2 UNIVERSITA' DI PISA
DIPARTIMENTO DI SCIENZE VETERINARIE
Corso di Laurea Magistrale in
SCIENZE E TECNOLOGIE DELLE PRODUZIONI ANIMALI
TESI DI LAUREA
Physiological and behavioural effects in honeybees
and mason bees fed with single protein and non-protein
amino acids enriched diet
Candidato: Relatore:
Elena Tafi Dott. Antonio Felicioli
Correlatore:
Dott.ssa Lucia Casini
3 INDEX SUMMARY ... 6 RIASSUNTO ... 8 1. INTRODUCTION ... 11 1.1 PLANTS REPRODUCTION ... 12 1.1.1 Pollination ... 13 1.1.2 Pollen vectors ... 17 1.2 APOIDEA AS POLLINATORS ... 24
1.2.1 Bees ecological role ... 28
1.2.2 Bees agricultural role ... 30
1.2.3 Main Apoidea species managed as pollinators ... 31
1.2.4 Apis mellifera L. life cycle ... 35
1.2.5 Osmia bees life cycle ... 39
1.3 PLANT-BEES MUTUALISTIC INTERACTIONS ... 42
1.3.1 Plant’s rewards for bees ... 44
1.4 NECTAR ... 49
1.4.1 Nectar production and secretion ... 49
1.4.2 Nectar composition ... 57
1.4.3 Nectar amino acids ... 63
1.4.4 The ecological role of non-protein amino acids ... 66
1.5 BEES HAEMOLYMPH ... 72
2. AIM OF THE WORK ... 73
3. MATERIALS AND METHODS ... 76
3.1 THE EXPERIMENTAL DESIGN ... 77
4
3.2.1 Mason bees collection ... 78
3.2.2 Honeybees collection ... 80 3.3 REARING CONDITIONS ... 81 3.3.1 Feeding conditions ... 81 3.3.2 Rearing in tubes ... 82 3.3.3 Rearing in cages ... 84 3.3.4 Rearing in bowls ... 86
3.4 SURVIVAL RATE AND FOOD CONSUMPTION RECORDING ... 91
3.5 BEHAVIOURAL OBSERVATIONS ... 91
3.6 HAEMOLYMPH SAMPLING AND ANALYSIS ... 92
3.6.1 Haemolymph sampling ... 92
3.6.2 Haemolymph analysis by gradient HPLC ... 95
3.7 STATISTICAL ANALYSIS ... 96
4. RESULTS ... 97
4.1 SYRUP CONSUMPTION ... 98
4.1.1 Syrup consumption by O. bicornis ... 98
4.1.2 Syrup consumption by O. cornuta ... 100
4.1.3 Syrup consumption by newemerged honeybees ... 101
4.1.4 Syrup consumption by foragers honeybees ... 103
4.2 SURVIVAL ... 105
4.2.1 O. bicornis survival ... 105
4.2.2 O. cornuta survival ... 107
4.2.3 Newemerged honeybees survival ... 108
4.2.4 Foragers honeybees survival ... 110
4.3 BEHAVIOURAL OBSERVATIONS ... 113
5
4.3.2 Results of behavioural observations in newemerged honeybees ... 120
4.3.3 Results of behavioural observations in foragers honeybees ... 125
4.4 HAEMOLYMPH AMINO ACIDS ... 130
4.4.1 Amino acids in the O. bicornis haemolymph ... 131
4.4.2 Amino acids in the O. cornuta haemolymph ... 134
4.4.3 Amino acids in the newemerged honeybees haemolymph ... 136
4.4.4 Amino acids in the forager honeybees haemolymph ... 139
5. DISCUSSION ... 143
5.1 DIET AND SYRUP CONSUMPTION ... 144
5.1.1 Diet and syrup consumption by mason bees ... 144
5.1.2 Diet and syrup consumption by honeybees ... 146
5.1.3 Diet and syrup consumption by mason bees and honeybees: an interfamily comparison ... 149
5.2 DIET AND BEES SURVIVAL ... 152
5.2.1 Diet and mason bees survival ... 152
5.2.2 Diet and honeybees survival ... 154
5.2.3 Diet and mason bees and honeybees survival: an interfamily comparison ... 158
5.3 DIET AND BEES BEHAVIOUR ... 163
5.3.1 Diet and mason bees and honeybees behaviour: an interfamily comparison .. 164
5.4 DIET AND HAEMOLYMPH COMPOSITION ... 168
5.4.1 Mason bees haemolymph composition ... 168
5.4.2 Honeybees haemolymph composition ... 175
5.5 HISTORY OF SINGLE AMINO ACIDS ... 179
6. CONCLUSIONS ... 186
6 SUMMARY
Nectar is the main food resource of adult bees and it fuels their flight. Amino acids are the most abundant solutes present in nectar, second only to sugars. All the essential amino-acids have been found almost ubiquitously in nectar. Among them, the protein amino acid proline has received more attention because of its role in the insect flight metabolism and because it is the most abundant amino-acid found in the honeybee haemolymph.
Also several non-protein amino acids have been found in nectar, within the so-called class of “nectar secondary compounds”. Among the non-protein amino acids, γ-amino butyric acid (GABA) and β-alanine are the most common and abundant in floral nectar.
Amino acids affect bees physiology, thereby their pollination activity, but their role is still unclear. Bees amino acids requirements are still to be fully investigated, especially for wild bees. Honeybees are one of the most efficient and wide-spread pollinators on a world scale. Among the solitary bees, mason bees are important pollinators of fruit trees and they are managed in several countries for orchards pollination. Due to the possibility to be easily kept in cages, both honeybees and mason bees are suitable genera for laboratory experiments.
Experiments were performed in order to investigate effects of a single amino acids enriched diet on food consumption, survival, behaviour and haemolymph composition on three bees species. Proline, GABA at natural concentration, GABA at twentyfold increased concentration (GABA 20x) and -alanine were tested separately on Apis mellifera (L.) workers (both newemerged and foragers), just emerged Osmia cornuta (Latreille) females and just emerged
Osmia bicornis (L.) females and males, all reared in laboratory conditions using two types of
7 Syrup consumption and survival rate were recorded daily in all the experiments. Behavioural observations were performed using the scan sampling method for bees reared in bowls. Haemolymph was sampled from bees at different time and analysed by gradient HPLC.
Different responses to a diet enriched with single amino acids were observed among the different bees species and also, within the same species, between different sexes and ages. A “cage-dependent” effect was also hypothesized.
Proline increased food consumption by O. cornuta females and newemerged honeybees, both reared in cages. This amino acid positively affected survival of newemerged honeybees reared in bowls while it decreases the lifespan of newemerged honeybees reared in cages, as well as the one of O. cornuta females reared in tubes.
High concentration of GABA (GABA 20x) increased syrup consumption by foragers honeybees reared in bowls and positively affected survival of O. cornuta females reared in bowls. GABA 20x showed also an interesting affect on forager honeybees behaviour, resulting in an increased motor activity of these bees during the first week of rearing. GABA at natural concentration negatively affected the lifespan of newemerged honeybees reared in cages.
-alanine increased the newemerged honeybees lifespan when they were reared in cages. Haemolymph composition differed among different bees species, and also within the same species, and resulted to be affected by diet. Especially proline and GABA showed curious trends in both mason bees and honeybees haemolymph suggesting the existence of some unexpected metabolic interactions that need to be further investigated.
