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Article

Impact of Direct-Fed Microorganism Syrup on Honey Bee (Apis mellifera L.) Hypopharyngeal Gland Development, Protein Digestibility and Gut Microbiota Composition

by
Pichet Praphawilai
1,2,
Khanchai Danmek
3,*,
Tippapha Pisithkul
4,
Saeed Mohamadzade Namin
5,
Sampat Ghosh
6,
Chuleui Jung
7,8 and
Bajaree Chuttong
2,*
1
Office of Research Administration, Chiang Mai University, Chiang Mai 50200, Thailand
2
Meliponini and Apini Research Laboratory, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
3
Biotechnology Program, School of Agriculture and Natural Resources, University of Phayao, Phayao 56000, Thailand
4
Program in Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
5
Department of Horticulture, College of Agricultural Science, Oregon State University, Corvallis, OR 97331, USA
6
Department of Life Science, Sardar Patel University, Balaghat 481331, Madhya Pradesh, India
7
Department of Plant Medicals, Gyeongkuk National University, Andong GB 36729, Republic of Korea
8
Agricultural Research Institute, Gyeongkuk National University, Andong GB 36729, Republic of Korea
*
Authors to whom correspondence should be addressed.
Animals 2026, 16(5), 704; https://doi.org/10.3390/ani16050704
Submission received: 11 January 2026 / Revised: 9 February 2026 / Accepted: 20 February 2026 / Published: 24 February 2026
(This article belongs to the Special Issue Functional Prebiotics and Probiotics: Impact on Animal Growth, Development and Health)
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Simple Summary

Honey bees are essential insects that help produce valuable foods like honey and royal jelly, which also provide health benefits for humans. However, bee health and productivity face various environmental challenges. This study investigated the beneficial bacteria naturally found in the digestive system of worker bees. Lactic acid bacteria were isolated, identified, and incorporated into sugar syrup in direct-fed microorganism (DFM) form to test their potential effects on bee health. The results demonstrated that bees fed with DFM syrup developed hypopharyngeal glands in their heads that are crucial for royal jelly production. These findings suggest that supplementing bee diets with beneficial bacteria can enhance protein digestibility and gut microbiota composition, which benefits overall health. This approach could help beekeepers improve their colony care and support sustainable production.

Abstract

Honey bees (Apis mellifera L.) are considered highly significant economic insects. It is a source of valuable food and medicinal products such as honey, bee pollen, royal jelly, bee brood, and beeswax, which possess excellent nutritional and pharmacological properties. Nevertheless, honey bee health and productivity were often challenged by various environmental factors. Therefore, bee colony management is of the utmost importance. In this light, bee supplements and gut microbiota are crucial to ensure that bees receive sufficient nutritional value to maintain their health and productivity. In this study, we isolate and characterize lactic acid bacteria from the hindgut of the worker bee. 16S rRNA sequencing revealed that three isolated bacteria were Apilactobacillus kunkeei (AK), Lactiplantibacillus sp. (LP), and Lactobacillus brevis (LB). Three species of lactic acid bacteria were investigated for potential probiotic properties by supplementing 50% (w/w) sucrose syrup in the form of a direct-fed microorganism (DFM). The supplement with DFM had no negative effect on average lifespan. Examination took place of the impact of probiotics on the development of the hypopharyngeal glands (HPGs) in the bee’s head at days 3, 6, and 9 post-treatments. The cage-bees fed by pollen and DFM syrup exhibited acini surface areas ranging from 0.020 to 0.023 mm2. The L. brevis (LB) group exhibited enhanced HPG development, with an average acini size of 0.027 ± 0.007 mm2 at day 6, while the non-treatment control had an average acini size of 0.023 ± 0.006 mm2. The significant size differences were maintained throughout the 9-day period. In addition, the DFM syrup enhanced microbial protein content in the bee head, digestibility, and community complexity compared with the negative control groups. Therefore, the DFM syrup with a potential strain of probiotic may enhance overall honey bee health status.

1. Introduction

Honey bees (Apis mellifera L.) are vital to global ecosystems and production of high-value products such as honey, royal jelly, and propolis, which provide significant nutritional and medicinal benefits. These contributions support food security, biodiversity, and human well-being [1,2]. The importance of honey bees extends far beyond the production of the bee products. They play a pivotal role in global agriculture, with pollination services estimated to contribute over USD 199 billion to the global economy annually [3]. However, honey bee populations are increasingly affected by environmental stressors such as monoculture practices, habitat degradation, pesticide exposure, pathogens, expanding urbanization, agricultural intensification, and the impact of climate change has often led to the destruction or fragmentation of flower-rich habitats. Collectively, these biotic and abiotic stressors, together with sensitivity, large foraging range, compromise colony health and productivity [4,5]. Monofloral pollen diets, particularly those deficient in protein or lacking a balanced amino acid profile, have been associated with adverse physiological effects in honey bees. It affects hemolymph vitellogenin levels, which are closely linked to immunity and decreased survival of worker bees [6]. Furthermore, the limitation of floral diversity and the ingestion of pollen with thick cell walls or anti-nutritional compounds may impair digestion and nutrient assimilation, thereby increasing the risk of malnutrition [7]. Such external stressors lead to declines in honey bee populations, which in turn limit the availability of their health-promoting products by disrupting gut microbiota balance, which is essential for supporting the health of honey bee colonies and offering therapeutic benefits and enhancing immune function. Ultimately, the interaction between monofloral diet and a compromised balance of gut microbiota can result in decreased nutrient availability and increased susceptibility to diseases, which can then lead to colony weakness and colony collapse [8,9].
Recent research has highlighted the potential of microbial supplements as a promising tool in apiary management [10]. Hence, understanding and maintaining gut microbiota becomes crucial. Five omnipresent bacteria are remarkably consistent and dominate the honey bee gut microbiota, including Snodgrasella alvi, Gilliamella apicola, Lactobacillus (Firm-4 and Firm-5), Bifidobacterium asteroides, and Frischella perrara [11,12,13]. It plays a fundamental role in restoring and supporting honey bee health, including potential benefits for hypopharyngeal gland development, crucial for royal jelly production [14]. However, gut microbiota is significantly influenced by environmental changes such as urbanization, landscape alterations, and agrochemical exposure [15]. There is an increasing imperative to explore probiotic solutions that can effectively restore and maintain gut health in honey bees. Although several studies have explored probiotic effects in honey bees, targeted investigations on specific organs remain limited. and the need for targeted research to identify key microbial taxa for probiotic formulations remains a significant challenge that could offer new strategies to enhance honey bee resilience against environmental stressors [15].
Probiotic supplementation, especially with lactic acid bacteria (LAB), has shown promising benefits for honey bee health by enhancing the immune response of bees, eliminating pathogens, maintaining gut microbiota homeostasis, and improving stress tolerance and disease resistance, particularly during off-floral seasons when natural forage is scarce [16,17,18,19,20]. This approach not only supports colony health by promoting beneficial bacteria like Lactobacillus spp. but also helps mitigate the impacts of environmental degradation and intensive agricultural practices, boosting the productivity and economic returns from honey bee products such as honey and beeswax [21]. Specific probiotic strains, like Lactobacillus plantarum and Bacillus subtilis, enhance digestive enzyme activity, facilitating better nutrient absorption and supporting colony health [22]. Furthermore, it can be applied to honey bee feed for enhancing the effectiveness of inhibiting pathogenic microorganisms in the digestive tract [23,24]. A mixture of beneficial bacteria can colonize the gut and outcompete harmful microbes. It helps the restoration process of gut microbiota disrupted by Vairimorpha spp. (formerly Nosema spp.) infection; this infection often shows a decline in beneficial bacteria such as Bifidobacterium. The key activity of probiotics is increasing the abundance of helpful genera like Lactobacillus, Bifidobacterium, and Snodgrassella alvi, thereby re-establishing a healthy microbial balance. Consequently, the beneficial microbes occupy niches in the gut, limiting the space and resources available for pathogens to thrive. This natural competition helps suppress the growth of harmful organisms [10,15]. Moreover, it could protect the honey bee from environmental stressors, such as exposure to agricultural chemicals like glyphosate by strengthening the gut microbiota and immune resilience [25]. However, the efficacy of commercial probiotics is variable, with native strains showing better results in colonizing honey bee guts, highlighting the need for further research to optimize probiotic formulations for honey bees [14].
The objective of this study is to isolate potential strains of probiotics and evaluate their effects on honey bee health, focusing on the impact of probiotic supplementation on overall health, longevity, hypopharyngeal glands (HPGs) development, and changes in gut microbiota. By assessing the ability of direct-fed microorganism syrup to address the nutritional stress caused by mono-floral pollen diets and disrupted gut microbiota, this study contributed to the development of sustainable beekeeping practices by improving bee health and the production of bee-derived substances that are a source of bioactive compounds beneficial to human health.

