.mellifera returned with pollen loads at 1200 hours (192.33 pollen forager/10minutes) followed by that at 1400 (174.3 pollen forager/10minutes) and

at 1000 hours (64.00 pollen forager/10minutes). At 300m hive distance, maximum colony foraging rate of A. mellifera returned with pollen loads were found at 1200 hours (130.00 pollen forager/10minutes) followed by that at 1400 hours (109.67 pollen forager/10minutes) and at 1000 hours (4833.00 pollen forager/10minutes).

Interaction of hives distance and time interval showed highly significant differences for colony foraging rate of A. mellifera returned with pollen loads on 6th week of study (F(4, 16) =99.02, P<0.00) (Appendix 31). Data regarding colony foraging rate of Apis mellifera was not possible to record on 17-03-2016 (7th weekly interval) because of rain. Colony foraging rate of A. mellifera returned with pollen loads at different hives distance on 24-03-2016 (8th weekly interval) also showed significant differences (F(2,16)=305.79, P<0.00) (Appendix 32). Fig. 4.4.6 illustrated that at hive distance 100m, maximum colony foraging rate of A. mellifera returned with pollen loads was observed at 1200 hours (137.33 pollen forager/10minutes) followed by that at 1400 hours (95.67pollen forager/10minutes) and at 1000 hours (56.33 pollen forager/10minutes). In the same week (24-03-

2016) at hive distance 200m from B. napus, maximum numbers of A. mellifera returned with pollen loads were observed at 1200 hours (82.00 pollen forager/10minutes) followed by that at 1400 hours (61.33 pollen forager/10minutes) and at 1000 hours (44.33 pollen forager/10minutes). However at 300m distance from B. napus, maximum colony foraging rate of A. mellifera returned with pollen loads was observed at 1200 hours (61.33 pollen forager/10 minutes) followed by that at 1400 hours (49.33 pollen foragers/10minutes and at

1000 hours (25.33 pollen forager/10minutes). Interaction of hives distance and time interval showed highly significant differences for colony foraging rate at different

hive distance (F(2,16) =25.47, P<0.00) (Appendix 32).

Colony foraging rate of A. mellifera with pollen loads per 10 minutes was assessed by different hive distance from B. napus throughout flowering season revealed that density of A.mellifera pollen collecting foragers declined significant with distance from the hives. There was a significant difference between pollen foragers at hive distance 100m and 200m from B. napus and a clear drop in numbers of A. mellifera was observed for 300m hives distance. Present findings is in line with outcomes of Fries and Stark (1983) who stated that density of honeybees in their B. rapa crop declined significantly with distance from hive, especially at hive distances greater than 300m from the edge of the crop. In another study of Manning and Wallis (2005) indicated that crop yield of B. napus was declined in plots located more than 200m from the apiary. Present findings also indicated that density of A. mellifera relates with time interval and season of the

B.napus crop. At 1000 hours in all three different hive distance, colony foraging rate of A. mellifera were found minimum as compared to 1200 hours and 1400 hours. Maximum colony foraging rate in all three different beehives distance was found at 1200 hours of the day. Colony foraging rate of A. mellifera were low at the time of commencement and cessation of the flowering but it remained high during mid flowering period. Present results indicated strong interaction between hive distance and time interval of foraging activity of A. mellifera bees.

4.4.2 Colony Condition with Different Beehives Distance from Brassica napus

Results about colony condition of A. mellifera at different hives distance on

(02-02-2016) (1st weekly interval) depicted that the area of egg brood was only

Time1000h Time1200h Time1400h 500

450

400

350

300

250

200

150 colony foraging rate/ minutes 10

100

Apismellifera 0 100 m 200 m 300 m Hive distance from Brassica napus crop

Fig. 4.4.1.Colony foraging rate of Apis mellifera (Mean ± SE) at different

distances from Brassica napus crop on 04-02-2016 (1st weekly

interval) at URF Koont, Gujar Khan during 2016.

100

Time1000h Time1200h Time1400h 500

450

400

350

300

250

200

150 colony foraging rate/ minutes 10

100

Apismellifera 0 100 m 200 m 300 m Hive distance from Brassica napus crop

Fig. 4.4.2. Colony foraging rate of Apis mellifera (Mean ± SE) at different

distances from Brassica napus crop on 18-02-2016 (3rd weekly

interval) at URF Koont, Gujar Khan during 2016.

101

Time1000h Time1200h Time1400h 500

450

400

350

300

250

200

150 colony foraging rate/ minutes 10

100

Apismellifera 0 100 m 200 m 300 m Hive distance from Brassica napus crop

Fig. 4.4.3. Colony foraging rate of Apis mellifera (Mean ± SE) at different

distances from Brassica napus crop on 25-02-2016 (4th weekly

interval) at URF Koont, Gujar Khan during 2016.

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significantly different between hives distance 100m (2903) and 300m (1366). The area under beehives distance 200m (2404) was not significantly different from

100m and 300 m from B. napus. Similarly, the results about open brood was also significant between treatments (F2, 6 =05.24, P=0.04). The area of open brood was significantly higher at hives distance 100m (3357) followed by that at 200m (2362) and at 300m (1560) from B. napus. The open brood area under hives distance 200m was not significantly different from 100m and 300m. The area of capped brood was also significantly different between treatment groups (F2, 6 =07.38, P=0.02). The area of capped brood was significantly higher at 100m (5501) followed by that at

200m (3665) and at 300m (1971). Non significant difference was found in area of stored honey (F2, 6 =03.52, P=0.09). The area of stored honey at 100m was (6802) followed by that at 200m (4900) and at 300m (3806). The area of stored pollen was significantly different between treatment groups (F2, 6 =05.17, P=0.04). The area of stored pollen was only significantly different between 100m (946) and 300m (428).