8 RIASSUNTO
Il nettare è il principale alimento delle api adulte e costituisce il “carburante” per la loro attività di volo. Tra i componenti del nettare, gli amino acidi sono tra i soluti più abbondanti, secondi solo agli zuccheri. Tutti gli amino acidi essenziali sono stati trovati nella quasi totalità dei nettari. Tra questi, l’amino acido proteico prolina ha ricevuto particolare attenzione a causa del suo ruolo nel metabolismo del volo degli insetti e perché risulta essere l’amino acido più abbondante nell’emolinfa delle api da miele.
All’interno della cosiddetta classe dei “composti secondari” del nettare sono stati trovati anche molti amino acidi non proteici. Tra questi, l’acido γ-amminobutirrico (GABA) e la -alanina sono i più comuni e abbondanti.
Gli amino acidi mostrano effetti sulla fisiologia della api e, di conseguenza, sulla loro attività di impollinazione, ma il loro ruolo resta ancora da chiarire. Il fabbisogno aminoacidico di questi insetti non è ancora stato pienamente indagato, specialmente per quanto riguarda le api selvatiche. Le api da miele sono tra gli impollinatori più efficienti e più diffusi su scala mondiale. Tra le api selvatiche, le osmie sono importanti impollinatori degli alberi da frutto e vengono allevate e commerciate in molti paesi per l’impollinazione dei frutteti. Sia le api da miele che le osmie risultano specie idonee all’allevamento in condizioni di laboratorio, data la loro adattabilità alla vita in gabbiette.
Gli esperimenti sono stati eseguiti al fine di investigare gli effetti di una dieta arricchita con singoli amino acidi sul consumo di alimento, sopravvivenza, comportamento e composizione dell’emolinfa in tre specie di api. Prolina, GABA a concentrazione naturale, GABA a concentrazione venti volte superiore (GABA 20x) e -alanina sono stati testati separatamente su operaie di Apis mellifera (L.) (sia neosfarfallate che foraggiatrici), femmine neosfarfallate
9 di Osmia cornuta (Latreille), e maschi e femmine neosfarfallati di Osmia bicornis (L.), tutti allevati in condizioni di laboratorio utilizzando due tipi di gabbie (gabbiette o tubi di piccole dimensioni e palle di grandi dimensioni).
La quantità di sciroppo consumato e la sopravvivenza sono state registrate quotidianamente in tutti gli esperimenti. Le osservazioni del comportamento sono state eseguite con il metodo del campionamento a scansione sulle api allevate nelle palle. L’emolinfa è stata prelevata in tempi diversi e analizzata con l’HPLC.
Sono state osservate risposte diverse a diete arricchite con singoli amino acidi a seconda della specie e, all’interno della stessa specie, a seconda del sesso e dell’età dell’ape. È stato anche ipotizzato un effetto dovuto al tipo di gabbia utilizzato.
La prolina ha indotto un maggior consumo di alimento nelle femmine di O. cornuta e nelle api mellifere neosfarfallate, entrambe allevate nelle gabbiette piccole. Questo amino acido ha influenzato positivamente la sopravvivenza delle api mellifere neosfarfallate allevate nelle palle, mentre ha avuto un effetto negativo sulla sopravvivenza delle stesse api allevate nelle gabbiette così come sulle femmine di O. cornuta allevate nei tubi.
Il GABA in alta concentrazione (GABA 20x) ha indotto un maggior consumo di sciroppo nelle api mellifere foraggiatrici allevate nelle palle e ha avuto un effetto positivo sulle femmine di O. cornuta allevate nelle palle. Il GABA 20x ha inoltre mostrato un effetto interessante sul comportamento delle api foraggiatrici, risultante in un aumento della loro attività motoria durante la prima settimana di allevamento. Il GABA a concentrazione naturale ha influenzato negativamente la sopravvivenza delle api mellifere neosfarfallate allevate nelle gabbiette.
La -alanina ha avuto un effetto positivo sulla sopravvivenza delle api mellifere neosfarfallate allevate nelle gabbiette.
10 La composizione aminoacidica dell’emolinfa varia tra le specie e anche all’interno della stessa specie, e risulta essere influenzata dalla dieta. La prolina e il GABA, in particolare, mostrano un curioso andamento sia nelle api mellifere che nelle osmie, suggerendo l’esistenza di inaspettati processi metabolici che necessitano di ulteriori studi.
11
1. INTRODUCTION
12 1.1 PLANTS REPRODUCTION
Plants can produce offspring by vegetative or sexual reproduction.
Vegetative reproduction occurs when the new individual grows from a fragment of the parent plant by mitotic division. This asexual offspring is a clone of the parent plant (Mc Gregor, 1976; Caporali, 2011). The main vegetative reproduction modes are:
- binary scission (the division of the mother cell into two daughter cells genetically identical to each other by mitosis);
- budding (the new organism develops from an outgrowth or bud due to cell division at one particular site of the mother cell body);
- fragmentation (a fragment splitted from the mother plant develops into a mature, fully grown individual);
- spores production (the plant produces spores by mitosis which are able to originate a new organism) (Caporali, 2011).
Sexual reproduction produces offspring by the fusion of two reproductive cells (gametes), resulting in a new individual genetically different from the parents. Sexual reproduction involves two fundamental processes: meiosis and fertilization. Meiosis consists in the production of haploid gametes. During fertilization, two gametes (a male and a female one) fuse leading to the formation of a zygote, which will develop into a new plant (Caporali, 2011).
Spermatophytes (seeds-bearing plants) are the dominant plant form on land, as the result of two principal adaptations: pollen and seeds. Spermatophytes include Gymnospermae (spermatophytes with naked seeds) and Angiospermae (spermatophytes with flowers). Angiosperms are the most advanced and complex plants, and they are the most widespread all
13 over the world. In angiosperms, sexual reproduction occurs in a dedicated structure: the flower.
Angiosperms have an aplodiplonte life cycle, in which an haploid generation (gametophyte) alternates with a diploid generation (sporophyte). The spermatophytes have the sporophyte as dominant generation. The sporophyte bears the flowers holding the male and female reproductive organs: anthers and ovary, respectively. Anthers and ovary constitute the sporanges. Inside the anthers, microspores are produced which develop into pollen. Inside the ovary, macrospores are produced which develop into ovules. The gametophyte generation is represented by the pollen germination and the embryo sac formation from the ovule, resulting in the gametes production (Caporali, 2011; Contessi, 2016).
1.1.1 Pollination
“Pollen grains are small, so small that they cannot be seen with the naked eye; nevertheless, each is a phase in the life cycle of the flowering plant, the point in the cycle where all the potentialities and characters of the plant, be they the beauty of the rose, the majesty and dignity of the oak or the grace and elegance of the vine, are distilled into a tiny cell and cast with millions of others to the winds” (Wodehouse, 1935).
Pollination is defined as the pollen transfer from the stamens of one flower to the stigma of the same or a different flower (Frediani, 2000; Goulson, 2010; Caporali, 2011).
The stamens constitute the male part of the flower which is called “androecium”. Each stamen is composed of a filament bearing the anther on the extremity. Pollen is the male multinucleate gametophyte, which contains male gametes (sperm cells), and it develops within the anthers, inside the pollen sacs (Taylor and Hepler, 1997; Caporali, 2011). Each anther has usually four pollen sacs, each consisting of a central sporogenous cell and an outer
14 anther wall. Sporogenous cells produce microsporocytes (or “pollen mother cells”). Through two meiotic divisions, each microsporocyte is transformed into a tetrad of haploid microspores encased in a callose wall. In some cases (e.g. the Ericaceae family) tetrads remain and do not proceed with mitotic division. In other families tetrads could turn in a dyad or transform in a polyad (e.g. the Mimosaceae family). Microspores undergo two meiotic divisions.