2. Materials and Methods

2.1. Sample Collection and Isolation of Lactic Acid Bacteria from Honey Bee Gut

Healthy colonies of Apis mellifera L. were maintained at the apiary located within the Faculty of Agriculture, Chiang Mai University (18°47′35.0″ N 98°57′40.0″ E), following standard beekeeping practices. Each colony typically consisted of an egg-laying queen and honey bee workers occupying eight frames of the hive, containing larvae, pupae, honey, and pollen. From each of three randomly selected hives, ten adult worker bees at the initial stage of development (collected directly from inside the hive) were collected using tweezers and then kept in centrifuge tubes. The bees were euthanized and their intestinal tracts were carefully dissected within 6 h using standard anatomical and dissection procedures for A. mellifera [26].
Lactic acid bacteria (LABs) were isolated from the hindguts of adult worker bees using a broth enrichment method [27]. Briefly, the hindgut was sampled and soaked in 3% (v/v) hydrogen peroxide for 1 min to surface-sterilize the samples, then suspended in de Man, Rogosa, and Sharpe (MRS) broth for 48 h. The samples were transferred onto MRS agar using the streak plate technique to isolate putative LABs colonies. After 48 h of incubation at 30 °C, a single colony was selected and subcultured in the MRS broth at 30 °C for 24 h. The isolates were characterized by biochemical properties and identified by using the 16S rDNA and stored at −20 °C until further use.

2.2. Primary Screening and Molecular Identification of Lactic Acid Bacteria

The morphology of the isolated bacteria was examined by Gram staining, and a catalase test was performed for primary screening of LABs [28]. Briefly, a single colony from MRS agar was fixed on a glass slide and stained with crystal violet for 1 min, then mordanted with iodine solution for 1 min, followed by treatment with ethanol as a decolorizer. Lastly, the slide was stained with safranin O for 30 s and washed with distilled water. The slide was viewed under a light microscope. The catalase test was performed by dropping 3% (v/v) hydrogen peroxide onto a single colony culture on a glass slide. The slide was observed for the formation of bubbles, which indicated a positive catalase reaction [29]. For molecular identification of LABs, bacterial DNA was isolated using the DNA purification kit according to the manufacturer’s instructions. Subsequently, polymerase chain reaction (PCR) was carried out to amplify the 16S rRNA sequencing with overlapping regions, employing two set of primers 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3 and 907R 5′ (CCG TCA ATT CMT TTR AGT TT) 3′, as well as 785F 5′ (GGA TTA GAT ACC CTG GTA) 3′ and 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′. The PCR was conducted under the following thermal conditions: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 20 sec, and extension at 72 °C for 1 min; with a final extension at 72 °C for 5 min. Each PCR mixture consisted of 10X PCR buffer, 0.1 µM of each primer pair, 10 mM dNTPs, 25ng of DNA, and 0.25 µL. All amplified 16S rRNA fragments were sequenced according to the manufacturer’s instructions, and the sequences were compared with data obtained from the GenBank database for the classification of the LAB strains. Multiple sequence alignment was performed using MEGA 11 software employing the neighbor-joining method. Bootstrapping was performed with 1000 replicates, and evolutionary distances were computed using the Kimura 2-parameter method [30].

2.3. Characterization of Lactic Acid Bacteria Under the Stress Conditions

To determine the survival of LABs in sucrose syrups, the concentration of sucrose syrups was simulated based on the concentrations used in beekeeping. Firstly, a single colony of LABs was grown in modified MRS for 12 h at 30 °C. The bacterial pellet was then collected by centrifugation (5000× g for 15 min at 4 °C) and washed twice with 0.85% NaCl. The bacterial cells were resuspended in 0.85% NaCl at a final concentration of 108 CFU/mL. The bacterial suspensions of each strain were mixed with three different sucrose concentrations, including syrup A (50% sucrose, 50% distilled water), syrup B (20% sucrose, 80% distilled water), and syrup C (10% sucrose, 90% distilled water). All syrups were sterilized by autoclave at 110 °C for 20 min. The samples were incubated for 24 and 48 h at 30 °C. After incubation, bacterial viability was evaluated using the drop plate technique. To evaluate pH tolerance properties, a single colony of LAB was inoculated into MRS broth and incubated at 30 °C for 12 h. The overnight culture (OD600 = 0.8–1.0) was transferred into MRS broth adjusted to different pH levels (3, 4, 5, 6, and 7), then incubated at 30 °C for 24 h. The treated cultures were quantified on MRS agar using the drop plate technique and incubated at 30 °C for 24–48 h. The number of viable cells was counted and expressed as (CFU/mL).