Pollen stored in beehives kept at 200m (720) was not significantly different from those kept at 100m and 300m distance (Fig. 4.4.7).

Results regarding colony condition at different hives from B. napus on 11-

02-2016 (2nd weekly interval) demonstrated significant results for area of egg brood

(F2,6=10.28, P=0.01). Area of laid egg at 100m hives distance (3274) was statistically significant different as compared to other two beehives distance from

200m to 300m (2431 to 1419) respectively. Area of open brood was found statistically significant at different hives distance from B. napus (F2,6=08.35,

P=0.01). At 100m, the area of open brood was significantly different (3630) from others followed by that at hives distance 200m (2514) and at 300m (1688).

103

Time1000h Time1200h Time1400h 500

450

400

350

300

250

200

150 colony foraging rate/ minutes 10

100

Apismellifera 0 100 m 200 m 300 m Hive distance from Brassica napus crop

Fig. 4.4.4. Colony foraging rate of Apis mellifera (Mean ± SE) at different

distances from Brassica napus crop on 03-03-2016 (5th weekly

interval) at URF Koont, Gujar Khan during 2016.

104

Time1000h Time1200h Time1400h 500

450

400

350

300

250

200

colonyforaging rate/ minutes 10 150

100

Apismellifera 0 100 m 200 m 300 m Hive distance from Brassica napus crop

Fig. 4.4.5. Colony foraging rate of Apis mellifera (Mean ± SE) at different

distances from Brassica napus crop on 10-03-2016 (6th weekly

interval) at URF Koont, Gujar Khan during 2016.

105

Time1000h Time1200h Time1400h 500

450

400

350

300

250

200

150 colony foraging rate/ minutes 10

100

Apismellifera 0 100 m 200 m 300 m Hive distance from Brassica napus crop

Fig. 4.4.6. Colony foraging rate of Apis mellifera (Mean ± SE) at different

distances from Brassica napus crop on 24-03-2016 (8th weekly

interval) at URF Koont, Gujar Khan during 2016.

106

Similarly, area of capped brood was significantly different between distances (F2,6=8.06, P=0.01). Area of capped brood at 100m was found maximum

(5705) followed by that at 200m (3847) and at 300m (2096). Colony condition regarding stored honey was statistically significant between treatments (F2, 6 =4.60,

P=0.06). Area of stored honey at 100m was maximum (7732) followed by that at

200m (5697) and at 300m beehives distance (4624). Area of stored pollen was significantly different between different hives distance (F2,6=05.29, P=0.04). Stored pollen area was only significantly different between 100m (1097) and 300m (561).

The area under 200m (880) was not significantly different from 100m and 300m

(Fig. 4.4.8).

Results of colony conditions at different hives distance from B. napus on

18-02-2016 (3rd weekly interval) showed significant differences for eggs

(F2,6=31.65, P=0.00). At hives distance 100m from B. napus, the area of eggs were found maximum (5818) followed by that at 200m beehives distance (4633) and at

300m (3259). Area of open brood was found statistically significant (F2,6=10.11,

P=0.012). Area of open brood at hives distance 100m was found high (4119) followed by that at 200m (2702) and at 300m beehives distance (1716). Similarly, area of capped brood showed significant results (F2,6=8.65, P=0.01). At beehives distance 100m from B. napus, area of capped brood was high (5984) followed by that at 200m (4136) and at 300m beehives distance (2295). Area of stored honey at different hives distance from B. napus showed significant results (F2,6=5.08,

P=0.051). At different hives distance from B. napus, the area of stored honey was found maximum at 100m distance (8836) followed by that at 200m (6779) and at hives distance 300m (5390). Area of stored pollen was found significantly different

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between treatments (F2,6=5.6380, P=0.04). At hives distance 100m from B. napus, the area of stored pollen was maximum (1297) followed by that at 200m (1080) and at 300m beehives distance (754.6) (Fig. 4.4.9).

Data recorded about colony condition at different hives distance from

B.napus crop on 25-02-2016 (4th weekly interval) of study (Fig. 4.4.10) demonstrated significant results for area of eggs (F2,6=7.18, P=0.02). At 100m hives distance, the area of egg was found high (6251) followed by that at 200m

(5267) and at 300m (4329) hives distance. Area of open brood was found significantly different between treatments (F2,6=5.20, P=0.04). At 100m hives distance, area of open brood was found maximum (4507) followed by that at 200m

(2929) and at 300m hives distance (1851). Area of capped brood was found significantly different between treatments (F2,6=6.93, P=0.02). At hives distance

100m, capped brood was found maximum (6147) followed by that at 200m (4440) and at 300m (2521). In case of stored honey, its area was also found significant between treatments (F2,6=5.11, P=0.05). At hives distance 100m, the area of stored honey was found maximum (10695) followed by that at 200m (8678) and at 300m

(7368). However, the area of stored pollen depicted significant results between beehives distance 100m and 300m (F2,6=5.22, P=0.04). At 100m, maximum area of stored pollen was found (1430.7) followed by that at 200m (1246) and at 300m

(888) hives distance. Results in terms of colony condition at different hives distance (Fig. 4.4.11) illustrated significant results (F2,6=7.47, P=0.023) for the area of egg brood on 03-03-2016 (5th weekly interval). Area of egg brood was found high at hives distance 100m (6413) followed by that at 200m (5393) and at 300m