After that, glucanases secreted by the tapetum (the inner layer of the pollen sac) dissolve the callose wall and microspores are released into the anther locule. Then, pollen wall (exine) deposition starts, utilizing materials secreted from the microspore itself and tapetal cells. After cytoplasmic reorganization resulting in the formation of a nucleus adjacent to the wall, each microspore undergo a first asymmetrical mitotic division, forming a big vegetative cell and a small generative cell. For a majority of plants species, the mature pollen grain (called “bicellular pollen”) originates directly from this bicellular structure, whereas for the rest of the botanical species the microspore has to proceed through a second symmetric mitotic division, forming two sperm cells. This second mitosis results in a “tricellular pollen”. Bicellular pollen undergoes the second mitotic division at a later stage during pollen germination on the stigma of the pollinated flower (Goldberg et al., 1993; Dai et al., 2007). Hence the term “pollen grain” refers to the microspore containing the vegetative nucleus and the sperm cells, as result of the mitotic division (Vasil, 1967).
Anthers opening leads to the release of the mature pollen grains outside. A variety of vectors, including wind, water and animals, transfer the pollen from the anthers to the female reproductive structure of the flower, called “gynoecium”. Gynoecium is constituted by one or multiple carpels, forming the ovary. Each carpel contain an ovule. The set of carpels forms the pistil, consisting of an ovary and, extending from the ovary, one or multiple styles with the pollen receptive portion, the stigma, on the tip. The stigma often has papillae covered by
15 the stigmatic fluid, which facilitate pollen adhesion and germination (Taylor and Hepler, 1997; Caporali, 2011).
Pollen arrives on the receptive stigma, comes in contact with the stigmatic fluid, takes in moisture and germinates (on the pollen grain surface, the outgrowth of a tube occurs) (Taylor and Hepler, 1997; Caporali, 2011). Hundreds of pollen grains are usually deposited on a stigma, and germination is known to be density dependent, a phenomenon called “the mentor effect” or “the pollen population effect” (Chen et al., 2000; Lord and Russel, 2002).
The “pollen tube” penetrates and grows within the style bearing the two sperm nuclei and arrives into the ovule inside the ovary. Fertilization follows this pollination process. In all angiosperms double fertilization occurs: one of the two sperm nuclei fertilizes the female egg cell, which is contained in the embryo sac within the ovule, and the other one fertilizes another cell which will develop into the endosperm (the food reserve tissue). Fertilization of the egg cell leads to the zygote formation. In gymnosperms, only one of the two sperm cells fertilizes the ovule, whereas the other one degenerates (Taylor and Hepler, 1997; Frediani, 2000; Caporali, 2011).
It is important to distinguish fertilization from pollination. The pollen grain is a microspore, not a gamete, and it does not directly contact the ovule. In order to obtain fertilization, pollen must arrive on the stigma and then germinate to produce gametes. Fertilization results in the production of a seed from the ovule and a fruit from the ovary (Caporali, 2011).
Spermatophytes usually have hermaphroditic flowers (or “perfect flowers”), containing both male and female reproductive organs. Only few species have unisexual flowers (or “imperfect flowers”). These latter plants are called “monoicus”, when they bear both male and female flowers on the same plant (e.g. corn, hawthorn, strawberry tree), or “dioecious”, when they bear male and female flowers on separate individuals (e.g. willow, nettle, kiwi, common
16 yew). Polygamous plants also exist, bearing both unisexual and bisexual flowers on the same individual (e.g. sycamore) (Caporali, 2011).
The pollen transfer from the stamens of one flower to the stigma of the same flower or a different flower belonging to the same plant, and the following ovule fertilization, is called “self-pollination” or “autogamy”. On the contrary, the pollen transfer from the stamens of one flower to the stigma of a flower belonging to a different individual, and the following ovule fertilization, is called “cross-pollination” or “heterogamy” (Frediani, 2000; Caporali, 2011). Compared to autogamy, heterogamy generally leads to the production of a better progeny. Cross-pollination enhance genetic variability in populations, resulting in the production of a more vigorous offspring, more capable to adapt to different environmental conditions and more resistant to diseases. Furthermore, plants generated from cross-pollinated parents produce fruits which are more uniform in size and shape, larger and earlier-maturing than the parents one. Hence, heterogamy is more advantageous for plants than autogamy, and it is the reproduction mode favoured by nature (Solbrig, 1976; Frediani, 2000; Holsinger, 2000). In numerous plants, self-pollination is permitted only after all efforts at cross-pollination have failed. Mc Gregor (1976) wrote that it would seem as if Nature orders the plant: “Become
fertilized, cross-fertilized if you can, self-fertilize if you must” (Mc Gregor, 1976).
The appearance of plants bearing unisexual flowers could be considered an attempt to avoid self-pollination. Nevertheless, if in dioecious plants self-pollination is not allowed, in monoicus species it is possible. It is likely the reproduction costs in dioecious plants are too high in relation to the amount of seeds which are produced only by female individuals. Therefore, both in monoicus and dioecious plants, pollination relies on any vector which is able to bring the pollen in contact with the ovule (Caporali, 2011). In hermaphroditic plants, fertilization can occur without the participation of external agents, but it is rare. In order to hinder this self-fertilization, hermaphroditic flowers have stamens and pistils with different length and direction, anthers do not mature simultaneously, stigmas could be not receptive to
17 the pollen and pollen grains could be not able to fertilize ovules produced by the same flower (Bateman, 1952).
1.1.2 Pollen vectors
Pollen transfer can be performed by abiotic or biotic pollinating agents.
Abiotic pollination
In abiotic pollination pollen transfer occurs via physical agents, mainly wind (anemophily) and water (hydrophily). About 20% of all Angiospermae rely on anemophilic or hydrophilic pollination (Ackerman, 2000). Anemophily is much more common than hydrophily, given that about 98% of the plants that pollinate abiotically are pollinated by wind (Faegri and van der Pijl, 1979; Ackerman, 2000).
Wind
Anemophilic pollination is predominant in gymnosperms (e.g. conifers), catkin-bearing trees (e.g. Betulaceae and Fagaceae), and herbaceous plants including important cereal crops used in human and animal feeding (e.g. Poaceae and Cyperaceae) (Faegri and van der Pijl, 1979; Regal, 1982). Anemophilic pollination is less precise and more wasteful in comparison with biotic pollination and traditionally it has been viewed as an inefficient process because of the high pollen to ovule ratios in anemophilous plants (more than 106:1) (Faegri and van der Pijl, 1979; Ackerman, 2000). In spite of this, in many entomophilous plant families there are some species that have become anemophilous. Within angiosperms, entomophily seems to be the primitive condition and anemophily is derived from it, probably to cope with environmental changes which render biotic pollination less advantageous than anemophily. By contrast,
18 wind pollination is the ancestral state in gymnosperms (Faegri and van der Pijl, 1979; Culley
et al., 2002).
Wind-pollinated plants have particular reproductive structures, including strobuli (cones), catkins and spikelets, which have brush or feather-like stigmas to facilitate pollen capture from the air (Caporali, 2011). These reproductive structures are not showy, nor do they offer the typical rewards (e.g. nectar, oil, odour, temperature and sexual attraction etc.) found in biotically-pollinated plants (Ackerman, 2000). In anemophilic plants anthesis does not occur unless the weather is favourable (i.e. warm and dry). Pollen release, transport and deposition depend mainly on wind, humidity, rainfall and temperature (Culley et al., 2002).
