In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis.

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78-16.02 µg/mL), although this was less effective than doxorubicin (IC50 of 0.56-1.55 µg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 µg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 µg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.

New regulatory role of Znf1 in transcriptional control of pentose phosphate pathway and ATP synthesis for enhanced isobutanol and acid tolerance.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production. Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

Individual honey bee tracking in a beehive environment using deep learning and Kalman filter.

The honey bee is the most essential pollinator and a key contributor to the natural ecosystem. There are numerous ways for thousands of bees in a hive to communicate with one another. Individual trajectories and social interactions are thus complex behavioral features that can provide valuable information for an ecological study. To study honey bee behavior, the key challenges that have resulted from unreliable studies include complexity (high density of similar objects, small objects, and occlusion), the variety of background scenes, the dynamism of individual bee movements, and the similarity between the bee body and the background in the beehive. This study investigated the tracking of individual bees in a beehive environment using a deep learning approach and a Kalman filter. Detection of multiple bees and individual object segmentation were performed using Mask R-CNN with a ResNet-101 backbone network. Subsequently, the Kalman filter was employed for tracking multiple bees by tracking the body of each bee across a sequence of image frames. Three metrics were used to assess the proposed framework: mean average precision (mAP) for multiple-object detection and segmentation tasks, CLEAR MOT for multiple object tracking tasks, and MOTS for multiple object tracking and segmentation tasks. For CLEAR MOT and MOTS metrics, accuracy (MOTA and MOTSA) and precision (MOTP and MOTSP) are considered. By employing videos from a custom-designed observation beehive, recorded at a frame rate of 30 frames per second (fps) and utilizing a continuous frame rate of 10 fps as input data, our system displayed impressive performance. It yielded satisfactory outcomes for tasks involving segmentation and tracking of multiple instances of bee behavior. For the multiple-object segmentation task based on Mask R-CNN, we achieved a 0.85 mAP. For the multiple-object-tracking task with the Kalman filter, we achieved 77.48% MOTA, 79.79% MOTSP, and 79.56% recall. For the overall system for multiple-object tracking and segmentation tasks, we achieved 77.00% MOTSA, 75.60% MOTSP, and 80.30% recall.

Pollination efficacy of stingless bees, Tetragonula pagdeni Schwarz (Apidae: Meliponini), on greenhouse tomatoes (Solanum lycopersicum Linnaeus).

The stingless bee Tetragonula pagdeni is distributed over a vast Southeast Asian territory. This species is commonly used as a commercial insect pollinator. Pollination efficacy of T. pagdeni was investigated with tomato (Solanum lycopersicum L.) cultivated in greenhouse environments. In the first experiment, the number of fruit sets, number of seeds, fresh weight, and fruit height were quantified in the greenhouse with stingless bees, without stingless bees, and with pollination by mechanical vibration by hand. In the second experiment, the treatments were conducted with tomatoes of indeterminate growth in the greenhouse with and without stingless bees to prevent variation among the different tomato plants. The obtained results showed that a greenhouse with stingless bees presented 85 ± 4.24 fruits per 100 flowers, more than a greenhouse with mechanical vibration (79.5 ± 2.12 fruits per 100 flowers) or a greenhouse without stingless bees (15 ± 0.00 fruits per 100 flowers). In addition, fruit produced in a greenhouse with stingless bees showed greater fruit weight and number of seeds than fruit produced in a greenhouse without stingless bees or pollinated by mechanical vibration. According to the obtained results, we suggest that T. pagdeni could be beneficial as an insect pollinator of greenhouse tomatoes in tropical regions, where the use of honeybees and bumblebees would be more difficult.

A model of infection in honeybee colonies with social immunity.