(4320). Similarly, area of open brood showed significant results between different

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hives distance (F2,6=5.34, P=0.04). At hives distance 100m, the area of open brood was found high (4737) compared to other two hives distance 200m and 300m ranging from (3120 to 2008). Area of capped brood depicted significant results between 100m and 300m hives distance from B. napus(F2,6 =6.39, P=0.03). At

100m hives distance from B.napus, area of capped brood was significantly high

(6319) followed by that at 200m (4648) and at 300m (2687). For area of stored honey showed significant results between treatments (F2,6=4.94, P=0.05). However, area of stored honey at 100m hives distance was found maximum (11602) followed by that at 200m (9781) and at 300m (8375). Area of stored pollen was found non significant between treatments (F2,6=5.31, P=0.04). At 100m hives distance, the area of stored pollen was high (1520) followed by that at 200m (1350) and at 300m

(968). Fig. 4.4.12 illustrated colony conditions of A. mellifera at different hives distance on 10-03-2016 (6th weekly interval). Thearea of egg showed significant results between treatments (F2,6=8.85, P=0.016). Area of egg was found significantly different (6580) at 100m hives distance followed by that at 200m

(5360) and at 300m (4587). Area of open brood was found significantly different between treatments (F2,6=5.39, P=0.04). At hives distance 100m, area of open brood was found significantly high (4937) followed by that at 200m (3320) and at

300m (2142). Area of capped brood also showed significant results (F2,6=6.419,

P=0.03). At 100m hives distance, area of capped brood was found maximum

(6604) followed by that at 200m (4820) and at 300m (2850) hives distance from B. napus. Area of stored honey during 6th week of study showed non significant results between hives distance 200m and 300m (F2,6 =4.66, P=0.06). At 100m hives distance from B. napus, area of stored honey was found maximum (12793)

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followed by that at 200m (10752) and at 300m (9473). Area of stored pollen depicted significant differences between treatments (F2,6=6.20, P=0.03). At hives distance 100m, area of stored pollen was found high (1624) than other two hives distance 200m and 300m ranging from 1390 to 1064.

Results regarding colony conditions of A. mellifera at different hives from

B. napus crop on 17-03-2016 (7th weekly interval) (Fig. 4.4.13) demonstrated significant differences for the area of eggs between treatments (F2,6=6.61, P=0.03).

At 100m beehive distance, area of eggs was found highe (6641) followed by that at

200m (5426) and at 300m (4687). Area of open brood was also found significantly different between treatments (F2,6=6.91, P=0.02). The area of open brood at 100m beehives distance from the main crop was found significantly highest (5304) compared to other two observation i.e. 200m and 300m ranging from (3520 to

2375). Area of capped brood during 7th week of observation showed significant difference between treatments (F2,6=6.75, P=0.02). At 100m, area of capped brood was found high (6882) followed by that at 200m (5085) and at 300m (3054). Stored honey depicted significant differences between different hives distance from

B.napus (F2,6=4.83, P=0.05). At hives distance 100m, area of stored honey was found maximum (13638) followed by that at 200m (11715) and at 300m hives distance from B. napus (10329). Similarly, area of stored pollen during 7th week of study was found significantly different between treatments (F2,6=5.35, P=0.04).

Area of stored pollen at 100m hives distance from B. napus was found maximum

(1757) followed by that at 200m (1490) and at hives distance 300m (1138).

Results about colony condition in terms of eggs of A. mellifera at different hives from B.napus on 24-03-2016 (8th weekly interval) (Fig. 4.4.14) illustrated

110

significant differences between treatments (F2,6=5.56, P=0.04). Area of eggs was only found significantly different between hives distance 100m (6746) and 300m

(4553). The area at hive distance 200m (5359) was not significantly different from

100m and 300m during 8th week. Area of open brood was found significantly different between treatments (F2,6=7.61, P=0.02). At hives distance 100m from B. napus, area of open brood was found maximum (5537) followed by that at 200m

(3720) and at 300m (2575). Area of capped brood was found significant between treatments during last week of study (F2,6=6.63, P=0.03). Area of open brood was only significantly different between hives distance 100m (7215) and 300m (3321).

Area of open brood at 200m hive distance (5485) was not significantly different from 100m. Area of stored honey showed significant results at different hives distance from B. napus (F2,6=6.46, P=0.03). At hives distance 100m from B. napus, area of stored honey was found maximum (14729) followed by that at 200m

(12370) and at 300m (11435). Area of stored pollen depicted significant results between treatments (F2,6=5.35, P=0.04). At 100m hive distance from B. napus, area of stored pollen on last week was found maximum (1857) followed by that at 200m

(1590) and 300m (1238).

Results of the present study demonstrated that colony conditions in terms of brood and stored food area at different hives distance from B. napus crop varied throughout the B. napus blooming period. Area of each group in population of

A.mellifera tends to be decrease as the distance of hives increases from B. napus.

Maximum brood (Eggs, open brood and capped brood) and stored food (Honey and pollen) was observed at hives distance 100m then it started to decrease at 300m hives distance throughout blooming period of B. napus. These results supports

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previous work of Fries and Stark (1983) that population dynamics of the honeybees in their Brassica rapa crop declined significantly with distance from the hive, especially at distances greater than 300m from the edge of the crop. Present results also showed that population dynamics of A. mellifera depends on the food availability. Maximum brood population was observed in March. Honey stored in last week of study was found to be significantly high than other weeks of study.

Population dynamics in terms of area of eggs, open brood, capped brood and stored pollen was found significantly between treatments. This significance of our results can be considered with respect to the effect of colony size and time of year on the relative values of nectar and pollen. These results are in line with the findings of

Eckert et al. (1994).