Anemophilous pollen grains are very light and have an optimal weight/volume ratio to guarantee the effectiveness of transport by wind. Furthermore, anemophilous pollen grains are smooth and present a little quantity of “pollenkitt” (adhesive material present around each pollen grain, mainly composed by lipids), hence they do not adhere to each other (Faegri and van der Pijl, 1979; Pacini and Hesse, 2005).
Water
Hydrophily is found primarily in monocotyledons (e.g. Najadales and Alismatales orders), which contain 80% of the families in which hydrophilous species have been reported. Only few dicotyledons families are water pollinated (e.g. Ceratophyllaceae and Plantaginaceae) (Ackerman, 2000).
There are two different hydrophilic pollination modes: ephydrophily and hyphydrophily. Ephydrophilic pollination occurs on the water surface. Pollen is transported directly on the water surface or by floating male flowers which are released by plants underwater. Then, floating female flowers contact pollen grains released on the water surface.
Hyphydrophilic pollination occurs underwater. In some species, filamentous pollen or pollen grains in mucilaginous strands are carried underwater by currents. In others, pollen denser
19 than water is released. Hyphydrophily is relatively uncommon in angiosperms and largely restricted to the monocotyledons, mainly seagrasses (Cox, 1993; Ackerman, 2000).
The pollen-ovule ratios of hydrophilous plants are not well characterized, but results indicate that the ratios are orders of magnitude less than anemophilous plants (103:1) (Philbrick and Anderson, 1987). Hydrophilous pollen is often filiform and shows a slimy or gelatinous coat consisting mostly of polysaccharides plus some proteins and lipids (Ackerman, 2000).
Biotic pollination
Biotic pollination involves animals as pollen vectors. It is the most widespread pollination mode among angiosperms and it is considered more efficient than abiotic pollination (Faegri and van der Pijl, 1979; Ackerman, 2000). The proportion of animal-pollinated plants range from about 78% in temperate zones to 94% in the tropical area (Ollerton et al., 2011).
Insects, mainly coleoptera, diptera, hymenoptera, and lepidoptera, constitute the great majority of known pollinators, but also some vertebrates (e.g. birds and bats) pollinate several plants.
Invertebrates
Not only bees, but also beetles, flies, wasps, ants and butterflies take part in the pollination process of many plants (the term “bee” refers to any member of the Apoidea superfamily). Beetles are thought to be the earliest animal pollinators of angiosperms (Faegri and van der Pijl, 1979; Young, 1986). Beetle pollination (cantharophily) is typical of the tropical zone, whereas it is rare in the European flora. Visits of beetles in blossoms are often considered accidental, but some species are habitual visitors and have acquired adaptations for flower visits (e.g. modification of mouth-parts for pollen chewing or changes in their position to better reach the nectar). However, beetles are primitive pollinators: they are not able to land
20 precisely on a flower, hence they can only pollinate the simplest flowers with very accessible nectar and pollen (Faegri and van der Pijl, 1979; Young, 1986).
Flies are considered regular visitors or pollinators of at list five-hundred species of flowers (Kearns and Inouye, 1994). Fly pollination (myophily) is performed by a great variety of species with different specialization degrees. Many flower-visiting flies have short mouthparts and are not able to extract nectar and pollen from flowers with a long and deep corolla tube. These unspecialized flies are restricted to flowers with very short tubes or a bowl shape (Faegri and van der Pijl, 1979; Kearns and Inouye, 1994). Other flies are highly evolved and have a long proboscis which enabling them to reach also deeply hidden nectar in flowers with long corolla tube. However, since flies do not nurse their brood, they have not as high foraging activity as those insects that collect food for their brood. As flies generally utilize many different food sources, their pollinating activity is considered irregular and unreliable (Faegri and van der Pijl, 1979).
Wasps belong to the Hymenoptera order, which comprises some of the most specialized and economically most important pollinating insects. Wasps are social insects and nurse their brood, but the diet of their larvae mainly consists of animal protein. Adult wasps need carbohydrates for maintaining their energy metabolism. Adults highest energy requirements occur when the brood-rearing season is over and the colonies have reached full strength. This means that wasps are especially active in pollination towards the end of the flowering season, when they have no more brood to feed. Some wasps species have mouth-parts modified into tube-like structures, through which nectar can be sucked up, and are regular pollinators (Faegri and van der Pijl, 1979).
Ants are closely related to wasps and bees, but their role as pollinators is much less important. There are only about a dozen examples of ant pollination throughout the world (Faegri and van der Pijl, 1971; Beattie et al., 1984). Ants are smooth, small and flightless, and little pollen can adhere to them. Furthermore, secretions from the ants metapleural glands causes a
21 reduction of pollen germination and viability (Beattie et al., 1984). These could be some reasons for the paucity of ant pollination systems.
Bees are the best adapted insects for flowers visits and are considered one of the most important pollinators of cultivated and wild plants (Faegri and van der Pijl, 1979; Rondinini and Pinzauti, 1994; Frediani, 2000; Goulson, 2010). The characteristic making Apoidea pre-eminently pollinators is the special diet of their larvae, fed with pollen or mixture of pollen and nectar. Adult bees, regularly looking for such food sources, visit a lot of flowerings which taking advantage of these specialized visits to reproduction aims (Ricciardelli d’Albore and Intoppa, 2000; Hatfield et al., 2012). The adaptations of bees to flower visits have their counterparts in the adaptations of flowers to visits by these insects: zygomorphic with great depth effect, mechanically strong with surfaces for landing, colour lively, nectar hidden but not very deeply, odours fresh (Faegri and van der Pijl, 1979).
Butterflies and moths do not feed their brood; all food collecting is for their own consumption. They have low foraging costs and can utilize flowers with small nectar rewards (Watt et al., 1974). Larvae of most lepidopters are phytophagous and feed on the plants on which eggs were laid. The presence of butterflies and moths is often dependent on the plants on which their larvae feed, but these plants are rarely those furnishing nectar to the adults. Primitive lepidopters have chewing mouth-parts and a varied diet, while in the more evolved species the mouth-parts are represented by a long, thin proboscis and the food is exclusively liquid, mainly nectar (Faegri and van der Pijl, 1979).
Many other invertebrates visit flowers, including snails and slugs, heteroptera, thysanoptera, grasshoppers and earwings. Flowers provide them pollen, nectar and vegetal tissues as excellent food sources. These unspecialized invertebrates are not “proper pollinators”, but they may cause pollination moving from one flower to another during feeding.
Entomophilous pollen is not smooth and dry as the anemophilous one. Entomophilous pollen grains are sticky and tend to adhere to each other. They are provided with pollenkitt as pollen
22 binding agent and often have a sculptured exine (e.g. the Asteraceae echinate exine) to facilitate pollen adhesion to insects body (Pacini and Hesse, 2005)
Vertebrates
Vertebrates (mainly birds and bats) rarely pollinate flowers in Europe, but their pollination activity is important in other continents. They have much higher food requirements than invertebrates and demand food all the year round. Vertebrate pollination is mainly a phenomenon of the tropical climates, where flowerings are always available (Faegri and van der Pijl, 1979).
About 900 are the birds species which regularly visit flowers. Some of the most important families of birds pollinators are Trochilidae (hummingbirds), Nectariniidae (sunbirds), Meliphagidae (honeyeaters) and Zosteropidae (white-eyes). Bird pollination takes place chiefly in America, Africa, Southeast Asia and Australasia. The number of plants pollinated by birds is estimated around 500, but information is incomplete (Anderson et al., 2016). Bird pollination (ornithophily) appears to have been evolved after entomophily. A variety of reasons has been proposed to explain this phenomenon. Birds are homeothermic and are active at a wider range of temperatures than insects, hence they may be more reliable pollinators in unfavourable climatic conditions. During a foraging flight, birds may travel further than insects, increasing the pollen flow between plants. Furthermore, birds feathers are excellent pollen carrying structures (Fleming et al., 2009). Flowers pollinated by birds usually have vivid coloured, often red, corolla tubes, abundant nectar, no odours and diurnal anthesis (Faegri and van der Pijl, 1979).
