Identify The Source of Pollen Feed in Honey and Bee Bread of Tetragonula biroi

This study aims to determine the types of feed pollen consumed by the bee species Tetragonula biroi. The research has the objective of providing information to the public regarding the sources of feed for T. biroi honey bees, which are located in Kuajang Village, Binuang District, Polewali Mandar Regency, West Sulawesi. The research was conducted between February and April 2020. A comparative method was employed to examine the shapes of plant pollen found around the meliponiary in comparison to the shapes of pollen discovered in honey and bee bread. Samples were prepared using the acetolysis method and stained using safranin. The research findings indicate the presence of 23 identified flowering plant species in the vicinity of the meliponiary, along with their identification in bee bread. However, only 12 species were identified in honey. Coconut pollen exhibited the highest percentage, accounting for 44.8% of honey and 38.13% of bee bread, categorizing it as a secondary pollen type.


Introduction
The bee species that has gained increasing popularity for cultivation is the stingless bee, commonly known as Trigona bees.Trigona bees belong to the subfamily Meliponinae in the order Hymenoptera, and they are distributed throughout almost all tropical and subtropical regions [1].In Asia, 42 species have been identified [2], with 37 species found in Indonesia, specifically in Java, Sumatra, Kalimantan, and Sulawesi [3].In Sulawesi, there are 8 species, 4 of which are found in South Sulawesi, namely Wallacetrigona incisa, Tetragonula laeviceps, Tetragonula biroi, and Heterotrigona itama [4].The Trigona bee species that are being cultivated in South Sulawesi, particularly in the North Luwu region, are W. incisa and T. biroi [5].Meanwhile, in the Polewali Mandar District of West Sulawesi Province, T. biroi is the species being cultivated.
Trigona bees easily adapt to their environment and are tame or rarely abscond (leave the nest).Trigona bees are more effective in visiting various types of plants, both cultivated and wild, to obtain sources of nectar and pollen.Several factors can influence Trigona bees in gathering their food, including environmental temperature and distance from the food source to the hive [6], as well as humidity [7].Some species of stingless bees tend to be more selective in choosing their food sources [8].
Abundant flowering plants as a source of nectar and pollen will have a significant impact on honey productivity.However, information regarding the plants as the most frequently visited food source for pollen collection is still insufficient in each beekeeping area.It is crucial for beekeepers to know the types of bee food sources and have a map of nectar and pollen sources in their area; this will assist in planning the management of their colonies [9].This includes the West Sulawesi region, where information on important food sources for Trigona bees of the species T. biroi is essential.Trigona beekeeping, also known as meliponiculture, has begun to form groups and independent cultivation within residential areas.Pollen can be used as an indicator to detect the origin of forage plants collected by bees.Pollen has distinctive characteristics, including different shapes from each plant species [10].Pollen also possesses cell walls with unique characteristics, such as a layer composed of sporopollenin.This layer is resistant to harsh 1315 (2024) 012064 IOP Publishing doi:10.1088/1755-1315/1315/1/012064 2 chemical processes and even enzymatic reactions during digestion, ensuring that the pollen's form remains unchanged [11].These properties enable pollen to be used as an indicator to identify the plants that serve as a food source for bees.
Other important information regarding the types of pollen in honey and bee bread is the frequency or percentage of pollen presence that determines the categorization of the honey.According to Wingenroth [12], honey can be grouped into three categories based on the frequency and types of pollen present in the honey: monofloral honey (a single type of pollen with a pollen frequency of >45%), bifloral honey (two types of pollen with a pollen frequency of >22.25%), and multifloral honey (three or more types of pollen with a pollen frequency of <16%).Knowledge of the types of bee forage can also serve as a database for tracing the origin of honey available in the market.

Research location
The sampling was conducted at the "Kampung Lebah Madu Trigona" meliponiary in Lemo Baru Hamlet, Kuajang Village, Polewali Mandar District, West Sulawesi Province (Fig 1).Kuajang Village is located 5.2 km from the city of Polewali and has an average daily temperature of 24-32 °C.Astronomically, it is situated at 3°25'50.6"South Latitude and 119°22'41.3"East Longitude, with an elevation ranging from 30 to 100 meters above sea level.

Sampling
Sampling of honey and bee bread was conducted from three different colonies.The selection of colonies was based on those that could be taken as samples, which had produced honey and bee bread within the hive and had reached the harvesting stage at the age of 4-5 months.The identification method used involved comparing the shape of pollen identified from honey and bee bread with the shape of fresh pollen obtained from flowering plants around the hive.The sampling radius for fresh flower pollen extended up to 500 meters and was performed in the morning or during flower blooming.Sampling of pollen from fresh flowers was done at 06:00 -07:00 or when the flowers were newly bloomed.All types of flowers were collected from plants at the research location.Flower pollen was placed in sample bottles containing 70% alcohol.Flowering plants that were not known for their types were sampled from leaves, buds, fruits (if present), and branches, and were preserved in 70% alcohol for identification purposes.