4.4.3 Correlation of Weather Factors with Foraging Rate of Apis mellifera

Weather factors had varied correlation with the colony foraging activity of

Apis mellifera in Brassica napus fields. Among these, temperature had significant, positive and strong correlation with the colony foraging rate of A. mellifera

(r=0.754; P= 0.0306*) whereas relative humidity and rainfall had significantly negative and strong correlation (r= -0.80274; P= 0.0165*, r= -0.73891; P=

0.0362*) with foraging activities of bees on B. napus (Table 4.4.1). Fig. 4.4.15 showed that on 2nd and 7th week of observation increase in R.H. combined with rainfall resulted in a drastic decrease in A. mellifera foraging activity (zero population). A. mellifera colony foraging activity was at maximum on 3rd week of observation (18-02-2016) when the temperature was 22 ̊C and average R.H. was

60%. This could be because of the favorable conditions particularly temperature and blooming period. Present results are partially in agreement with those of Tan et

112

al. (2012) who reported highest activity of A. mellifera at 20 C.̊ Our results are also in accordance with Kasper et al. (2008) who stated that temperature positively influenced the insect pollinators’ activity on flowers. Omoloye and Akinsola

(2006) also declared that bee activity was found to be significantly positive correlated with the temperature and significantly negative with the relative humidity in all the three honeybee species and on different cultivars of oilseed crops. After 3rd week of observation, foraging rate of A. mellifera tended to decrease up to last week of observation (24-02-2016). This may be due to variation in amount of nectar present in flowers. According to Zajáacz et al. (2008) amount of nectar produced in flowers influenced by temperature, relative humidity as well as rainfall during flowering period. Low visits of A. mellifera may be due to scarce availability of resources.

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E OB CB 16000 SH SP 15000 14000 13000

2 12000 11000 10000 9000 8000 7000 6000 5000 4000

3000 Brood and stored and Brood area food cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.7 Brood and stored food area of Apis mellifera at different hives distance (Mean±SE) on 02-02-2016 (1st weekly interval) throughout Brassica napus blooming period at URF Koont, Gujar Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

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E OB CB 16000 SH SP 15000 14000

13000 ) 2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 Brood and stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.8 Brood and stored food area of Apis mellifera at different hives distance

(Mean±SE) on (11-02-2016) (2nd weekly interval) throughout Brassica

napus blooming period at URF Koont, Gujar Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

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E OB CB 16000 SH SP 15000 14000

13000 ) 2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 Brood and stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.9 Brood and stored food area of Apis mellifera at different hives

distance (Mean±SE) on (18-02-2016) (3rd weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP=

Stored Pollen

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E OB CB 16000 SH SP 15000 14000

13000 ) 2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 Brood and stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.10 Brood and stored food area of Apis mellifera at different hives

distance (Mean±SE) on (25-02-2016) (4th weekly interval)

throughout Brassica napus blooming period at URF Koont,

Gujar Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

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E OB CB 16000 SH SP 15000 14000

13000 ) 2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 Brood and stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.11 Brood and stored food area of Apis mellifera at different hives

distance (Mean±SE) on (03-03-2016) (5th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

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E OB CB 16000 SH SP 15000 14000

13000 ) 2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 Brood and stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig 4.4.12 Brood and stored food area of Apis mellifera at different hives

distance (Mean±SE) on (10-03-2016) (6th weekly interval)

throughout Brassica napus blooming period at URF Koont, Gujar

Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

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16000 E OB CB SH SP 15000 14000

13000 )

2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.13 Brood and stored food area of Apis mellifera at different

hives distance (Mean±SE) on (17-03-2016) (7th weekly

interval) throughout Brassica napus blooming period at

URF Koont, Gujar Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

120

E OB CB 16000 SH SP 15000 14000

13000 ) 2 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 Brood and stored and Brood area food (cm 2000 1000 0 100m 200m 300m Hive distance from Brassica napus

Fig. 4.4.14 Brood and stored food area of Apis mellifera at different hives

distance (Mean±SE) on (25-03-2016) (8th weekly interval)

throughout Brassica napus blooming period at URF Koont,

Gujar Khan during 2016.

E= Eggs, OB= Open Brood, CB= Capped Brood, SH= Stored Honey, SP= Stored Pollen

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Table 4.4.1 The correlation between weather factors and Apis mellifera colony foraging

rate for Brassica napus L. at URF Koont, Gujar Khan during 2016.

Weather Factors r p

Avg. Temperature (̊C) 0.7542 0.0306*

Avg. Relative Humidity (%) -0.7389 0.0362*

Rainfall (mm) -0.80274 0.0165*

*Indicated significant

n= 12

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400 100

350 90 80 300 70 250 60

foragers/10min 200 50 Apis mellifera foragers 40 150 Temperature ( ͦC) 30 100 R.H (%) 20 Rainfall (mm)

50 10 Apis mellifera Apis 0 0

Weeks of observation

Fig. 4.4. 15. Effect of temperature (̊C), relative humidity (%) and rainfall (mm)

on Apis mellifera visits/ 10minutes per canola plant at weekly

interval on Brassica napus in URF Koont, Gujar Khan during 2016.

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CONCLUSION

In lights of above results, local beekeepers can get useful information regarding main crop (B. napus) and alternate foraging sources for their managed honeybee colonies of Apis mellifera L. at UKF, Gujar Khan. It is also recommended that A. mellifera is a major pollinator of canola (Brassica napus).