2.4. In Vivo Experimental Design and Direct-Fed Microorganism Syrup Preparation

According to the standard procedure for rearing adult worker bees in cages under laboratory conditions by Williams, et al. [31]. Adult worker bees were obtained from healthy colonies by placing capped brood frames in an incubator at 33 °C and 60% humidity for 24 h. Then, 30 newly emerged worker bees were transferred to individual cylindrical plastic cages with a diameter of 9 × 8.3 cm, and beeswax foundation sizes 4 × 4 cm were added to the cages. Air vents and 1.5 cm diameter holes were made on the top of each cage for syringe feeders. A syringe feeder with 5 mL capacity and 3 mm pores was used to provide syrup and water to the bees. Similarly, the maize (Zea mays) pollen patties were prepared by mixing 50% w/v syrup in the ratio of 2:1, and 2 g aliquots were fed to each cage every 3 days. The bee cages were kept in an incubator set at 33 °C and 60% humidity. For the preparation of DFM syrup, LABs were incubated in MRS broth for 24 h. Bacterial cells were harvested by centrifuge (5000× g, 15min, 4 °C) and subsequently washed twice with 0.85% (w/v) NaCl. The concentration of the LABs suspension was adjusted to an optical density (OD600) of 1.000 and mixed with 50% (w/v) sucrose syrup to achieve an initial inoculum of 108 CFU/mL. The adult worker bee in cages were fed with 2.5 g of DFM syrup, while control groups were fed with syrup (Table 1). The freshly prepared DFM syrup and syrup was replaced daily in each cage to maintain consistent feeding. The experiment was performed in 2025, from August to September.

2.5. Measurement of Consumption Rate and Longevity of Worker Bees

The consumption rate and longevity of adult bees were assessed using a caged-bee experiment. Five treatment groups were established, each consisting of three replicate cages, with 30 newly emerged adult bees per cage. Bees were maintained under controlled environmental conditions (33 ± 1 °C and 60 ± 5% relative humidity) to evaluate the efficacy of DFM syrup on bee’s consumption and longevity. The DFM were prepared and fed to the bee as previous described. The remaining diets were weighed, calculated, and presented as cumulative consumption (mg/bee/3 days). The longevity of adult bees in each treatment was daily recorded until all bees died, and the longevity was calculated following the Kaplan–Meier survival analysis.

2.6. Measurement of Worker Bee Hypopharyngeal Gland Development

The hypopharyngeal glands (HPGs) were dissected from five bees per treatment group, sampled from a single cage containing 30 bees, to assess gland development at 3, 6, and 9 days of age. The size measurement method was described by Corby-Harris and Snyder [32]. The worker bees’ heads and bodies were separated, and the heads were transferred into a glass disc. They were then dissected under a stereo microscope (Carl Zeiss AG, Oberkochen, Germany) and imaged with a stereo microscope camera (Leica Microsystems GmbH, Fluorescence Stereo Microscopes Leica M205 FCA and Leica M205 FA, Wetzlar, Germany). The maximum length (L) and width (W) were measured in pixels from 20 randomly selected acini per replicate, with borders clearly in focus. These measurements were then converted to millimeters using a scale bar in image analysis software, ImageJ (version 1.53k, National institutes of Health, Bethesda, MD, USA). The acinar surface area (SA) was calculated using the following formula [33]:
Acinar surface area = π × ((a × b)/2),
where a = maximum length, b = maximum width, and π = 3.14.
The averages of the 100 individual acini measured per five bee heads were used for statistical analysis [34].

2.7. Total Protein in Bee Heads and Digestibility in the Hindgut

The total protein content in bee heads was determined using a modified method based on Chakrabarti, et al. [35]. Briefly, three worker bee heads were dissected from each cage of every treatment group at 6 days post-feeding and pooled to form one sample per replicate cage. The bee heads were homogenized in the 600 µL of 0.25 M Tris-HCl buffer (pH 7.5) in a 1.5 mL microcentrifuge tube. The samples were then centrifuged at 18,000× g for 15 min, and 100 µL of the supernatant was transferred to a new tube for analysis. Similarly, to assess digestibility, the hindgut of the bee was dissected using sterile forceps for the determination of digestibility. The method was described by Kim et al. [36] with slight modification. Each 50 mg of pollen diet and hindgut was weighed, then homogenized in 250 µL 0.25 M Tris-HCl buffer (pH 7.5). Subsequently, centrifuge at 18,000× g for 15 min and transfer 100 µL of supernatant to a 1.5 mL microcentrifuge tube and dilute with 300 µL 0.25 M Tris-HCl buffer. The soluble protein concentration in bee heads, pollen diet, and hindgut was evaluated by Bradford Protein Assay Kit (Himedia Laboratories, Mumbai, India), and the absorbance at 595 nm was measured on a microplate reader (Synergy H1, BioTek instruments, Winooski, VT, USA).
The approximate digestibility (%) was calculated using the equation: Digestibility (%) = [(Protein concentration of diet − Protein concentration in hindgut)/Protein concentration of diet] × 100 [37,38].

2.8. Microbial Community Structure in the Gut of Worker Bees via Metagenomic Analysis

To investigate the effects of supplementing direct-fed microorganisms on the diversity of gut microbiota in worker bees, compared to non-treatment control groups that did not receive probiotics (NTC), and the D0 group. The bee cages were prepared as previously described but separated for specifically Microbiota analysis. Gut samples were collected at 6 days post-feeding from three cages per treatment group, using ten worker bees per replicate. Total DNA was extracted directly from pooled hindgut samples comprising ten individual bees from the same experimental group without prior microbial enrichment. This approach was chosen to preserve the native gut microbial community structure. DNA extraction was performed using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, GmbH, Hilden, Germany) following the manufacturer’s protocol. Briefly, 250 mg of each pooled sample was transferred into the bead-containing tubes supplied with the kit, and the provided extraction buffer was added. Samples were incubated at 70 °C for 20 min and then subjected to mechanical disruption by bead beating. The extracted DNA was assessed for both quantity and quality using spectrophotometric and fluorometric methods. Then, the bacterial 16S rRNA gene was amplified using the 16S Barcoding Kit 24 V14 (SQK-16S114.24, Oxford Nanopore Technologies, Oxford, UK) in combination with the UltraRun LongRange PCR Kit (QIAGEN, GmbH, Hilden, Germany). Barcoded PCR products were purified with AMPure XP beads to eliminate contaminants and non-specific amplification products. DNA concentrations were subsequently quantified using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) to ensure optimal input for sequencing library preparation. Sequencing adapters were ligated to the purified amplicons following the manufacturer’s protocol. Sequencing was carried out on the MinION Mk1D platform using a MinION flow cell (Oxford Nanopore Technologies Ltd, Oxford, UK). The sequencing run was managed and monitored in real time using the MinKNOW™ software suite (version 25.05.14, Oxford Nanopore Technologies Ltd, Oxford, UK. Taxonomic classification and bacterial community profiling were performed using the EPI2ME 16S workflow (https://epi2me.nanoporetech.com/workflows/wf-16s/; accessed 6 September 2025). Downstream analysis, including microbial diversity assessment and statistical comparisons, was conducted using MicrobiomeAnalyst (https://www.microbiomeanalyst.ca/; accessed October 2025).