Bat pollination (chirophterophily) occurs in about 250 genera of plants. Among the eighteen known bat families, only two of them include species which are morphologically specialized for nectar gathering (i.e. Pteropodidae and Phyllostomidae) (Fleming et al., 2009). Mechanism of pollen transfer in bats is similar to that seen in birds: pollen grains adhere to
23 the body of the bats while they are lapping nectar and reach other flowers as bats move to a new plant. Bats specialized in visiting flowers to collect pollen and nectar have long pointed heads, a reduced number and size of teeth, and long tongues with soft papillae or brush-like tips to facilitate food gathering (Subramanya and Radhamani, 1993; Fleming et al., 2009). Flowers pollinated by bats usually have nocturnal anthesis, soft colours (often grey, greenish, purplish or creamy), strong odours at night (often musty or reminiscent of cabbage), and position outside the foliage (Faegri and van der Pijl, 1979).
Among vertebrates pollinators we could mention some small non-flying mammals (e.g. squirrels, lower primates and tree-shrews) which live on trees, eat flowers or suck nectar, and may accidentally transfer pollen from one flower to another. There are also some cases of rodents, small marsupials and small primates which regularly visit flowers, feed with nectar and, consequently, pollinate. This is the case of the Tarsipes spencerae (the “honey mouse”) and the Rattus fuscipes, a little Australian marsupial and an Australian rat respectively, which are specialized to visit flowers of different Proteaceae (Faegri and van der Pijl, 1979). Other non-flying mammals, including the sugar glider (Petaurus breviceps), the eastern pygmy possum (Cercartetus nanus) and the brown antechinus (Antechinus stuarthii), have been recorded regularly visiting flowers, chiefly in Australia and tropical regions (Goldingay et al., 1991).
24 1.2 APOIDEA AS POLLINATORS
Apoidea is a superfamily, within the Hymenoptera order and Apocrita suborder, which numbers more than 20000 species divided in 9 families worldwide. In Europe 6 Apoidea families are present: Apidae, Megachilidae, Colletidae, Andrenidae, Halictidae and Melittidae. In recent years, Antophoridae, which constituted the seventh family, was included in the Apidae family (Michener, 2007; Comba, 2015). According to the most recent checklist of Italian wild bees kept up to date by Comba (2015), 1119 species belonging to the Apoidea superfamily are present in Italy. Onwards, we refer to any species belonging to Apoidea with the general common term “bee”.
Bees depend on pollen collected from flowers as a protein source to feed their larvae and probably also for ovarian development by egg-laying females. Whereas, nectar is eaten by adult bees as an energy source and also mixed with pollen to make larval food. Bees morphology is adapted to this diet specialization: the body is covered with feathery hairs, some parts of the body are modified in order to allow pollen gathering and carrying, mouthparts are stretched out for nectar collection, wings are full grown and suitable for an efficient flight (Ricciardelli d’Albore and Intoppa, 2000; Michener, 2007).
Bees sociality
During their evolution bees have developed different social behaviours, so that now bee species exist showing almost all sociality levels known for insects, from solitary to eusocial species. In solitary bees (e.g. Colletes spp., Anthophora spp. etc.) each female builds her own nest and provides food for her offspring with no help from other bees, and it usually dies before the maturation of her brood. Aggregation of solitary bees (e.g. Halictus spp.) can occur when numerous individual nests are built in limited areas because of the inclination of
25 females to return to the birthplace or a mutual attraction among individuals of the same species (Michener, 2007).
Social bees live in colonies, consisting of two or more adult females living in a single nest. The females forming a colony can be divided into one to many workers, which are responsible for foraging, brood care, guarding, and are often unmated, and one queen, which is usually mated and is responsible for most or all of the egg laying. Among social bees we can distinguish highly eusocial species and primitively eusocial ones. Honey bees (Apis) and the stingless tropical honey bees (Melipona and Trigona genus) are highly eusocial bees and always live in colonies in which the queen is unable to live alone (she never forages), nor do workers alone form viable colonies (they cannot mate and produce female offspring). In these bees, new colonies are established by groups or swarms. Most bumblebees (Bombini), sweat bees (Halictinae) and carpenter bees (Xylocopinae) live in small colonies started by single females working as solitary individuals, which perform nest building, foraging, cells provisioning or larvae feeding and egg laying. Later, with the emergence of daughters, colonial life may arise, including division of labour between queen and workers. These are primitively eusocial colonies that usually break down with the production of reproductive individuals (Michener, 2007).
Both permanent honey bee colonies and temporary bumblebee colonies are called eusocial, meaning that they have division of labour (egg-layer and foragers) among cooperating adult females of two generations. Some bee colonies have no division of labour or castes and all colony members show a similar behaviour. Such colonies are “communal”, in which more females use the same nest but each of them makes and provisions her own cells (Michener, 2007).
According to the number of life cycles performed in a year, species can be divided in monovoltine (Osmia spp.), bivoltine and polivoltine (some Andrenidae). Some monovoltine
26 bees such as Bombus terrestris L. can become bivoltine in particular areas and climate conditions (Mediterranean scrub) (Gurel et al., 2008).
Bee nests
The nest is the place where bees rear their broods. Nests are always made by the mother or, in social species, by the workers and consist of brood cells. A cell protects the immature stages, and in most cases the food, of the growing larva. Nests can be set up in the most various places: rotten woods, hollow branches, reeds, cracks in walls, empty shells, cavities in trees etc. There are also digger bees (e.g. Andrena, Anthophora and Halictus genus) which nest in the ground, digging tunnels connected with ramifications where progeny is placed. The most developed bees (i.e. the corbiculate tribes) built cells with wax secreted by their wax glands mixed with other materials such as resin or pollen. These cells are in clusters or in “combs”, usually in a cavity in a tree or in the ground, or in a cavity in a larger nest (Ricciardelli d’Albore and Intoppa, 2000; Michener, 2007).
Foraging activity and pollination
The pollinators are primarily female bees, which collect pollen as the principal protein source in their own diet and to feed their larvae. The pollen that is suitable for ovules fertilization is that which bees lose on floral stigmata during their foraging activity on flowers. Pollen moistened by bees with nectar or oil for transport is not more suitable for fertilization. Male bees and the females of parasitic species gather nectar from flowers but carry only the pollen that stick to their body, thus they play a less important role in pollination (Michener, 2007). As for their floral preferences, bees can be defined “strictly oligoleptic” (when they visit only few species of one botanical genus), “widely oligoleptic” (when they visit more genus of only one botanical family) or “polyleptic” (when they visit several species of different family). However this division is not so categorical, since some species considered strictly oligoleptic
27 have been often found on an unusual botanical species. Moreover oligoleptic bees can develop a temporary interest towards strange food sources when the usual one is not available. In Europe the greater part of pollinator’s species are polyleptic. Oligoleptic ones are only 10% of all species (Ricciardelli d’Albore and Intoppa, 2000).
A characteristic bee behaviour, related to pollination activity, is the “floral constancy”: on any one trip, or during a longer period of time, individual bees tend to visit flowers belonging to the same species. Constancy is learned by each bee and may change as function of flowerings availability, or may differ among individuals of the same species at the same time and place (Michener, 2007).