On the basis of the research findings for managed pollination of B. napus,

Apis mellifera played an important role in the enhancement of seed quality of B. napus and recommended for the farmers to increase the productivity of their canola crop with managed pollination of A. mellifera by introducing their managed honeybees colonies near the vicinity of canola crop resulted in considerable increase in yield as well as quality.

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124

SUMMARY

Canola (Brassica napus L.) is an important oil seed crop, highly dependent on insect pollinators’especially A. mellifera to increase its seed productivity. Apis mellifera L. is considered as the most important flower visitors and most efficient B. napus pollinators. Keeping in view its economic important, present study was planned and conducted at University Research Farm Koont,

Gujar Khan (2015-2016) to determine the pollinator’s diversity and abundance in

B. napus alongwith managed Apis mellifera pollination, determination of colony foraging sources of A. mellifera and pollen quantification, effect of hives distance on foraging rate and colony condition of A. mellifera and influence of A. mellifera on yield of B. napus.

Current study comprised four parts. In the first part of study, investigations were made on pollen forage sources of A. mellifera; they comprised of 18 species belonging to 11 families. Asteraceae contributed 6 plant species as pollen sources followed by Brassicaceae and Solanaceae with two plant species as pollen sources. The other families had one pollen sourceeach.

About 11.11% of the identified plants were crops species while 33.33%, 22.22%,

22.22% and 11.11% were weeds, shrubs, herbs and ornamental plants respectively. Total number of pollen species observed from A. mellifera body were belongs to families of Brassicaceae, Asteraceae, Malvaceae, Solanaceae,

Primulaceae, Convolvulaceae, Euphorbiaceae, Bignoniaceae, Xanthorrhoeaceae,

Fabaceae and Amavanthaceaea. Among these observed families, maximum numbers of B. napus pollens from family Brassicaceae were observed from

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125

pollen samples of 5th (03-02-2015) (78.10%), 6th (10-02-2015) (77.43%) and 7th

(17-02-2015) (76.81%) week of study and minimum numbers of B. napus pollens were found on 10th (10-03-2015) (24.86%), 11th (17-03-2015) (25.32%) and 12th

(24-03-2015) (5.53%) week of the study. After Brassicaceae, Asteraceae played an important role in foraging of A. mellifera. From Asteraceae, maximum pollens of Sonchus asper were observed on 10th (10-03-2015) (25.5%), 11th (17-03-2015)

(26.4%) and 12th (24-03-2015) (30.1%) week of the study. After B. napus and S. asper, pollens of Calendula officinalis were found maximum from A. mellifera body on 10th (24.3%), 11th (27.8%) and 12th (29.1%) week of study. A. mellifera foragers also used Taraxacum officinale as a pollen source. Collection of T. officinale started in the first week of study (06-01-2015) and continued until last week (24-03-2015) of observation. In the study area, pollens of other plant species (P. hysterophorus, H. autumnale, C. indicum, A. rosea, A. esculentus, S. nigrum, Petunia spp., A. arvensis, C. arvensis, Euphorbia spp., T. stans, A. tenuifolius, L. perennis and Celosia spp.) found meager in numbers.

The 2nd experiment was designed to investigate the colony level pollination efficiency of A. mellifera on B. napus crop. The results revealed that maximum foraging activity was observed at 1200 hours during the whole period of experiment from 6th January to 24th March, 2015. Results indicated that on average maximum colony foraging rate of A. mellifera (281.2 forager bees/ ten minutes) was observed at 1200 hours on 10-02-2015 (6th weekly interval) of study. Similarly, analysis of variance revealed significant differences between two time intervals from 1000 hours to 1400 hours. Although in most of weeks, foraging rate at 1200 hours was maximum and significantly different from others,

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however, there were some weekly intervals when foraging rate at 1000 hours was statistically similar with that of at 1400 hours interval i.e., 3rd, 4th and 5th weekly observation showed significant differences and the rest weekly observations showed non significant differences among two intervals of observation. Among weather factors, temperature was only positively related factor with the colony foraging rate of A. mellifera (r= 0.744; P= 0.0055**) whereas relative humidity and rainfall had significantly negative correlation (r= -0.7287; P= 0.0072**; r= -

0.7094; P= 0.0098**) with visits of bees on B. napusflowers during 2015. On

2nd, 8th and 9th weeks of study, R.H. increased combined with rainfall resulted in a zero colony foraging activity of A. mellifera. A. mellifera foragers attained maximum activity during 6th week (10-02-2015) of study when the temperature was 21.5 ̊C and average R.H. was 60%. The results about colony condition of A. mellifera during blooming period of B. napusin 2015 showed that area of eggs were found minimum (1767) on 08-01-2015 (first weekly interval) and increased with blooming progression of B. napus up to (6414) cells per colony in the last week (24-03-2015) of study. Similarly, open brood was also gradually increased with evident and found maximum (7365) during last week of study (24-03-2015) while minimum open brood (2978.3) was found on the first week of study (06-

01-2015). The area of capped brood was significantly higher (7800.8) on the last week of study (24-03-2015) while minimum (2957) was observed during first week of study (06-01-2015). The results about colony condition in terms of food stores collected by A. mellifera during blooming period of B. napus illustrated that significant difference was also found in area of stored honey from first week

(7619.6) up to last week of study (10830). Similarly, increasing trend in area of

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stored pollen was also observed. Maximum stored pollen was observed during last week of flowering (1940.6) while minimum was observed during first week

(557.8) of study.