2.9. Statistical Analysis

Statistical analyses were performed using IBM® SPSS® Statistics version 24. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was applied to compare the syrup and pH tolerance properties of LABs, effects of direct-fed microorganism syrup feeding on consumption rate, hypopharyngeal gland size development, protein concentration in bee head and percentage of digestibility in hindgut. Kaplan–Meier survival analysis and log-rank test were used to determine the effect of direct-fed microorganism syrup on the longevity of honey bees. For all comparisons, the level of significance level was set at p < 0.05. Micriobial community analyses were conducted using QIIME2 (version 2024.2, Knight Lab, University of California San Diego, CA, USA) and R studio software (version 4.5.1, Posit PBC, Boston, MA, USA). Alpha diversity were performed using one-way ANOVA, with significant determined at p < 0.05. Pairwise analyses were further performed, with significance set at p < 0.001. Beta diversity was assessed using Bray–Curtis dissimilarity, and principal coordinates analysis (PCoA) was applied to visualize differences in microbial community structure among dietary treatments. Statistical significance of microbial community composition differences was tested using permutational multivariate analysis of variance (PERMAOVA).

3. Results

3.1. Identification of Lactic Acid Bacteria

The three isolates distinct from LABs in the gastrointestinal tract of honey bees were characterized and identified. All of the bacteria exhibited cream or white spherical colonies with a small diameter ranging from 0.1 to 0.4 mm. When tested for catalase enzyme production by applying 3% H2O2 onto the colonies to observe bubble formation, all isolates showed a negative catalase reaction. Furthermore, Gram staining revealed that all bacteria were Gram-positive, with the majority exhibiting a rod-shaped morphology. The isolates were subjected to 16S rRNA sequencing. The nucleotide sequences were subject to nucleotide BLAST (version 2.13.0, National Center for Biotechnology Infornation, Bethesda, MD, USA). The analysis revealed that the isolated bacteria were classified into family: Lactobacillaceae, characterized by their distinct cell shapes, which were rod-shaped. The isolates were categorized into the following genera: Apilactobacillus, Lactiplantibacillus, and Levilactobacillus. On the basis of molecular identification results, we identified LAB species as Apilactobacillus kunkeei with 99.80% identities (MBG-01 AK), Lactiplantibacillus sp. with 99.93% identities (MBG-02 LP), and Lactobacillus brevis with 99.79% identities (MBG-03 LB), which are notable species found in the honey bee gut. (Figure 1). The three LAB isolates were selected and characterized for their sucrose syrup tolerance and pH tolerance.

3.2. Stress Tolerance Characterization of Lactic Acid Bacteria

To explore the potential use of probiotics in honey bees, it is essential to assess the survival of isolated strains after exposure to application methods and conditions within the bee gut. Providing probiotics to the worker bee by mixing them into sucrose syrup is the most convenient. Hence, in this study, LABs isolated from the bee gut were tested in different sucrose syrup concentrations, including 50, 20, and 10% w/w. The high osmotic pressure in 50% w/w sucrose syrup rapidly reduces the cell viability of AK and LP by nearly 99.9% (5.05 and 5.22 log CFU/mL respectively), LB exhibits more resistance, with a decrease of 98.56 ± 0.89% (6.42 log CFU/mL) after 24 h of incubation, whereas at lower syrup concentrations, cell viability was maintained at higher levels, ranging from 6 to 7 log CFU/mL (Table 2). These osmophilic properties of LABs would be useful for further application in bee colonies. In the bee gut environment, a combination of enzymes and low pH gastrointestinal juice creates a unique habitat for probiotics, while also being harmful to various microorganisms. To confirm the viability of LABs under low pH conditions, in vitro pH tolerance testing is essential. The results revealed that A. kunkeei is very sensitive to low pH conditions. Cell viability was reduced by more than 1 log CFU/mL after incubation in MRS broth containing a pH lower than 6. On the other hand, Lactiplantibacillus sp. and L. brevis were able to maintain cell viability at 9 log CFU/mL at pH 4 after 24 h of incubation (Table 3). These results suggest that Lactiplantibacillus sp. and L. brevis can efficiently survive in the hindgut of honey bees.

3.3. Effect of Direct-Fed Microorganism on Consumption Rate and Longevity of Adult Bees

In the initial phase of experiment, the consumption of adult bees for both syrup and pollen showed no significant differences among the treatments. The result revealed that in the first 3–6 days, syrup intake by bees was lower than the later stages, whereas pollen intake was higher during the early period, consistent with the age-related development of the adult bees (Table 4).
The survival analysis revealed comparable mean and median longevity values across treatment groups, with overlapping 95% confidence intervals. No statistically significant differences were observed (p = 0.374) (Table 4). The bees cage fed with LB had the longest average longevity of 26.04 ± 1.88 days. This was followed by NTC, LP, and AK, with average lifespans of 25.16 ± 2.02, 25.16 ± 1.98, and 25.03 ± 1.97 days, respectively. In contrast, the negative control group fed only syrup (NEG) had a shortened lifespan of 22.77 ± 1.81 days (Table 5 and Figure 2).

3.4. Effect of Direct-Fed Microorganism on Hypopharyngeal Gland (HPG) Development

The results indicated that the size of HPGs tended to increase with age and nutritional status, with the peak of development observed on day 6 after adult emergence. The bee cages fed with pollen and syrup inoculated with direct-fed microorganism exhibited acini surface areas ranging from 0.020 to 0.023 mm2, showing a significant difference compared to the control group fed only sucrose syrup (S), which ranged from 0.013 to 0.018 mm2. The bee cages supplemented with syrup and L. brevis (LB) demonstrated enhanced HPGs development, with an average acinus size of 0.027 ± 0.007 mm2 on day 6, whereas the non-treatment control group (NTC) averaged 0.023 ± 0.006 mm2. Significant differences in size were maintained throughout day 9 when compared to the control group (Figure 3). The other cages, supplemented with A. kunkeei (AK) and Lactiplantibacillus sp. (LP), showed HPG development comparable to the negative control group (NEG) only at 6 days. These findings suggest that direct-fed microorganism, particularly L. brevis, positively influence the development of HPGs.