Honeybees (Apis mellifera) play a significant role in the pollination of various food crops and plants. In the past decades, honeybee management has been challenged with increased pathogen and environmental pressure associating with increased beekeeping costs, having a marked economic impact on the beekeeping industry. Pathogens have been identified as a contributing cause of colony losses. Evidence suggested a possible route of pathogen transmission among bees via oral-oral contacts through trophallaxis. Here we propose a model that describes the transmission of an infection within a colony when bee members engage in the trophallactic activity to distribute nectar. In addition, we examine two important features of social immunity, defined as collective disease defenses organized by honeybee society. First, our model considers the social segregation of worker bees. The segregation limits foragers, which are highly exposed to pathogens during foraging outside the nest, from interacting with bees residing in the inner parts of the nest. Second, our model includes a hygienic response, by which healthy nurse bees exterminate infected bees to mitigate horizontal transmission of the infection to other bee members. We propose that the social segregation forms the first line of defense in reducing the uptake of pathogens into the colony. If the first line of defense fails, the hygienic behavior provides a second mechanism in preventing disease spread. Our study identifies the rate of egg-laying as a critical factor in maintaining the colony's health against an infection. We propose that winter conditions which cease or reduce the egg-laying activity combined with an infection in early spring can compromise the social immunity defenses and potentially cause colony losses.

Genetic Characterization of Exotic Commercial Honey Bee (Hymenoptera: Apidae) Populations in Thailand Reveals High Genetic Diversity and Low Population Substructure.

Domestication of animal species is often associated with a reduction in genetic diversity. The honey bee, Apis mellifera Linnaeus, 1758, has been managed by beekeepers for millennia for both honey and wax production and for crop pollination. Here we use both microsatellite markers and sequence data from the mitochondrial COI gene to evaluate genetic variation of managed A. mellifera in Thailand, where the species is introduced. Microsatellite analysis revealed high average genetic diversity with expected heterozygosities ranging from 0.620 ± 0.184 to 0.734 ± 0.071 per locus per province. Observed heterozygosities were generally lower than those expected under Hardy-Weinberg equilibrium, both locally and across the population as a whole. Mitochondrial sequencing revealed that the frequency of two evolutionary linages (C-Eastern European and O-Middle Eastern) are similar to those observed in a previous survey 10 yr ago. Our results suggest that Thai beekeepers are managing their A. mellifera in ways that retain overall genetic diversity, but reduce genetic diversity between apiaries.

Genetic Structure of the Aphis craccivora (Hemiptera: Aphididae) From Thailand Inferred From Mitochondrial COI Gene Sequence.

The cowpea aphid, Aphis craccivora Koch (Hemiptera: Aphididae), is one of the most destructive insect pests of legume plants worldwide. Although outbreaks of this pest occur annually in Thailand causing heavy damage, its genetic structure and demographic history are poorly understood. In order to determine genetic structure and genetic relationship of the geographic populations of this species, we examined sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene of 51 individuals collected from 32 localities throughout Thailand. Within the sequences of these geographic populations, 32 polymorphic sites defined 17 haplotypes, ranging in sequence divergence from 0.2% (1 nucleotide) to 2.7% (16 nucleotides). A relatively high haplotype diversity but low nucleotide diversity was detected in the populations of A. craccivora, a finding that is typical for migratory species. Phylogenetic analysis revealed a weak phylogeographic structuring among the geographic populations and among the haplotypes, indicating their close relationship. Considering the distance between the sampling sites, the occurrence of identical haplotypes over wide areas is noteworthy. Moreover, the low genetic distance (FST ranging from -0.0460 to 0.3263) and high rate of per-generation female migration (Nm ranging from 1.0323 to 20.3333) suggested population exchange and gene flow between the A. craccivora populations in Thailand.

Decision-making in honeybee swarms based on quality and distance information of candidate nest sites.

In the nest-site selection process of honeybee swarms, an individual bee performs a waggle dance to communicate information about direction, quality, and distance of a discovered site to other bees at the swarm. Initially, different groups of bees dance to represent different potential sites, but eventually the swarm usually reaches an agreement for only one site. Here, we model the nest-site selection process in honeybee swarms of Apis mellifera and show how the swarms make adaptive decisions based on a trade-off between the quality and distance to candidate nest sites. We use bifurcation analysis and stochastic simulations to reveal that the swarm's site distance preference is moderate>near>far when the swarms choose between low quality sites. However, the distance preference becomes near>moderate>far when the swarms choose between high quality sites. Our simulations also indicate that swarms with large population size prefer nearer sites and, in addition, are more adaptive at making decisions based on available information compared to swarms with smaller population size.

