Population Growth and Insecticide Residues of Honey Bees in Tropical Agricultural Landscapes

: Global decline of pollinators, especially bees, has been documented in many countries. Several causes such as land-use change and agricultural intensiﬁcation are reported to be the main drivers of the decline. The objective of this study was to investigate the e ﬀ ect of land use on honey bee and stingless bee populations. Research was conducted in Bogor and Malang to compare between two di ﬀ erent geographical areas. Managed bees such as honey bees ( Apis cerana and A. mellifera ) and stingless bees ( Tetragonula laeviceps ) were investigated to examine the e ﬀ ect of agricultural intensiﬁcation. Field experiments were conducted by placing beehives in selected habitats (i.e., beekeeper gardens, forests areas, and agriculture areas). Population growth and neonicotinoid residue analysis of bees in di ﬀ erent hive locations were measured to study the e ﬀ ect of habitat type. Population growth of bees represents the forager abundance and colony weight. Based on the analysis, we found that habitat type a ﬀ ected forager abundance and colony weight of honey bees ( p < 0.05), although the patterns were di ﬀ erent between species, region, as well as season. Forests could support the stingless bee colony better than agriculture and home garden habitats. Insecticide (neonicotinoid) was barely recorded in both honey bees and stingless bees.


Introduction
In the agroecosystem, pollinators are a pivotal component of biodiversity that provide an important ecosystem service through crop pollination [1] and increasing fruit set [2]. Pollinators also can be used as indicators of ecosystem health because of their sensitiveness to environmental stressors [3], for instance, the negative impacts of pesticide application [4]. There is growing concern relating to declines found in pollinators around the world [5]. In Europe and the US, a decline in wild bee species richness has been recorded, where the declining trends are in the abundance of honey bees (Apis mellifera) and a small number of wild pollinators [6]. Although high diversity of bees is found in the tropics (e.g., [7,8]), there is a lack of information about this phenomenon. Therefore, investigation needs to be undertaken into the scale, magnitude, and causes of the decline and the effects on pollination services.

Observation of Bees in the Hives and Residue Analysis
To study the effect of habitat type on bees, we observed the hives on each experimental plot, measured the population growth of bees, and performed a residue analysis. Population growth of bees was measured by counting the forager abundance (foraging activity) and colony weight (weighing full hive). The method of foraging activity monitoring was based on [21] by counting the foragers departing or returning to the colony for thirty minutes per hive. In each plot, the observation was conducted from 7 am until 11 am. Weighing full hives was conducted to calculate the colony weight that included the summed weight of the box, combs with food stores, and the bees [22]. Both forager abundance and colony weight were observed every two weeks during two months. Observations were conducted in different seasons (i.e., rainy and dry seasons). Observations in the dry season were done from March to May 2019, while for the rainy season, observations were from July to September 2019.
In addition, insecticide residue analysis was conducted by collecting honey and bee (foragers) samples in three habitat types. We collected 5 mg of bees and 5 mg of honey per plot and initially froze it before being analyzed using the QuEChERS protocol [23]. Insecticide residue analysis was conducted in the medical laboratory of Jakarta (https://labkesda.jakarta.go.id) and was focused on imidacloprid content as the representation of neonicotinoid insecticide.

Data Analysis
The difference of forager abundance and colony weight of bees between habitat types was analyzed using a Kruskal-Wallis test. If we found significant differences, a post-hoc test was done using Fisher's least-significant difference with α = 0.05. To analyze the relationship between forager abundance as well as colony weight of bees and observation time, we analyzed using fitting median-based linear models based on the Theil-Sen single median. All analyses were performed using R statistical software [24] and package "mblm" for fitting median-based linear models [25].

Effect of Different Habitat Types and Season on Honey Bees
Based on the analysis, we found that habitat type and season affected the forager abundance and colony weight of bees (P < 0.05), although the patterns were different between species and

Observation of Bees in the Hives and Residue Analysis
To study the effect of habitat type on bees, we observed the hives on each experimental plot, measured the population growth of bees, and performed a residue analysis. Population growth of bees was measured by counting the forager abundance (foraging activity) and colony weight (weighing full hive). The method of foraging activity monitoring was based on [21] by counting the foragers departing or returning to the colony for thirty minutes per hive. In each plot, the observation was conducted from 7 am until 11 am. Weighing full hives was conducted to calculate the colony weight that included the summed weight of the box, combs with food stores, and the bees [22]. Both forager abundance and colony weight were observed every two weeks during two months. Observations were conducted in different seasons (i.e., rainy and dry seasons). Observations in the dry season were done from March to May 2019, while for the rainy season, observations were from July to September 2019.
In addition, insecticide residue analysis was conducted by collecting honey and bee (foragers) samples in three habitat types. We collected 5 mg of bees and 5 mg of honey per plot and initially froze it before being analyzed using the QuEChERS protocol [23]. Insecticide residue analysis was conducted in the medical laboratory of Jakarta (https://labkesda.jakarta.go.id) and was focused on imidacloprid content as the representation of neonicotinoid insecticide.