In order to collect pollen, bees are provided with dedicated hairy structures on different parts of their body according to the mode of pollen transport. “Podilegid” bees have a scopa in the hind legs, whereas “gastrilegid” species have a ventral scopa. The so-called “corbiculate” bees (i.e. Apini, Bombini, Euglossini and Meliponini tribes) have a distinctive structure for pollen collection and transport on the tibia of hind legs called “corbicula” or “pollen basket”. These bees gather pollen from a flower, then leave and, keeping in flight above it, they begin to perform a series of motions in quick sequence, involving legs provided with bristles and hairs, to prepare pollen loads which are transported on pollen baskets. Some more primitive bee species (e.g. those belonging to Hylaeinae and Euryglossinae subfamilies) have no scopae for carrying pollen externally and thus carry pollen in the crop (a specialized part of the gut in which food, mainly nectar, honeydew and water, is transported and stored) (Michener, 2007; Contessi, 2016).
As for the morphology of their mouthparts, modified to facilitate nectar sucking, bees can be divided in “short-tongued” (i.e. Colletidae, Andrenidae, Melittidae and Halictidae) and “long-tongued” (i.e. Apidae and Megachilidae). The different length of the proboscis allows bees to collect nectar from flowers with different corolla shapes (e.g. only the long-tongued bees are able to visit flowers with a long corolla tube and deep nectar) (Faegri and van der Pijl, 1979).
28 Nectar thieves and nectar robbing
A great number of bee visits to flowers do not result in pollination. This commonly happens if there is a mismatch between the morphology of the insect and that of the flower. For example if the insect is small, it may be able to enter a flower and collect nectar without contacting either the stamens or the stigma. These bees which are able to gather nectar from flowers without perform any pollen transfer are called “nectar thieves”. Both bumblebees and honey bees sometimes extract nectar by pushing in between the petals at the base of the flower corolla and by-passing the reproductive structures of the flower (Goulson, 2010).
Other bees make holes in sympetalous flower corollas (where the petals are fused into a tube) to obtain a direct access to the nectaries without contact the pollen. This behaviour is called “nectar robbing”. Nectar robbers are either primary robbers (bees which make holes in the flower corolla by piercing or biting it) or secondary robbers (individuals which use the holes made by primary nectar robbers). If flowers have previously been robbed, primary nectar robbers may re-use holes and act as secondary robbers. Bumblebees (Bombus spp.), carpenter bees (Xylocopa spp.), stingless bees (Trigona spp.) and some Megachilidae are common nectar robbers of many flower species. Honey bees often act as secondary nectar robbers (Irwin et al., 2010).
1.2.1 Bees ecological role
Pollination is considered a key “ecosystem service” (Ricciardelli d’Albore and Intoppa, 2000; Klein et al., 2007; Potts et al., 2010). Ecosystem services are defined as the benefits to human welfare provided by organisms interacting in ecosystems (Daily, 1997).
Pollination is essential to preserve biodiversity, ensuring reproduction and propagation of wild plants (Kearns et al., 1998; Ricciardelli d’Albore and Intoppa, 2000; Potts et al., 2010). Spontaneous vegetation characterize the landscape, prevent soil erosion and provide food,
29 refuge and nesting sites to several species of wild animals. Hence, preservation of spontaneous botanical species is also important to guarantee wild fauna survival (Mc Gregor, 1976).
Around 80% of all flowering plant species rely on animal pollinators, mainly insects (FAO, 2004; Hatfield et al., 2012). Insects provide pollination service delivering pollen in sufficient quantity and quality at the appropriate time and place for ovule fertilisation (Klein et al., 2007).
The most important group of insects pollinators is formed by bees: honeybees (Apis mellifera L.) and several species of wild bees, including bumblebees (Bombus spp. Latreille), mason bees (Osmia spp. Panzer), leafcutter bees (Megachile spp. L.) and the alkali bee (Nomia
melanderi Cockerell). 25.000 is the estimated number of bees species involved in pollination.
This great number of different bee species shows significantly differences in size and habit requirements, and diverge accordingly in the plants they visit and pollinate. The variability of wild plant depends on this diversity (FAO, 2004).
In recent decades insects pollinators populations have declined, threatening ecosystem diversity. Many pollinator population densities are being reduced below levels at which they can sustain pollination services in natural ecosystems and for the maintenance of wild plant reproductive capacity (FAO, 2004; Gallai et al., 2008; Potts et al., 2010; Hatfield et al., 2012; Garibaldi et al., 2013; Bartomeus et al., 2014). Where populations of pollinators have declined, there is also a decline in insect-pollinated plants (Biesmeijer et al., 2006). The loss of wild plants leads to the loss of others animal species, mainly birds, small mammals and herbivores, that directly or indirectly rely upon them (Biesmeijer et al., 2006; Goulson, 2010). There are several causes of this decline, among which the main ones are: loss or fragmentation of habitat, pesticide and herbicides use, climate change, intensive farming
30 practices, parasites and diseases, and introduction of alien species (FAO, 2004; Williams and Osborne, 2009; Goulson, 2010; Hatfield et al., 2012).
In order to contrast pollinators decline, the conservation strategies should enhance the spontaneous vegetation diversity and abundance to guarantee food and nesting sites to insects. These strategies should include the restoring of uncropped margins among cultivated fields sown with wild flowers seed mixes, land set-aside, the use of pesticides and herbicides in lesser extent, and the develop of a more extensive and sustainable agriculture overall (Felicioli, 2000a; Williams and Osborne, 2009; Goulson, 2010; Hatfield et al., 2012).
1.2.2 Bees agricultural role
“The grower may fertilize, and cultivate the soil, prune, thin and spray the trees, in a word, he
may do all of those things which modern practice advocates, yet without his pollinating agents, chief among which are the honey bees, to transfer the pollen from the stamens to the pistil of the blooms, his crop may fail” (Gates, 1917).
About 75% of the crop species used for food depends on insect pollinators, especially bees (Klein et al., 2007; Bartomeus et al., 2014). Of the hundred or so animal-pollinated crops which provide most of the world’s food supply, 15% are pollinated by honeybees, while at least 80% are pollinated by wild bee species and other wildlife (FAO, 2004). Gallai and colleagues (2008) calculated an annual economic value for the contribution of insect pollinators to the production of crops used directly for human food of €153 billion, which is about 9,5% of the total value of the production of human food worldwide. Only in Europe, this economic value amount about to €22 billion (Gallai et al., 2008).
Although a great part of the worldwide crop production originate from wind-pollinated species (e.g. wheat, rice and maize), many vegetables, fruit trees and fodder plants depend on
31 entomophilous pollination. Hence, insect pollinators play, both directly and indirectly, a very important role in human nutrition, since all the animal products consumed by humans (eggs, meat, milk and their derived products) derive, in some way or other, from insect-pollinated crops used in animal feeding (Mc Gregor, 1976).
While some crops depend entirely on insect pollinator visits to set fruit, many others are only partly dependent on animal pollination, but entomophilous pollination is generally advantageous (Klein et al., 2007). There are many works asserting insects visits enhance fruit set and increase fruits size and quality. Bartomeus and colleagues (2014) found that insect pollination increased quantity and quality yield of buckwheat (Fagopyrum esculentum Moench.) and field bean (Vicia faba L.) compared to self- and anemophilous pollination (Bartomeus et al., 2014). Similar results were found in sunflower (Helianthus annuus L.) (Greenleaf and Kremen, 2006), strawberry (Fragaria x ananassa) (Andersson et al., 2012; Bartomeus et al., 2014), oilseed rape (Brassica napus L.) (Stanley et al., 2013; Bartomeus et
al., 2014), sweet pepper (Capsicum annuum L.) (Roldàn Serrano and Guerra-Sanz, 2006),
apple (Malus domestica Borkh) (Garrat et al., 2013) and tomato (Lycopersicon esculentum Mill) (Palma et al., 2008; Ahmad et al., 2015; Sowmya et al., 2015).