Third experiment was designed to find out the contribution of pollination by managed A. mellifera on yield of B. napus. For this, observations were madeon insect pollinators throughout the flowering period of B. napus during which thirty five species belonging to five orders and twenty families were recorded visiting canolacrop, out of these Hymenoptera constituted 4557 (89.8%) of insect pollinators. Managed A. mellifera was most frequent visitor comprising

87.66%. Maximum activity was observed during 1200 hours followed by that at

1400 hours and at 1000 hours; this trend was similar for majority of insect pollinators. Maximum visitation frequency performed by A. mellifera on

Brassica napus flowers during 1200 hours of observation on 6th week of study

(5±0.19) however, minimum number of visits per plant per minute were recorded on 12th week (2.41± 0.12), however comparison of 1000 hours depicted highest activity during 3rd (2.58±0.06) and 4th week (2.58± 0.18) of study and lowest during 12th week (1.48±0.11). Similarly data of 1400 hours showed greater activity on 6th week (3.55± 0.10) while lowest activity during 12th week

(1.95±0.08) of observation. Maximum pods plant-1 expressed significant difference between open bee pollinated (350.5) and without insect pollinated

(166.25) canola plants. Maximum seeds per plant were resulted from open bee pollination (3408.5) as compared to withoutinsect pollinated canola plants

(1589.75). Highest seed weight (5.41g) resulted from managed honeybee and open pollination followed by caged canola plants (3.75g) where as oil content

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percentage (49.5) resulted from insect pollination followed by caged canola plants (45.6). Protein content was significantly high (24.6) in open pollinated canola plants followed by caged plants (21.4). Fatty acids like Oleic acid and

Linolenic acid was 48.7 and 10.3 respectively in open bee pollinated plants as compared to without insect pollinated canola plants, these fatty acids were 44.3 and 8.8 respectively. Canola seed germination rate expressed significant difference between non caged canola plants (94.5%) and caged canola plants

(61.3%).

In the 4th experiment, effect of beehives distance on colony level pollination efficiency of A. mellifera on B. napus crop was evaluated. Results showed that maximum colony foraging rate by Apis mellifera at 100m hives distance from B. napus) during 3rd week (18-02-2016) of study at 1000 hours

(342.33±3.7) however, minimum number of bees with pollen per 10 minutes were recorded on last week of observation (56.33±2.2), similarly data of 1200 hours showed greater activity of pollen bees on 3rd week (431±3.1) while lowest activity during last week (137.33±2.3) of studyat 1400 hours maximum pollen bees were observed during 3rd week (381.67±2.2) of study while minimum activity was found in last week (95.67±3.8) of study. However comparison of

200m beehives distance from B. napus depicted highest activity of pollen bees at

1000 hours during 3rd week of observation (248.33±2.4) and lowest during last week (44.33±2). Similarly at 1200 hours maximum activity was found during 3rd week (356±2.6) while lowest during last week (82±2.1) of study. At 1400 hours, greater activity of bees with pollen was found during 3rd week (302.67±1.8) while minimum during last week (61.33±2.4) of observation. Mean comparison of

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300m hives distance showed maximum activity of pollen bees at 1000 hours during 3rd week of observation (193.3±1.7) and minimum during last week

(25.33±1.8) of study while at 1200 hours, showed greater activity (61.33±2.8).

Maximum colony foraging rate of A. mellifera with pollen was observed at 1400 hours (220.67±2.3) during 3rd week of observation while minimum (49.33±1.7) during last week of study.

Maximum colony conditions of A. mellifera at 100m hives distance for eggs was observed on last week of study (24-03-2016) (6746) while minimum was recorded during first week of observation (2903±437.3) however, comparison to 200m hives distance, maximum brood and stored food was also observed during last week of study (5359) and minimum was observed during first week (2404) of observation. Similarly at 300m, maximum brood and food stored was observed during last week (4553) and minimum (1419) was during first week. Results about open brood of A.mellifera at 100m showed maximum population during last week (5537) of study while minimum was observed during first week (3357) however, at 200m highest population dynamics of A. mellifera open brood was observed during last week (3720) while minimum was recorded during first week (2362) of study. Open brood at 300m showed maximum colony condition during last week (2575) while minimum was observed during first week (1560). Maximum capped brood of Apis mellifera at 100m was observed during last week (7215) of study, while minimum was observed during first week

(5501) of study. At 200m beehives distance, maximum capped brood was observed on last week (5485) of study while minimum was observed during first week (3665) of study. Comparison to 300m, maximum colony conditions about

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capped brood was recorded during last week (3321) of study, while minimum was observed during first week (1971) of study. Maximum area of stored honey at 100m hives distance was observed during last week of study (14729) and minimum (6802) was recorded on first week of study. At 200m hives distance, maximum stored honey was observed on last week (12370) of study while minimum was observed during first week (4900) of study. Comparison to 300m, maximum colony conditions about stored honey was recorded during last week

(11435) of study, while minimum was observed during first week (3806) of study. Results about stored pollen of A.mellifera at 100m showed maximum population dynamics during last week (1857) of study while minimum was observed during first week (946) however, at 200m highest population dynamics of A. mellifera stored pollen was observed during last week (1590) while minimum was recorded during first week (720) of study. Stored pollen at 300m showed maximum colony condition during last week (1238) while minimum was observed during first week (428).