3.5. Effect of Direct-Fed Microorganism on Total Protein in the Bee Head and Digestibility in the Hindgut

The total protein in the bee head indicated that the direct-fed microorganism syrup group had a potential effect in increasing soluble protein levels in the bee head in the range from 686.68 to 733.25 µg/bee after 6 days of feeding (Figure 4A), while the non-treatment control (NTC) and negative control (NEG) had 642.84 and 497.08 µg/mL, respectively. Furthermore, the digestibility in the hindgut supports this interpretation, as all direct-fed microorganisms in the syrup group had a higher percentage of digestibility in the range of 83.30–87.75% compared to the non-treatment control (NTC) had 80.25% (Figure 4B). This result indicated that direct-fed microorganism syrup, particularly L. brevis (LB), increased the protein synthesis in the bee head, which is closely linked to royal jelly production in adult worker bees. Additionally, it improved the efficiency of nutrient breakdown and absorption, contributing to overall health and productivity.

3.6. Effect of Direct-Fed Microorganism on Gut Microbiota Alterations

To explore whether the physiological improvements observed in probiotic-supplemented bees were associated with changes in the gut microbial environment, we characterized the hindgut bacterial communities using 16S rRNA gene metagenomic sequencing. Across different species of direct-fed organism syrup, both richness and evenness varied based on dietary supplementation. Consistent with an immature microbial community, the newly emerged bees (D0) exhibited the lowest richness and reduced evenness. Lactobacillus was the most dominant bacteria in all treatment groups. Among treatment groups, the negative control (NEG) had the lowest abundance of Lactobacillus and displayed moderate richness and relatively high evenness. The balanced communities were dominated by core bacteria such as Frischella, Snodgrassella, and Commensalibacter. Additionally, these groups tended to have more higher abundances of genera that include opportunistic or potentially pathogenic species, such as Salmonella, Klebsiella, and Escherichia and an imbalanced microbial community. In the non-treatment control group (NTC), Lactobacillus was found to be the most dominant taxa following by Commensalibacter, Snodgrassella and Gilliamella. While supplementation with AK and LB increased the taxa richness in these microorganism-fed groups, LP fed group demonstrated an increase in core gut microbial taxa such as Frischella and Bombilactobacillus and suppressed the presence of pathogenic taxa, including Escherichia, Klebsiella and Salmonella (Figure 5). Overall, probiotic supplementation influenced gut microbial community complexity, enabling them to be distinct from the syrup controls. In alpha-diversity, statistical comparisons revealed significant differences in richness (Chao1, p = 0.030), but not when considering evenness altogether (Shannon, p = 0.164). Among different treatments, LP achieved the most balanced profile (Figure 6A). In beta-diversity analysis, principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarity illustrated differences in gut microbial community structure among the same dietary treatments (Figure 6B). Community composition differed significantly among groups (PERMANOVA, p = 0.003), with probiotic-supplemented groups (LP and LB) clustering distinctly from the syrup-only control and the newly emerged bees (D0).

4. Discussion

Probiotic microorganisms refer to the group of microorganisms that are consumed by the host in appropriate amounts and confer health benefits [39]. The application of probiotics in honey bees to enhance bee health has recently become increasingly popular. Understanding the characterization and identification of probiotics isolated from the honey bee gut is necessary for further investigations into their functionality in improving bee health. Studies on the gut microbiota of honey bees have shown that the diversity and quantity of probiotics vary across different stages of development and maturation into adult bees [12]. The roles of gut microbiota in honey bees include providing protection against various pathogens and contributing to the processing of refractory components of the pollen coat and dietary toxins. When the gut microbiota is absent or disrupted, it leads to changes in the expression of genes related to immunity, metabolism, behavior, and development [40]. Currently, disruptions in the balance of gut microbiota are caused by crop monocultures, which have poor pollen quality, and the use of agrochemicals that adversely affect honey bees [37]. Hence, feeding honey bees with potential probiotic strains is a good strategy to maintain the health of bee colonies under stressful conditions. To assess the effective use of probiotic supplements in honey bee feed, it is essential to evaluate the viability of isolated strains after exposure to the application method and the conditions within the bee gut, such as high osmotic pressure in sucrose syrup and the mildly acidic environment [41]. High concentrations of sucrose syrup induce osmotic stress in bacterial cells and disrupt both the cell membrane and proteins, leading to rapid cell lysis in many bacteria. The direct-fed microorganisms’ cell viability was reduced by sucrose syrup more than 90% within 96 h at 30 °C [42]. Although the selected strain in this study, including A. kunkeei, Lactiplantibacillus sp., and L. brevis, had reduced viability, these strains survived in a 50% sugar syrup within 48 h, which was used for honey bee feeding. After the probiotics were incorporated into the bee gut, it should resist exposure to gastrointestinal juices, as the pH value in the midgut and hindgut is approximately 6.0 and 5.2, respectively [43]. Based on the in vitro pH tolerance of selected strains, it can be preliminarily confirmed that they efficiently survive in the bee hindgut. The bee cage experiments indicated that bees fed pollen and syrup supplemented with L. brevis had a longer longevity compared with a group fed only syrup. There are several studies that have proven that probiotics not only produce an antimicrobial compound that inhibits the proliferation and survival of pathogens [17,24,44]. But they are also used for enhancing the bee health by boosting their immune system and leading to resistance to bee diseases [45]. The development of essential organs in adult bees is influenced by both the received diet and the health status of bees. The bee health could be improved by supplementing with probiotics and prebiotics [46]. The HPG is crucial for the honey bee health status and royal jelly production. Generally, a larger gland size indicates a better health and a greater capacity for royal jelly secretion [32,47]. We observed that all the caged bees treated with maize pollen, with or without microorganism supplementation, exhibited a significantly larger acini size compared to the control group that fed only sucrose syrup. This finding suggests that even low-quality pollen provides essential protein and nutrients that support HPG development more effectively than a carbohydrate-only diet. However, the acini enlargement induced by maize pollen remained inferior when compared to the bee fed by mixed pollen or high-quality pollen such as Brassica napus, Rhus chinensis, which promote superior gland growth and enhance genes associated with royal jelly production [48]. In subsequent analyses, among pollen-fed groups, the caged bees that received syrup supplemented with L. brevis demonstrated the highest enhancement of HPGs development on day 6 and 9. This finding highlights the synergistic role of pollen nutrition and probiotic feeding in enhancing glandular activity. Similar observations have been reported previously, where worker bees fed with syrup and artificial pollen supplemented with probiotics isolated from the bee gut, including Enterococcus faecalis, L. brevis, and L. casei, show enlarged HPGs and increased royal jelly production compared to control groups without probiotic supplementation. Additionally, our finding suggests that DFM syrup can modulate protein synthesis in the bee head, which secretions by the hypopharyngeal and mandibular glands of the nurse bees also reflect. Our results indicate that probiotic feeding enhances glandular activity by day 6, supporting the bees’ ability to produce essential food for larvae and increase colony population growth. Previous study of syrup supplement with Lactobacillus rhamnosus and their possible metabolites (organic acids, e.g., lactic acid and acetic acid) has been shown to promote bee growth, improve digestibility, and enhance overall health [49]. Moreover, probiotic mixtures containing L. plantarum, L. rhamnosus, and A. kunkeei can also be applied in syrup via spraying, which delivers probiotics directly to bees on the comb. This method has been reported to strengthen immune responses, modulate gut microbiota, reduce pathogen loads, and improve brood development, colony size, and overall productivity in beekeeping [50]. Metagenomic profiling further supports these findings, showing that diets enriched with beneficial fermenters, such as pollen or probiotic supplementation, are associated with higher microbial richness and balanced Bacillota–Pseudomonadota ratios. Shannon diversity was greatest in one of the L. plantarum-supplemented groups, followed by the L. brevis-supplemented groups, while sucrose controls exhibited lower values. Newly emerged bees had the lowest diversity indices, confirming that hindgut colonization requires dietary input. Together, these results demonstrate that direct-fed microorganism syrup promotes richer and more evenly distributed microbial communities compared with non-supplemented bees.
The colonization of gut microbiota, particularly Lactobacillus Firm-5, has been reported to be associated with an increased level of metabolites in adult bee brain and transcriptional process related to amino acid metabolism and biosynthesis. These changes may reflex to sensory perception or colony social network of honeybees [51]. At the transcriptional level, probiotics in hindgut have been shown to regulate the expression of brain genes associated with royal jelly protein (MRJP1, MRJP2, MRJP7) and learning behavior. This highlights a mechanistic link between gut microbial metabolism, gene regulation in the bee brain, and cognitive performance [52]. In addition, there are benefits of probiotics on host health, primarily through their metabolic activities. One of the most important mechanisms involves the production of organic acid, which directly influences the gut environment and microbial community structure [53,54]. Homofermentative bacteria, such as Lactiplantibacillus sp., metabolize carbohydrates predominantly into lactic acid. This process results in a strong acidification of the gut environment, lowering the pH to levels that are unfavorable for the growth of opportunistic and pathogen microorganisms. By creating this condition, LP effectively inhibits the colonization of harmful taxa and enhances the advantage of beneficial of lactic acid bacteria. The acidification also strengthens gut barrier function, further protecting the host against microbial invasion [55,56]. On the other hand, heterofermentative bacteria such as A. kunkeei and L. brevis produce a wider range of organic acid, including lactic acid, acetic acid, succinic acid, and ethanol. These metabolic activities maintain a moderately acidic to near-neutral pH, which supports greater microbial diversity within the bee gut ecosystem [21]. While this diversity may include opportunistic or pathogen bacteria, our study also detected bacteria such as Salmonella, Escherichia and Klebsiella in the bee gut, particularly in non-pollen-fed groups. These genera have occasionally been reported in honey bees from rural environments and may originate from environmental contamination or secondary colonization [57,58]. The bees used in this study were sourced from colonies at Chiang Mai University, which is situated in a peri-urban setting that integrates urban communities with close proximity to natural environments. Therefore, the presence of these bacteria is not unexpected. Importantly, their abundance was markedly reduced in pollen-fed groups, suggesting that dietary enrichment with pollen and probiotics promotes a more balanced microbiota capable of suppressing potentially harmful taxa.
Therefore, direct-fed microorganism syrup can be used as a tool to induce changes in gut microbial composition, particularly by increasing beneficial bacteria, which may affect the health and performance of bees in the long term. The use of probiotics is thus a promising strategy for the sustainable development of the beekeeping industry. Particularly, direct-fed microorganism syrup containing the potential strain Lactiplantibacillus sp. and L. brevis could be used to enhance the overall health of worker bees. It would help the bees to maintain the colony strength in poor environments and could also serve as a foundational strategy to mitigate the negative impacts of environmental stressors like pesticide exposure. However, this study has limitations; future research should investigate the long-term effects of probiotic supplements and assess the efficacy of these strains in field settings with larger colony sizes. Additionally, exploring the specific metabolic pathways and genetic expression changes induced by these probiotics would provide a more comprehensive understanding of their beneficial mechanisms.