Economics of comb wax salvage by the red dwarf honeybee, Apis florea.

Colonies of Apis florea, which only abscond a short distance, usually return to salvage old nest wax; but, those colonies, and all other honeybee species which go considerably further, do not. Wax salvage would clearly be counter-productive unless the energy input/energy yield threshold was a profitable one. There are two possible trade-offs in this scenario, the trade-off between the energy expended to recover the wax (recovering hypothesis) as against that of replacing the wax by new secretion (replacing hypothesis). In order to compare the two hypotheses, the fuel costs involved in salvaging wax on one return trip, the average flower handling time, flight time and relative values for substituting the salvaged wax with nectar were calculated. Moreover, the energy value of the wax was determined. Net energy gains for salvaged wax were calculated. The energy value of the salvaged wax was 42.7 J/mg, thus too high to be the limiting factor since salvaging costs are only 642.76 mJ/mg (recovering hypothesis). The recovery costs (642.76 mJ/mg) only fall below the replacement costs for absconding distance below 115 m thus supporting the replacing hypothesis. This energetic trade-off between replacing and recycling plus the small absconding range of A. florea might explain why A. florea is probably the only honeybee species known to salvage wax and it parsimoniously explains the underlying reasons why A. florea only salvages wax from the old nest if the new nesting site is less than 100-200 m away-energetically, it pays off to recycle.

Sun angle time windows for absconding by the dwarf honeybee, Apis florea.

In Asia, the red dwarf honeybee, Apis florea, is notorious for its absconding habit. Interestingly, such colonies show a bimodal frequency distribution about a noonday lull throughout the year. Because slight errors in reading the relative position of the sun near its zenith results in very large orientation errors in the waggle dances of other honeybees in the tropics, we postulated that the frequency distribution of absconding in the red dwarf honeybee relative to local clock time could be explained in similar fashion. The frequency distribution of absconding by the red dwarf honeybee with respect to time was found to be bimodal with a pronounced lull at noonday, which in turn is related to the altitude angle of the sun. So, these bees largely avoid flying off between 12:00h and 13:00h on the one hand and that their preferred departure angle of the sun is between 55 degrees and 65 degrees on the other. Given the difficulties of taking an accurate reading of the sun at angles +/-6 degrees of the sun's zenith (resulting in a 1h loss around noon) and the 2h required to reach consensus over the final direction to be flown, the bees are simply left with two time windows, morning and afternoon, in which to abscond and, indeed some 90% of the red dwarf honeybee colonies do so. The noonday lull is not associated with high temperatures for any given day. Absconding is not inhibited by high temperatures.

Interspecific utilisation of wax in comb building by honeybees.

Beeswaxes of honeybee species share some homologous neutral lipids; but species-specific differences remain. We analysed behavioural variation for wax choice in honeybees, calculated the Euclidean distances for different beeswaxes and assessed the relationship of Euclidean distances to wax choice. We tested the beeswaxes of Apis mellifera capensis, Apis florea, Apis cerana and Apis dorsata and the plant and mineral waxes Japan, candelilla, bayberry and ozokerite as sheets placed in colonies of A. m. capensis, A. florea and A. cerana. A. m. capensis accepted the four beeswaxes but removed Japan and bayberry wax and ignored candelilla and ozokerite. A. cerana colonies accepted the wax of A. cerana, A. florea and A. dorsata but rejected or ignored that of A. m. capensis, the plant and mineral waxes. A. florea colonies accepted A. cerana, A. dorsata and A. florea wax but rejected that of A. m. capensis. The Euclidean distances for the beeswaxes are consistent with currently prevailing phylogenies for Apis. Despite post-speciation chemical differences in the beeswaxes, they remain largely acceptable interspecifically while the plant and mineral waxes are not chemically close enough to beeswax for their acceptance.