Weather conditions 2016 have influenced the colony foraging activity of

Apis mellifera at different hives distance in the field conditions. Among weather factors, temperature was only positively related factor with the colony foraging rate of A. mellifera during eight weeks of study (r= 0.75418; P= 0.0306*) whereas relative humidity and rainfall had significantly negative correlation (r= -

0.80274; P= 0.0165*,r= -0.73891; P= 0.0362*) with inflorescence visits of bees on B. napus. On 2nd and 7th date of observation increase in R.H. combined with rainfall resulted in a drastic decrease in A. mellifera foraging activity. A. mellifera colony foraging activity was at maximum on 3rd week of observation (18-02-

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2016) when the temperature was 22 ̊C and average R.H. was 60%.

The overall findings of present study demonstrated that insight into the importance of managed A. mellifera to help in B. napus pollination, an important crop in the Gujar Khan region. Our results indicate diversity and abundance of pollinator insects, especially abundance managed A. mellifera, plays a significant role in seed quantity and quality of Canola crop.

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APPENDICES

Appendix 1: Variance analysis of data regarding colony foraging rate of Apis

mellifera during 1st week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 6468.4 3234.2 6.4060 0.0218 * Replications 4 3241.06 810.266 1.6049 0.2635 ns Error 8 4038.933 504.866 Total 14 13748.4

Appendix 2: Variance analysis of data regarding colony foraging rate of Apis mellifera during 3rd week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 25829.733 12914.8 39.211 0.0001 *** Replications 4 4450.6666 1112.66 3.37820 0.0672 ns Error 8 2634.933 329.366 Total 14 32915.333

Appendix 3: Variance analysis of data regarding colony foraging rate of Apis

mellifera during 4th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 4941.733 2470.866 39.5127 0.0001 *** Replications 4 497.7333 124.4333 1.9898 0.1891 ns Error 8 500.266 62.5333 Total 14 5939.733

157

158

Appendix 4: Variance analysis of data regarding colony foraging rate of Apis mellifera during 5th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 82743.333 41371.66 28.3788 0.0002 *** Replications 4 1995.333 498.833 0.34217 0.8424 ns Error 8 11662.666 1457.833 Total 14 96401.333

Appendix 5: Variance analysis of data regarding colony foraging rate of Apis

mellifera during 6th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 7744.533 3872.266 8.79694 0.0095 ** Replications 4 3025.733 756.4333 1.718450 0.2384 ns Error 8 3521.466 440.1833 Total 14 14291.73

Appendix 6: Variance analysis of data regarding colony foraging rate of Apis

mellifera during 7th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 68212.8 34106.4 13.36510 0.0028 **

Replications 4 16175.6 4043.9 1.584662 0.2682 ns Error 8 20415.2 2551.9 Total 14 104803.6

159

Appendix 7: Variance analysis of data regarding colony foraging rate of Apis mellifera during 10th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 9360.4 4680.2 78.6147 0.0000 *** Replications 4 451.73333 112.933 1.89697 0.2045 ns Error 8 476.26666 59.5333 Total 14 10288.4

Appendix 8: Variance analysis of data regarding colony foraging rate of Apis

mellifera during 11th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 980.933 490.466 5.41154 0.0326 *

Replications 4 497.73333 124.4333 1.37293 0.3249 ns Error 8 725.066 90.63333 Total 14 2203.733

Appendix 9: Variance analysis of data regarding colony foraging rate of Apis mellifera during 12th week of study

Source of Variance d.f. SS MS F Value P Calculated Treatments 2 23102.533 11551.26 20.73463 0.0007 *** Replications 4 441.6 110.4 0.19816 0.9324 ns Error 8 4456.8 557.1 Total 14 28000.933

160

Appendix 10:Variance analysis of data regarding visitation frequency of Apis mellifera L. during 1st week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 3.096 1.54 19.29 0.0006*** Error 9 0.722 0.0802 Total 11 3.818

Appendix 11: Variance analysis of data regarding visitation frequency of Apis mellifera L. during 3rd week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 4.6096 2.3048 23.134 0.0003*** Error 9 0.8966 0.0996 Total 11 5.5063

Appendix 12:Variance analysis of data regarding visitation frequency of Apis mellifera L. during 4th week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 11.049 5.525 77.424 0.000*** Error 9 0.642 0.0714 Total 11 11.691

161

Appendix 13:Variance analysis of data regarding visitation frequency of Apis mellifera L. during 5th week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 1.939 0.97 17.006 0.0009*** Error 9 0.513 0.057 Total 11 2.452

Appendix 14:Variance analysis of data regarding visitation frequency of Apis

mellifera L. during 6th week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 11.988 5.993 56.256 0.0000*** Error 9 0.958 0.106 Total 11 12.946

Appendix 15:Variance analysis of data regarding visitation frequency of Apis

mellifera L. during 7th week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 2.456 1.228 8.882 0.0074** Error 9 1.244 0.138 Total 11 3.700

162

Appendix 16:Variance analysis of data regarding visitation frequency of Apis

mellifera L. during 10th week of study

Source of Variation d.f. SS MS F Value P Calculated

Times (Factors) 2 3.042 1.521 15.439 0.0012** Error 9 0.886 0.098 Total 11 3.928

Appendix 17:Variance analysis of data regarding visitation frequency of Apis mellifera L. during 11th week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 342.167 171.08 7.349642 0.0128* Error 9 209.5 23.27 Total 11 551.67

Appendix 18: Variance analysis of data regarding visitation frequency of Apis

mellifera L. during 12th week of study

Source of Variation d.f. SS MS F Value P Calculated Times (Factors) 2 392 196 19.12195 0.0006*** Error 9 92.25 10.25 Total 11 484.25

163

Appendix 19: Variance analysis of data regarding total number of pods plant -1 of various pollination techniques

Source of Variance d.f. SS MS F Value P Calculated Replication 3 10269. 375 3423.125 0.51572 0.6999