5. Conclusions

The use of direct-fed microorganism syrup, particularly with strains such as Lactiplantibacillus sp. and L. brevis were associated with enhanced HPG development, head protein content, hindgut protein digestibility, and altered gut microbiota profiles without compromising worker bee longevity. These strains favored beneficial taxa like Lactobacillus while suppressing potential contaminants such as Escherichia, Salmonella, and Shigella. This approach is especially beneficial in poor environmental conditions where traditional food sources may be lacking.
These results highlight promising applications of direct-fed microorganisms syrup in apiculture; further field-based studies are required to validate their long-term impact on colony health and sustainability.

Author Contributions

Conceptualization, P.P., K.D. and B.C.; methodology, P.P., K.D., T.P., S.M.N. and S.G.; software, T.P.; validation, C.J. and B.C.; investigation, P.P., K.D. and T.P.; data curation, P.P.; writing—original draft preparation, P.P., K.D., S.M.N. and B.C.; writing—review and editing, P.P., K.D., S.G., C.J., T.P., S.M.N. and B.C.; visualization, C.J.; supervision, C.J.; funding acquisition, K.D., C.J. and B.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Agricultural Research Development Agency (Public Organization) grant no. CRP6805031090, partially supported by Chiang Mai University and Proactive Researcher Program, Chiang Mai University. Thailand Science Research and Innovation Fund and the University of Phayao (grant no. 2275/2568) and the National Research Foundation of Korea, NRF (grant no. 2018R1A6A1A03024862).

Institutional Review Board Statement

All experimental procedures involving honey bees were conducted in accordance with the ethical guidelines for animal research at Chiang Mai University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Agriculture, Chiang Mai University (Approval No. RAGIACUC021/2568).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data/findings of the study are available upon request to the corresponding authors.

Acknowledgments

We would like to express our gratitude to Thanchanok Auearchin and Supakhom Klaitanoad, Research assistant from Meliponini and Apini Research Laboratory, Faculty of Agriculture, Chiang Mai University, for their assistance with laboratory work. We extend our thanks to the researchers from Gyeongkuk National University, Republic of Korea, for their support in this research.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DFMDirect-Fed Microorganisms
HPGsHypopharyngeal glands
16S rRNA16S Ribosomal Ribonucleic Acid
CFU/mLColony Forming Units per Milliliter
dNTPsDeoxynucleotide Triphosphates
H2O2Hydrogen Peroxide
LABsLactic Acid Bacteria
MRJP1Major Royal Jelly Protein 1
MRSde Man, Rogosa, and Sharpe medium
OD600Optical Density at 600 nm
PCoAPrincipal Coordinates Analysis
PERMANOVAPermutational Multivariate Analysis of Variance