Pollen extraction
Samples of honey taken from each of the three colonies, totaling 250 ml each, are then homogenized.Then, 1 ml was taken from each of these samples and diluted with 1 ml of glycerin and stained with 1 ml of safranin.Four grams of bee bread samples from each bee colony were dissolved in 70% alcohol.Two ml of this solution were transferred to a reaction tube and supplemented with 1 ml of glycerin.Pollen staining was performed using 1 ml of safranin with a concentration of 2%.The samples were then placed on a glass slide and covered with a cover slip.The observation of the samples under a light microscope was carried out at a magnification of 400x, with three slides of the glass object for each colony sample.The number of observed pollen grains under the microscope was counted to analyze the percentage of pollen collected by bees.The extraction and observation of flower pollen were based on the acetolysis method used by Erdtman [13].The percentage of pollen count was calculated using the formula:

Σ‫ܤ‬
Were X was the percentage of the total amount of pollen from a single plant species in honey and Bee bread (%); A was the number of pollen grains from a single plant species, and B was the total number of pollen grains observed from all plant species in one preparation.The identified pollen is calculated in percentages and categorized as Predominant Pollen Type (PPT) (>45%), Secondary Pollen Type (SPT) (16-45%), Important Minor Pollen Type (IMPT) (3-15%), and Minor Pollen Type (MPT) (<3%) [14].The determination of pollen is based on measuring the longest diameter of the equatorial pollen.There are five-grain diameter types based on the diameter: very small granules <10 μm, small grains 10-24 μm, medium grains 24-49 μm, large granules 50-99 μm, and giant grains >100 μm [15].

The Percentage of Pollen in Bee Bread and Honey
The percentage of plant pollen identified in honey and bee bread can be seen in Figure 2. Thirteen types of plant pollen were identified in honey, while the colony's bee bread contained 23 types of plant pollen.Coconut pollen was the most abundant type identified in both honey and bee bread, accounting for 44.8% and 38.13% respectively.The second most abundant pollen type accounted for 24.12% of bee bread and 24.65% in honey.The honey obtained from the research location is classified as bifloral honey, which means it contains two types of pollen with a frequency exceeding 22.25% [12].Coconut and leucaena pollen fall under the category of secondary pollen type (SPT), while the pollen from other plant types ranges from 0.4% to 5.43% in bee bread and 0.85% to 8.08% in honey.Pollen from plant types other than coconut and leucaena are categorized as minor pollen type (MPT) to important minor pollen type (IMPT).
The highest percentage of pollen was identified from coconut flowers and lamtoro flowers.This is because the scent of coconut and lamtoro flowers produces a fragrant aroma that attracts bees to visit them.In addition, the color of the flowers also influences bees in choosing which flowers to visit.The yellow and white colors of coconut and lamtoro flowers are preferred by bees.Based on research conducted by Oktavia [16], it is revealed that the attraction for honey bees to visit flowers is the aroma and sweet taste of the flowers, as well as the color of the food, which is white and yellow.

Available Pollen around Meliponiary
The available pollen in the vicinity of Meliponiary can be seen in Table 1.Meliponiary is situated between residential areas and community plantations with diverse vegetation, including crops, forests, food crops, vegetables, and ornamental plants.The research location is an area where coconut trees thrive.Coconut trees are known to be abundant producers of both floral and extrafloral pollen and nectar.Although the distribution of lamtoro plants around Meliponiary is not as extensive as coconut trees, a single tree can produce an abundant amount of flowers.This is an attractive feature for bees to gather both types of food.Bees tend to maximize their time collecting food in areas with a plentiful supply and will decrease or relocate when the food becomes insufficient, searching for neighboring or nearby locations with food sources [17].Each pollinator species, including bees, tends to collect pollen from the same source, which is considered the best food source they have found.This makes it easier for them to remember the direction or location of the food source [18].
Many sources of pollen from plants are located around the hive with distances ranging from 1 to 20 meters (Figure 3).In addition to coconut trees and lamtoro, there are also types of papaya, langsat, sunflower, Zinnia sp, bridal tears, eight o'clock flowers, bashful princess, and rambutan.This indicates that bees, when searching for food, also tend to enrich their diet by finding the closest sources of food to their nest.This is supported by the research findings of [19], which suggest that the factors influencing bees in collecting food are not based on pollen size but on the distance, aroma, and color of the flowers.Many sources of pollen from plants are located around the hive with distances ranging from 1 to 20 meters (Figure 3).In addition to coconut trees and lamtoro, there are also types of papaya, langsat, sunflower, Zinnia sp, bridal tears, eight o'clock flowers, bashful princess, and rambutan.This indicates that bees, when searching for food, also tend to enrich their diet by finding the closest sources of food to their nest.This is supported by the research findings of [19], which suggest that the factors influencing bees in collecting food are not based on pollen size but on the distance, aroma, and color of the flowers.

The Forms of Pollen in Bee Bread and Honey
The identification of plant pollen by comparing the pollen from various sources of bee feed with the pollen from honey and bee bread revealed a total of 23 different types with diverse forms (Figure 4).The identification results showed various types of pollen with different sizes, which is consistent with previous studies conducted by Nugroho and Soesiloha [19], [20], and [21].These studies provided information that stingless bees collect pollen not based on size but rather on the distance of flowers from the nest, as well as the aroma and color of the flowers.The bees do not differentiate the types of their food based on the location's altitude above sea level, as revealed by the pollen identification results, except for a tendency towards different sizes due to altitude differences [22].Generally, pollen can be classified based on its physical appearance.Pollen essentially has a complex structure, shape, and pattern, requiring specific terminology for its identification, including the identification of bee feed sources.

Conclusions
Identification of plant pollen collected by bees in bee bread and/or trapped in honey pots can be used as an indicator of a bee colony's food source.The most frequently identified location in this meliponary study is coconut pollen (Cocos nucifera), accounting for 38.13% of bee bread and 44.8% of honey.The second plant is Leucaena leucocephala, with a percentage of 25.12% in bee bread and 24.65% in honey.Both types of pollen fall under the category of secondary pollen type and the honey produced from T. biroi cultivation is classified as biflora honey.

Figure 1 .
Figure 1.Map of research location.

Figure 2 .
Figure 2. The percentage of plant pollen found in honey and bee bread samples

6 Figure 3 .
Figure 3. Description of the meliponiary location