In Italy, in some regions characterized by specialized agriculture such as Emilia Romagna and Trentino Alto Adige, the “pollination service” is performed, putting beehives among crops during the flowering period.
1.2.3 Main Apoidea species managed as pollinators
Insect pollination is become a production practice used all over the world for crop production. Bees are purchased or rented by farmers in many countries to ensure and increase quantity and quality production of cultivated crops both in open field and under greenhouse (Mc
32 Gregor, 1976; Gallai et al., 2008). Among the thousands of bee species described worldwide, only few are commercially reared and managed as crop pollinators. These include both social and solitary species, mostly nesting in pre-existing cavities (Cane, 1997; Bosch and Kemp, 2002). Among social bees, Apis mellifera and some bumblebee species are used throughout the world on a vast array of cultivated plants (McGregor, 1976; van den Eijnde et al., 1991; Rondinini et al., 2000; Velthuis and van Doorn, 2006). Solitary species managed for pollination aim include the cavity-nesting leafcutter bee (Megachile rotundata Fabricius) and several species of mason bees, and the ground-nesting alkali bee (Torchio, 1966; Loi and Pinzauti, 2000; Sedivy and Dorn, 2014; Giejdasz and Fliszkiewicz, 2016).
Apis mellifera
Among Apoidea, Apis mellifera (Hymenoptera: Apidae) has been the first species managed for pollination service. It is used to enhance productivity of a large variety of crops, including fruit trees (e.g. apricot, apple, almond, cherry etc.), vegetables (e.g. cucumber, carrot, melon etc.), industrial crops (e.g. sunflower and oilseed rape) and forage crops (e.g. alfalfa, clovers, sulla, field bean etc.) (Mc Gregor, 1976; Morse and Calderone, 2000; Rondinini et al, 2000). Beekeepers traditionally move their hives to these crops at their flowering time, renting colonies to the grower. Sometimes the bees are supplied almost as a favour in exchange for apiary locations throughout the year. In the U.S.A. millions of colonies are rented each year for crops pollination, of which 900000 only for almonds in California (Morse and Calderone, 2000).
Nevertheless, the honeybee is not always the best pollinator of cultivated plants. It is not able to efficiently pollinate certain flowers such as the alfalfa, potatoes and tomatoes ones, which have a particular morphology and need to be “tripped” or vibrated to release pollen. Furthermore, honeybees show a low foraging activity with suboptimal climatic conditions (i.e. low temperatures and bad weather) (Goulson, 2010; Sedivy and Dorn, 2014). These
33 evidences and the increasing knowledge about the pollinator potential of some wild bees, lead to start commercial rearing of other species to complement Apis mellifera.
Bombus spp.
The potential value of bumblebees (Hymenoptera: Apidae) as pollinating insects in agriculture has been recognized for a long time and now they are extensively managed for pollination all over the world. Few multinational companies rear and sell bumblebee colonies to growers in all continents for crops pollination. Five species are commercially reared, among which Bombus terrestris L. is the most used one in several countries. Bombus
impatiens Cresson and Bombus occidentalis Greene are used in northern America, whereas Bombus ignitus Smith and Bombus lucorum L. are utilized in Asia (Velthuis e van Doorn,
2006; Winter et al., 2006; Ahmad et al., 2015).
The main crop that bumblebees pollinate is the greenhouse tomato. Worldwide, this involves about 95% of all bumblebees sales (around one million of colonies each year) (Velthuis e van Doorn, 2006; Yoon et al., 2013). The value of the bumblebee pollinated tomato crops is estimated to be about 12˙000˙000 per year, which guarantee to the bumblebees industry a yearly turnover of approximately 55˙000˙000 (Velthuis and van Doorn, 2006).
Other crops pollinated by bumblebees are chiefly berries, pepper, eggplant, melon, cucumber, courgette and some fruit trees (Velthuis and van Doorn, 2006; Sowmya et al., 2015). Bumblebee efficiency as pollinator of these crops, and of tomato above all, is due to its distinctive behaviour of sonication (“buzz pollination”) that vibrates pollen from the poricidal anthers of these plants (Winter et al., 2006; Sowmya et al., 2015).
Osmia spp.
Mason bees (Hymenoptera: Megachilidae) are reared on a commercial scale mainly as pollinators of rosaceous fruit trees, including almond, peach, apricot, plum, cherry, apple and
34 pear (Bosch et al., 2008; Bosch and Kemp, 2001; Sedivy and Dorn, 2014). Osmia ribifloris Cockerell is also used as an efficient pollinator of blueberries in the U.S.A. (Sampson et al., 2009).
The four most successfully managed mason bees species are Osmia cornifrons Radoszkowski,
Osmia lignaria Say, Osmia cornuta Latreille and Osmia bicornis L. which have been
developed as orchard pollinators in Japan, the U.S.A. and Europe respectively (Torchio and Asensio, 1985; Bosch and Kemp, 2001; Bosch and Kemp, 2002; Sedivy and Doorn, 2014). Commercial rearing is performed from mason bees populations obtained by trap-nesting (placement of artificial nesting materials in areas where wild mason bees populations are present) (Bosch and Kemp, 2002). For optimal pollination efficiency, mason bees nesting should be synchronised with the beginning of the orchard blooming period. Hence, few days before the first flower, mason bees are released in the orchards as already emerged adults or placed while still inside the cocoons (Bosch and Kemp, 2001; Bosch and Kemp, 2002).
Megachile rotundata
The leafcutter bee (Hymenoptera: Megachilidae) has been developed as the major alfalfa pollinator by the American growers since the 1950s (Pinzauti, 2000; Pitts-Singer and Cane, 2011). Alfalfa flowers have a peculiar “tripping” mechanism where the corolla needs to be forced open to allow the releasing of the stamens and, consequently, the pollen. When a pollinator “trips” the flower, stamens shoot forward and the anthers containing the pollen strike the head of the insect. Hence, pollinators must have the strength and the ability to force their way between the petals to reach nectar and pollen (Larkin and Graumann, 1954; Torchio, 1966). Differently from honeybees, the leafcutter bee is able to trip alfalfa flowers and this makes it the most efficient alfalfa pollinator (Torchio, 1966; Kemp and Bosch, 2000). Whereas in the U.S.A. the leafcutter bee revolutionized the alfalfa seed industry and began to
35 be managed intensively, this insect remains uncommon for alfalfa pollination in its native European area (Pitts-Singer and Cane, 2011).
Nesting shelters with artificial nesting materials and emerging bees are placed in alfalfa fields shortly after bloom initiation in June and July and removed after about six weeks. Bees emergence time must be synchronized with time of peak alfalfa bloom (Kemp and Bosch, 2000).
Nomia melanderi
The alkali bee (Hymenoptera: Halictidae) native to the western U.S.A. is the other most efficient alfalfa pollinator together with the leafcutter bee (Mc Gregor, 1976; Cane, 2008). Ground-nesting bees are not usually commercially reared as crop pollinators, for reasons of crops rotation and because nests sites cannot be readily prepared and colonized. The alkali bee is an exception (Cane, 2008). However, compared to the leafcutter bee, the alkali bee is restricted in its use as a managed alfalfa pollinator because it requires specific soil moisture, temperature and alkalinity (Pitts-Singer and Cane, 2011).