Data Analysis
The difference of forager abundance and colony weight of bees between habitat types was analyzed using a Kruskal-Wallis test. If we found significant differences, a post-hoc test was done using Fisher's least-significant difference with α = 0.05. To analyze the relationship between forager abundance as well as colony weight of bees and observation time, we analyzed using fitting median-based linear models based on the Theil-Sen single median. All analyses were performed using R statistical software [24] and package "mblm" for fitting median-based linear models [25].

Effect of Different Habitat Types and Season on Honey Bees
Based on the analysis, we found that habitat type and season affected the forager abundance and colony weight of bees (P < 0.05), although the patterns were different between species and

Observation of Bees in the Hives and Residue Analysis
To study the effect of habitat type on bees, we observed the hives on each experimental plot, measured the population growth of bees, and performed a residue analysis. Population growth of bees was measured by counting the forager abundance (foraging activity) and colony weight (weighing full hive). The method of foraging activity monitoring was based on [21] by counting the foragers departing or returning to the colony for thirty minutes per hive. In each plot, the observation was conducted from 7 am until 11 am. Weighing full hives was conducted to calculate the colony weight that included the summed weight of the box, combs with food stores, and the bees [22]. Both forager abundance and colony weight were observed every two weeks during two months. Observations were conducted in different seasons (i.e., rainy and dry seasons). Observations in the dry season were done from March to May 2019, while for the rainy season, observations were from July to September 2019.
In addition, insecticide residue analysis was conducted by collecting honey and bee (foragers) samples in three habitat types. We collected 5 mg of bees and 5 mg of honey per plot and initially froze it before being analyzed using the QuEChERS protocol [23]. Insecticide residue analysis was conducted in the medical laboratory of Jakarta (https://labkesda.jakarta.go.id) and was focused on imidacloprid content as the representation of neonicotinoid insecticide.

Data Analysis
The difference of forager abundance and colony weight of bees between habitat types was analyzed using a Kruskal-Wallis test. If we found significant differences, a post-hoc test was done using Fisher's least-significant difference with α = 0.05. To analyze the relationship between forager abundance as well as colony weight of bees and observation time, we analyzed using fitting median-based linear models based on the Theil-Sen single median. All analyses were performed using R statistical software [24] and package "mblm" for fitting median-based linear models [25].

Effect of Different Habitat Types and Season on Honey Bees
Based on the analysis, we found that habitat type and season affected the forager abundance and colony weight of bees (P < 0.05), although the patterns were different between species and Description Highland agroecosystem, dominated by vegetable crops with high insecticide application   We placed three beehives in each plot for observation. The sizes of beehives were different depending on the beekeeper's practice in rearing bees. In Malang, the hive size of A. cerana was 40 × 25 × 25 cm with four combs and A. mellifera was 50 × 40 × 25 cm with four combs. While in Bogor, the hive size of A. cerana was 35 × 30 × 25 cm with six combs and T. laeviceps was 30 × 10 ×10 cm without combs.

Observation of Bees in the Hives and Residue Analysis
To study the effect of habitat type on bees, we observed the hives on each experimental plot, measured the population growth of bees, and performed a residue analysis. Population growth of bees was measured by counting the forager abundance (foraging activity) and colony weight (weighing full hive). The method of foraging activity monitoring was based on [21] by counting the foragers departing or returning to the colony for thirty minutes per hive. In each plot, the observation was conducted from 7 a.m. until 11 a.m. Weighing full hives was conducted to calculate the colony weight that included the summed weight of the box, combs with food stores, and the bees [22]. Both forager abundance and colony weight were observed every two weeks during two months. Observations were conducted in different seasons (i.e., rainy and dry seasons). Observations in the dry season were done from March to May 2019, while for the rainy season, observations were from July to September 2019.
In addition, insecticide residue analysis was conducted by collecting honey and bee (foragers) samples in three habitat types. We collected 5 mg of bees and 5 mg of honey per plot and initially froze it before being analyzed using the QuEChERS protocol [23]. Insecticide residue analysis was conducted in the medical laboratory of Jakarta (https://labkesda.jakarta.go.id) and was focused on imidacloprid content as the representation of neonicotinoid insecticide.