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Animals 16 00704 g001
Figure 1. Phylogenetic tree of lactic acid bacteria isolated from the honey bee gut. The phylogenetic tree was constructed by applying the neighbor-joining method and tested by bootstrapping with 1000 replicates of data. Percentages are reported at nodes, and the scale bar represents 0.02% sequence divergence. The isolated of LABs strain is labeled as a MBG-01 AK, MBG-02 LP and MBG-03 LB. Sequences for the 16S rRNA phylogenetic analysis were obtained from the GenBank database for the following strains: Apilactobacillus kunkeei, Apilactobacillus ozensis, Lacticaseibacillus saniviri, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus plantarum, Lactobacillus brevis, Lactobacillus koreensis, Lentilactobacillus curieae, Lentilactobacillus rapi, Levilactobacillus hammesii, Levilactobacillus parabrevis, and Escherichia coli was used as an outgroup organism.
Figure 1. Phylogenetic tree of lactic acid bacteria isolated from the honey bee gut. The phylogenetic tree was constructed by applying the neighbor-joining method and tested by bootstrapping with 1000 replicates of data. Percentages are reported at nodes, and the scale bar represents 0.02% sequence divergence. The isolated of LABs strain is labeled as a MBG-01 AK, MBG-02 LP and MBG-03 LB. Sequences for the 16S rRNA phylogenetic analysis were obtained from the GenBank database for the following strains: Apilactobacillus kunkeei, Apilactobacillus ozensis, Lacticaseibacillus saniviri, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus plantarum, Lactobacillus brevis, Lactobacillus koreensis, Lentilactobacillus curieae, Lentilactobacillus rapi, Levilactobacillus hammesii, Levilactobacillus parabrevis, and Escherichia coli was used as an outgroup organism.
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Animals 16 00704 g002
Figure 2. Effect of direct-fed microorganism syrup feeding on longevity of adult bees. The Kaplan–Meier curve was adopted for comparisons between groups.
Figure 2. Effect of direct-fed microorganism syrup feeding on longevity of adult bees. The Kaplan–Meier curve was adopted for comparisons between groups.
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Figure 3. Effects of direct-fed microorganism syrup on hypopharyngeal gland size (A) and development of HPGs morphology (B) on days 3, 6, and 9. Different letters and symbols in box-plot graph indicate statistically significant differences between treatments and feeding times by Duncan’s Multiple Range Test (p < 0.05).
Figure 3. Effects of direct-fed microorganism syrup on hypopharyngeal gland size (A) and development of HPGs morphology (B) on days 3, 6, and 9. Different letters and symbols in box-plot graph indicate statistically significant differences between treatments and feeding times by Duncan’s Multiple Range Test (p < 0.05).
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Figure 4. Protein concentration in bee head (A) and percentage of digestibility in hindgut (B) when treated by direct-fed microorganism syrup for 6 days. Different letters in bar graph indicate statistically significant differences by Duncan’s Multiple Range Test. (p < 0.05).
Figure 4. Protein concentration in bee head (A) and percentage of digestibility in hindgut (B) when treated by direct-fed microorganism syrup for 6 days. Different letters in bar graph indicate statistically significant differences by Duncan’s Multiple Range Test. (p < 0.05).
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Figure 5. Comparison of the effects of probiotic supplementation on the hindgut micro biome of worker bees. The average of relative abundance of the top 10 most abundant bacterial genera is shown at the genus level. Samples include newly emerged bees (D0) and bees at day 6 fed with different supplementary diets: A. kunkeei (AK), Lactiplantibacillus sp. (LP), L. brevis (LB), non-treatment control (NTC), and negative control (NEG). Genera are grouped into (i) core bacteria (Apilactobacillus, Lactobacillus, Gilliamella, Frischella, and Snodgrassella), (ii) non-core bacteria (Commensalibacter and Bombilactobacillus), and (iii) potential contaminants/pathogens (Escherichia, Salmonella, and Shigella). This comparison highlights the shifts in gut microbiota composition from newly emerged bees to day 6 workers under different probiotic treatments.
Figure 5. Comparison of the effects of probiotic supplementation on the hindgut micro biome of worker bees. The average of relative abundance of the top 10 most abundant bacterial genera is shown at the genus level. Samples include newly emerged bees (D0) and bees at day 6 fed with different supplementary diets: A. kunkeei (AK), Lactiplantibacillus sp. (LP), L. brevis (LB), non-treatment control (NTC), and negative control (NEG). Genera are grouped into (i) core bacteria (Apilactobacillus, Lactobacillus, Gilliamella, Frischella, and Snodgrassella), (ii) non-core bacteria (Commensalibacter and Bombilactobacillus), and (iii) potential contaminants/pathogens (Escherichia, Salmonella, and Shigella). This comparison highlights the shifts in gut microbiota composition from newly emerged bees to day 6 workers under different probiotic treatments.
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Figure 6. Effects of direct-fed microorganism supplementation on honey bee hindgut microbiota diversity. Alpha-diversity indices (Chao1 richness and Shannon diversity) of worker bee gut microbiota across with different supplementary diets: newly emerged bees (D0), A. kunkeei (AK), Lactiplantibacillus sp. (LP), L. brevis (LB), non-treatment control (NTC), and negative control (NEG). Boxes represent interquartile ranges, horizontal lines indicate medians, whiskers denote range, and black diamonds show means. Statistical comparisons revealed significant differences in richness (Chao1, p = 0.026), but not in diversity (Shannon, p = 0.164) *** indicates significance in pairwise comparison (p < 0.001) (A). Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarity, illustrating differences in gut microbial community structure among the same dietary treatments. Each point represents an individual sample, colored according to treatment. Community composition differed significantly among groups (PERMANOVA, p = 0.003), with probiotic-supplemented groups Lactiplantibacillus sp. (LP) and L. brevis (LB) clustering distinctly from the syrup-only control (B).
Figure 6. Effects of direct-fed microorganism supplementation on honey bee hindgut microbiota diversity. Alpha-diversity indices (Chao1 richness and Shannon diversity) of worker bee gut microbiota across with different supplementary diets: newly emerged bees (D0), A. kunkeei (AK), Lactiplantibacillus sp. (LP), L. brevis (LB), non-treatment control (NTC), and negative control (NEG). Boxes represent interquartile ranges, horizontal lines indicate medians, whiskers denote range, and black diamonds show means. Statistical comparisons revealed significant differences in richness (Chao1, p = 0.026), but not in diversity (Shannon, p = 0.164) *** indicates significance in pairwise comparison (p < 0.001) (A). Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarity, illustrating differences in gut microbial community structure among the same dietary treatments. Each point represents an individual sample, colored according to treatment. Community composition differed significantly among groups (PERMANOVA, p = 0.003), with probiotic-supplemented groups Lactiplantibacillus sp. (LP) and L. brevis (LB) clustering distinctly from the syrup-only control (B).
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Table 1. Details of carbohydrate and protein dietary administration to honey bees in different experimental groups.
Table 1. Details of carbohydrate and protein dietary administration to honey bees in different experimental groups.
ExperimentsBee Feeding
CarbohydrateProtein
AKSucrose Syrup + A. kunkeeiPollen patty
LPSucrose Syrup + Lactiplantibacillus sp.Pollen patty
LBSucrose Syrup + L. brevisPollen patty
NTCSucrose SyrupPollen patty
NEGSucrose Syrup-
Table 2. Syrup tolerance properties of LABs when treated with sucrose syrup in different concentrations.
Table 2. Syrup tolerance properties of LABs when treated with sucrose syrup in different concentrations.
Syrup
Concentration (w/w)		Incubation
Time (h)		Cell Viability (Log CFU/mL)
AKLPLB
50%07.64 ± 0.42 a7.85 ± 0.21 a8.32 ± 0.42 a
245.05 ± 0.36 b5.22 ± 0.44 b6.42 ± 0.36 b
484.28 ± 0.15 c4.28 ± 0.20 c4.79 ± 0.15 c
20%08.28 ± 0.11 a8.12 ± 0.16 a8.34 ± 0.09 a
247.58 ± 0.14 b7.48 ± 0.20 b7.57 ± 0.14 b
487.47 ± 0.24 b7.35 ± 0.17 b7.39 ± 0.28 b
10%08.12 ± 0.14 a8.21 ± 0.18 a8.38 ± 0.08 a
247.21 ± 0.24 b7.51 ± 0.06 b7.89 ± 0.13 b
486.05 ± 0.20 c7.34 ± 0.15 c7.48 ± 0.04 c
Note: Each value in the table represents the mean ± standard deviation (SD) from three replications. Different letters in column indicate statistically significant differences by Duncan’s Multiple Range Test. (p < 0.05).
Table 3. pH tolerance properties of LABs when treated with culture media (MRS broth) at different pH levels.
Table 3. pH tolerance properties of LABs when treated with culture media (MRS broth) at different pH levels.
MRS Broth at
Different pH Level		Cell Viability After 24 h of Incubation
(Log CFU/mL)
AKLPLB
MRS broth pH 7.09.05 ± 0.03 a9.49 ± 0.07 a9.45 ± 0.06 a
MRS broth pH 6.08.93 ± 0.09 b9.46 ± 0.02 a9.52 ± 0.02 a
MRS broth pH 5.08.09 ± 0.08 c9.37 ± 0.03 b9.40 ± 0.05 a
MRS broth pH 4.07.11 ± 0.06 d9.09 ± 0.01 c9.04 ± 0.15 b
MRS broth pH 3.0No growth7.60 ± 0.01 d7.42 ± 0.14 c
Note: Each value in the table represents the mean ± standard deviation (SD) from three replications. Different letters in column indicate statistically significant differences by Duncan’s Multiple Range Test. (p < 0.05).
Table 4. The cumulative consumption of pollen and syrup by adult bees fed with different direct-fed microorganisms.
Table 4. The cumulative consumption of pollen and syrup by adult bees fed with different direct-fed microorganisms.
ExperimentsCumulative Consumption of Syrup (mg/Bee/3 Days)
Day 3Day 9Day 12
AK61.66 ± 13.04 a87.03 ± 9.92 a126.93 ± 28.8 a
LP60.72 ± 16.67 a70.86 ± 9.39 a111.74 ± 11.64 ab
LB58.07 ± 6.96 a71.06 ± 11.27 a97.11 ± 8.50 ab
NTC51.53 ± 5.19 a63.42 ± 18.15 a69.12 ± 28.10 b
NEG45.17 ± 2.06 a62.37 ± 19.81 a67.53 ± 24.02 b
Cumulative consumption of pollen (mg/bee/3 days)
AK16.40 ± 1.31 a10.18 ± 1.11 a4.94 ± 1.81 a
LP12.92 ± 7.14 a8.28 ± 3.63 a4.64 ± 2.82 a
LB19.22 ± 5.93 a9.42 ± 7.48 a4.16 ± 2.26 a
NTC19.22 ± 4.34 a8.63 ± 2.00 a2.83 ± 0.32 a
Note: Each value in the table represents the mean ± standard deviation (SD) from three replications. Different letters in column indicate statistically significant differences by Duncan’s Multiple Range Test. (p < 0.05).
Table 5. Median longevity and 95% confidence interval of adult bee fed with different direct-fed microorganism supplements syrup.
Table 5. Median longevity and 95% confidence interval of adult bee fed with different direct-fed microorganism supplements syrup.
Experiments95% Lower BoundMean
Longevity
(Days)		95% Upper Bound95% Lower BoundMedian Longevity
(Days)		95%
Upper Bound
AK21.1825.03 ± 1.97 a28.8916.93824.00 ± 3.60 a31.062
LP21.2725.16 ± 1.98 a29.0516.47125.00 ± 4.35 a33.529
LB22.3626.04 ± 1.88 a29.7220.32926.00 ± 2.89 a31.671
NTC21.2125.16 ± 2.02 a29.1117.92326.00 ± 4.12 a34.077
NEG19.2222.77 ± 1.81 a26.3217.60925.00 ± 3.77 a32.391
Note: Different letters indicate statistically significant differences by Kaplan–Meier survival analysis and log-rank test (p < 0.05).
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MDPI and ACS Style