Small soil cubes cut from dense nesting aggregations are used to populate newly prepared nesting sites among alfalfa fields. Nesting sites which have been heavily populated need a periodically renovation. In order to produce good numbers of alkali bees, growers must maintain soil moisture throughout the nesting area, compact soil without a superficial crusty or fluffy layer, bare soil surface with sparse vegetation and a fine, sandy loam soil (Johansen, 1971; Cane, 2008).
1.2.4 Apis mellifera life cycle
Honeybees are eusocial bees belonging to the Apoidea superfamily and Apidae family. Apis genus includes four species: Apis florea Fabricius, Apis dorsata Fabricius, Apis cerana
36 Fabricius and Apis mellifera L. (Contessi, 2016). In the present thesis we deal with the latter species.
Honeybees live in colony composed of tens of thousands of individuals (from 10˙000 to 100˙000). The colony live on waxy honeycombs built inside natural pre-existing cavities or in hives provided by humans. The honeybees society is one of the four “superorganisms” recognized among the Animalia kingdom, together with termites, ants and the naked mole rat (Heterocephalus glaber Ruppel) (Moritz and Southwick, 1992).
Wilson and Sober (1989) define a superorganism as “a collection of single creatures that
together possess the functional organization implicit in the formal definition of organism”
(Wilson and Sober, 1989). An organism is defined as a “metazoan animal with cells arranged
in at least two non-uniform layers and differentiated into somatic and reproductive cells with different functions” (Kaestner, 1969). By just replacing “cell” with “organism” we can
transform the same definition to the following definition for a superorganism: “superorganismic units with organisms arranged in at least two non-uniform types and
differentiated into sterile and reproductive organisms with different functions” (Moritz and
Southwick, 1992).
Moritz and Southwick (1992) outlined the features a superorganism must have:
- Sessility: the superorganism is confined to a defined nesting site which is commonly occupied by most of the individuals most of the time and where food is stored and brood is reared.
- Intraorganismic homeostasis: within the colony tasks are divided among different groups of individuals but each member can change its own activity into the most urgent one. Furthermore, food supplies are stored in the nest to cope with periods of unfavourable climatic conditions.
- Nest cripticity: superorganisms nesting sites are always set up in hidden places and/or equipped with defensive arms, because the stored food supplies are attractive for predators.
37 - A large number of colony members: many thousands of insects are necessary to form a functioning superorganism. Members function as a cooperative unit and there are no solitary existences in the colony.
- Compartmentalization: specialized groups of colony members deal with specific functions. - Budding reproduction: the swarming.
- Natural selection acts upon the superorganism and not at individual level (Moritz and Southwick, 1992).
Honeybees are a matriarchal, monoginic and pluriannual society. Female individuals are divided in two castes: queen and workers. Males (drones) are not a caste. Female honeybee larvae are bipotential and are able to develop both queens and workers (polyphenismis) (Evans and Wheeler, 1999; Evans and Wheeler, 2001). The larva future caste is determined by the diet it receives from the third day after hatching until emergence. During the first three days after hatching, all females larvae are fed with royal jelly produced by nurses workers hypopharingeal glands. Then, the future queens only are fed with royal jelly, whereas future workers receive a pollen-honey mixture, as well as future drones (Frediani and Pinzauti, 1993; Contessi, 2016).
In each honeybees colony, the queen lays eggs in the brood cells. The egg could be fertilized, yielding a female, or unfertilized, yielding a male (arrhenotoky) (Cook, 1993). Hatching occurs three days after oviposition and, since that moment, larvae are regularly fed by nurses. Larvae develop throughout five stages in the days following hatching. From the second stage, larvae bend, taking the typical “C” shape (Frediani and Pinzauti, 1993; Contessi, 2016). On the fifth day after hatching, workers close the brood cell with a waxy operculum. Inside the operculated cell, the larva spins a silky envelope and pupates. Adult workers eclose from the envelope and emerge from their brood cell 21 days after oviposition. Queens eclosion and emergence occur 16 days after oviposition, whereas drones emerge after 24 days (Frediani
38 and Pinzauti, 1993; Contessi, 2016). When a colony is fully operative, it includes: one queen, about 300 drones, 10˙000 foragers working outside the hive, 25˙000 workers working inside the hive, 900 larvae, 20˙000 pupae and 6˙000 eggs (Frediani and Pinzauti, 1993).
About a week after emergence, the queen performs the nuptial flight (Schlüns et al., 2005; Contessi, 2016). The queen meets drones in specific meeting points where males gather waiting for a queen (Contessi, 2016). Mating takes place in mid-air and a queen have to mate with many males (polyandry) to ensure a complete filling of her spermatheca (Palmer and Oldroyd, 2000; Schlüns et al., 2005; Contessi, 2016). A completely full spermatheca holds about 6˙000˙000 of spermatozoa (Contessi, 2016). From 4 to 21 days after mating, the queen begins eggs laying (Frediani and Pinzauti, 1993). A queen lays up to 300 eggs every day during Summer. During Autumn oviposition decreases until to stop in Winter. In its lifetime (4/5 years), a queen lays more than 2˙000˙000 of eggs (Frediani and Pinzauti, 1993; Contessi, 2016).
Honeybee workers are involved in all the colony duties. In their first week after emergence, workers spend their time cleaning cells and feeding the brood, the queen and the drones with honey and pollen. In the second week after emergence, workers became “nurses” and produce royal jelly as the hypopharingeal glands start work. In the third week, wax glands develop and workers start to build honeycombs. Then they perform guard duty, protecting the hive entrance as “guardians”. At last, workers became “foragers” and spend their time collecting pollen and nectar outside the hive. A worker lives about forty days in summer, while Autumn emerging workers live until the following spring (Frediani and Pinzauti, 1993; Contessi, 2016).
39 1.2.5 Osmia bees life cycle
Mason bees are solitary bees belonging to the Apoidea superfamily and Megachilidae family.
Osmia Panzer genus includes about 213 species living in the New and Old World, in the
Palearctic and Nearctic regions, and in the Ethiopian zone. Mason bees are not present in the Indo-Australian and Neotropical regions (Sinha, 1958). In Italy, 40 Osmia species have been found (Pagliano, 1995). In the present thesis we deal with two of the most common Osmia species: O. cornuta and O. bicornis.
Mason bees are univoltine, with only one generation per year, and are the earliest emerging bees in spring (Sedivy and Dorn, 2014). Adults overwinter inside their cocoons in a state of dormancy called diapause. Each cocoon lies within the nest in a pedotrophic cell where egg hatching, larval development, cocoon spinning, pupation and adult eclosion occur (Raw, 1972; Sedivy and Dorn, 2014).
Osmia bees nests generally consist of a series of cells built end to end and separated by mud
partitions. Nests are built in pre-existing cavities, which could be holes in walls, empty snail shells, empty nests of other insects, reeds inner cavities, dried branches or stems of dead plants (Raw, 1972; Bosch, 1994). In spring, generally from February to May, adult mason bees emerge from cocoons. Males emerge earlier than females (protandry) and start nectar foraging to feed themselves waiting for females to copulate (Raw, 1972; Bosch, 1994). After few days, females emerge and produce pheromones which attract males. Mating may occurs on flowers, on the soil or on a fixed support in the proximities of the nest. Mason bees never mate during flight (Felicioli and Pinzauti, 1994).
After mating, females start to forage for pollen and nectar provisions, and search for a nesting site. O. cornuta build nest in cavities with a diameter of 8-10 mm. O. bicornis choose diameter between 6 and 8 mm (Raw, 1972; Felicioli, 2000b). Both O. cornuta and O. bicornis show a gregarious nesting behaviour (Rosenheim, 1990; Felicioli, 2000b).