Data Analysis
The difference of forager abundance and colony weight of bees between habitat types was analyzed using a Kruskal-Wallis test. If we found significant differences, a post-hoc test was done using Fisher's least-significant difference with α = 0.05. To analyze the relationship between forager abundance as well as colony weight of bees and observation time, we analyzed using fitting median-based linear models based on the Theil-Sen single median. All analyses were performed using R statistical software [24] and package "mblm" for fitting median-based linear models [25].

Effect of Different Habitat Types and Season on Honey Bees
Based on the analysis, we found that habitat type and season affected the forager abundance and colony weight of bees (p < 0.05), although the patterns were different between species and region. In general, habitat types showed to be the most important factor that affected both forager abundance and colony weight of bees compared to season and observation time ( Table 2). Effect of habitat type on forager abundance and colony weight of A. cerana differed between Malang and Bogor. In Malang, the difference of habitat type significantly influenced the forager abundance and colony weight of A. cerana, which were found higher in home gardens and forests compared to the agricultural areas (Figures 2a and 3a). In Bogor, forager abundance of A. cerana was not significantly different among habitat type (Figure 2b), while the colony weight of A. cerana in forests was significantly higher than in home gardens (Figure 3b). The same pattern was observed with A. cerana in Malang for forager abundance of A. mellifera in Malang and T. laeviceps in Bogor, which were also found higher in home gardens and forests compared to agricultural areas (Figure 2c,d and Figure 3c,d).      Based on non-parametric regression, we found that the forager abundance of A. cerana in Malang was prone to decrease with increasing observation time (p = 0.001) ( Table 3). In addition, the colony weights of A. cerana and T. laeviceps in Bogor as well as A. mellifera in Malang tended to increase with increasing observation time (p < 0.05).

Detection Results of Insecticide Residue in Honey and Body of Honey Bees
Based on residue analysis, we barely detected insecticide residue (imidacloprid content) both in the honey and the body of bees ( Table 4). Two of 18 honey samples (11.11%) contained a small amount of insecticide residue (<5 µg/kg). Insecticide residues were also detected in three bee samples (16.66%). Surprisingly, we did not detect the insecticide residue in the agriculture habitat, yet it was detected in forest and home garden habitats.

Discussion
Our research provided the evidence that habitat type significantly affects both forager abundance and colony weight of honey bees as well as stingless bees in Indonesia. The lowest forager abundance and colony weight was shown in agricultural areas, which indicated that agricultural areas had a negative effect on bees. Research by [26] showed that bee species have distinct preferences for different plant communities, and their abundance is related to the abundance of their host plants. Agricultural areas that are dominated by certain crop plants might affect the fitness and population growth of bees. This is due to plant diversity and is a key driver of bee fitness. Bees were found to be fitter and their populations grew faster in more florally diverse environments because of a continuous supply of food resources [27].
Landscape diversity also influences the growth and reproduction of honey bees, besides the availability of pollen in agricultural landscapes. For instance, A. mellifera compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amounts and diversity [28]. This indicates the importance of agri-environmental schemes to support pollinators and not just the plant diversity and pollen availability. In Malang, A. mellifera is handled by beekeepers following "migratory management", which causes bees undue stress. In order to ease access to food source, beekeepers move the hives to flowering areas. However, this management may affect the population growth of bees. Research by [29] showed that the lifespan of migratory adult bees tends to decrease compared to stationary bees.
In this research, we only focused on environmental stressors (e.g., habitat condition) and did not investigate other potential drivers that affect population growth of bees, such as pests and pathogens, and genetic diversity as well as vitality of bees [9]. However, habitat type did not guarantee that bees were unhampered from insecticide. This might be related to the foraging range of bees as well as the food source, which is not only pollen but also honeydew. Honey bee presence was positively affected by the presence of honeydew and source of insecticide residue that affected honey quality [30]. Neonicotinoid (imidacloprid), which was found in our study, was also detected in 11% samples of honey from apiaries located in Poland [31]. However, the death of honey bees from Bologna were reported from different active ingredients of insecticide [32]. Thus, in this research, neonicotinoid was detected in the body of bees and honey, although it was in a small amount of residue.

Conclusions
Our research results showed the factor that affected forager abundance and colony weight of bees the most was habitat type. The agricultural habitat had lower bee forager abundance and colony weight compared to forest and home garden habitats. This indicates that the hypothesis from the beekeepers' perspective is accepted in relation to the negative effect of agriculture (especially pesticide application) on their honey production. However, our experiment revealed that habitat type, especially forest and home garden, did not guarantee that honey bees were unhampered from pesticide.