Praphawilai, P.; Danmek, K.; Pisithkul, T.; Namin, S.M.; Ghosh, S.; Jung, C.; Chuttong, B. Impact of Direct-Fed Microorganism Syrup on Honey Bee (Apis mellifera L.) Hypopharyngeal Gland Development, Protein Digestibility and Gut Microbiota Composition. Animals 2026, 16, 704. https://doi.org/10.3390/ani16050704

AMA Style

Praphawilai P, Danmek K, Pisithkul T, Namin SM, Ghosh S, Jung C, Chuttong B. Impact of Direct-Fed Microorganism Syrup on Honey Bee (Apis mellifera L.) Hypopharyngeal Gland Development, Protein Digestibility and Gut Microbiota Composition. Animals. 2026; 16(5):704. https://doi.org/10.3390/ani16050704

Chicago/Turabian Style

Praphawilai, Pichet, Khanchai Danmek, Tippapha Pisithkul, Saeed Mohamadzade Namin, Sampat Ghosh, Chuleui Jung, and Bajaree Chuttong. 2026. "Impact of Direct-Fed Microorganism Syrup on Honey Bee (Apis mellifera L.) Hypopharyngeal Gland Development, Protein Digestibility and Gut Microbiota Composition" Animals 16, no. 5: 704. https://doi.org/10.3390/ani16050704

APA Style

Praphawilai, P., Danmek, K., Pisithkul, T., Namin, S. M., Ghosh, S., Jung, C., & Chuttong, B. (2026). Impact of Direct-Fed Microorganism Syrup on Honey Bee (Apis mellifera L.) Hypopharyngeal Gland Development, Protein Digestibility and Gut Microbiota Composition. Animals, 16(5), 704. https://doi.org/10.3390/ani16050704

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