Pollinating agent 1 71631.125 71631.125 10.791 0.0462* (factor) Error 3 19912.375 6637. 4583

Total 7 101812.875

Appendix 20: Variance analysis of data regarding total number of seeds plant -1 of various pollination techniques

Source of Variance d.f. SS MS F Value P Calculated Replication 3 485177.375 161725.79 0.7197 0.6033 Pollinating agent 1 6615703.125 6615703.12 29.44 0.0123* (factor) Error 3 674112.375 224704.13

Total 7 7774992.875

Appendix 21: Variance analysis of data regarding seed weight 100 pods-1 of various pollination techniques

Source of Variance d.f. SS MS F Value P Calculated Replication 3 0.0342375 0.0114125 0.5346 0.6900 Pollinating agent 1 5.5278125 5.5278125 258.964 0.0005*** (factor) Error 3 0.0640375 0.0213458 Total 7 5.6260875

164

Appendix 22: Variance analysis of data regarding oil content (%) of two different pollination techniques

Source of Variation d.f. SS MS F Value P Calculated Treatments 1 30.8112 30.8112 25.8674 0.09 Replications 3 39.1537 13.0512 2.48536 .237 Error 3 15.753 5.2512 Total 7 85.7187

Appendix 23: Variance analysis of data regarding protein content (%) of two different pollination techniques

Source of Variation d.f. SS MS F Value P Calculated Treatments 1 20.48 20.48 19.5047 0.0215 Replications 3 3.27 1.09 1.0380 0.488 Error 3 3.15 1.05 Total 7 26.9

Appendix 24: Variance analysis of data regarding Oleic acid (%) of two

different pollination techniques

Source of Variation d.f. SS MS F Value P Calculated Treatments 1 40.0512 40.0512 3.66951 0.151 Replications 3 36.3737 12.1245 1.11086 0.4666 Error 3 32.7437 10.9145 Total 7 109.1687

165

Appendix 25: Variance analysis of data regarding Linoleic acid (%) of various

pollination techniques

Source of Variation d.f. SS MS F Value P Calculated Treatments 1 4.205 4.205 1.1504 0.362 Replications 3 1.53 0.51 0.1395 0.9300 Error 3 10.965 3.655 Total 7 16.7

Appendix 26: Variance analysis of data regarding germination rate (%) of two different pollination techniques Source of Variation d.f. SS MS F Value P Calculated Treatments 1 2211.125 2211.125 1234.116 0.0001 *** Replications 3 20.375 6.79166 3.79069 0.1514 Error 3 5.375 1.79166 Total 7 2236.875

Appendix 27: Variance analysis of data regarding colony foraging rate of A. mellifera at different beehives distance during 1st week of study Source of variation d.f SS MS F Value P Calculated Replications 2 33.85 16.92 0.447 ns Distance (factor) 2 11315.851 5657.925 149.512 0.0000*** Time (factor) 2 21636.740 10818.37 285.878 0.0000*** Interaction 4 1967.7037 491.925 12.9992 0.0001*** Error 16 605.481 37.8425 Total 26 35559.629

166

Appendix 28: Variance analysis of data regarding colony foraging rate of Apis mellifera at different beehives distance during 3rd week of study

Source d.f SS MS F P Value Replications 2 0.66666 0.33333 0.01581 ns Distance (factor) 2 113824.66 56912.33 2699.399 0.0000*** Time (factor) 2 35748.666 17874.33 847.794 0.0000*** Interaction 4 1101.3333 275.3333 13.0592 0.0001*** Error 16 337.33333 21.083 Total 26 151012.66

Appendix 29: Variance analysis of data regarding colony foraging rate of Apis mellifera at different beehives distance during 4th week of study Source of Variation d.f SS MS F Value P Value Replications 2 0.666666 0.33333 0.01793 ns

Distance (factor) 2 103586 51793 2787.067 0.0000*** Time (factor) 2 6849.555 3424.77 184.292 0.0000***

Interaction 4 1078.444 269.611 14.5082 0.0001*** Error 16 297.333 18.5833 Total 26 111812

167

Appendix 30: Variance analysis of data regarding colony foraging rate of Apis mellifera at different beehives distance during 5th week of study Source of Variation d.f SS MS F Value P Value Replications 2 97.5555 48.7777 3.2914 ns Distance (factor) 2 25390.22 12695.11 856.6523 0.0000*** Time 2 40750.22 20375.11 1374.8903 0.0000*** (factor) Interaction 4 3909.555 977.3888 65.9531 0.0000*** Error 16 237.1111 14.8194 Total 26 70384.66

Appendix 31: Variance analysis of data regarding colony foraging rate of Apis mellifera at different beehives distance during 6th week of study Source d.f SS MS F Value P Value Replications 2 54.888 27.444 1.8783 ns Distance (factor) 2 29217.55 14608.77 999.840 0.0000*** Time (factor) 2 77846.88 38923.44 2663.96 0.0000*** Interaction 4 5787.555 1446.888 99.02661 0.0001*** Error 16 233.777 14.6111 Total 26 113140.66

168

Appendix 32: Variance analysis of data regarding colony foraging rate of Apis mellifera at different beehives distance during 8th week of study

Source d.f SS MS F Value P Calculated Replications 2 2.888888 1.4444 0.0725 ns Distance 2 12172.222 6086.11 305.7920 0.0000*** (factor) Time 2 11966.8888 5983.444 300.633 0.0000*** (factor) Interaction 4 2028.2222 507.0555 25.4766 0.0001*** Error 16 318.4444 19.90277 Total 26 26488.666