Effects of Queen Rearing Technology of Apis cerana by Cutting Comb on Reproductive Capacity and Productive Performance

Simple Summary

This study focused on Apis cerana to address the challenges associated with traditional artificial queen rearing methods, including the difficulty of larval grafting. Additionally, it aimed to provide a theoretical basis for the broader application of queen rearing using comb-cutting techniques. It compared the effects of two queen-rearing technologies—comb cutting and larval grafting—on queen and colony performance. The key measurements included egg morphological indicators (length, width, and weight), number of capped brood cells, worker morphological traits (forewing length, hindwing width, and tergite length), honey sac weight, and colony foraging efficiency. Results showed that queens from the comb-cutting group had superior egg quality (greater length and heavier weight) and more capped brood cells (p < 0.05). Their offspring also exhibited improved morphological traits and high daily colony foraging rates (p < 0.05), with no significant difference in forager honey sac weight between groups (p > 0.05). In conclusion, queen rearing by cutting the comb effectively enhanced the reproductive and productive performance of A. cerana colonies. This approach represents a promising method for rearing high-quality queens and holds substantial value for optimizing beekeeping practices and ensuring sustainable colony development.

Abstract

The queen, as the reproductive core of a honeybee colony, has declining reproductive capacity with age, making it necessary to rear new queens to replace older ones. Traditional artificial queen-rearing methods face challenges, such as difficulties in larval grafting, particularly for Apis cerana. To address these issues, we developed a queen-rearing technology by cutting the comb. This study compared queen-rearing technology using comb cutting (CC) with larval grafting in A. cerana, measuring egg traits (length, width, weight), capped brood number, worker offspring initial weight, forager honey sac weight, worker morphology traits, and colony foraging efficiency. Queens reared using comb-cutting technology exhibited superior egg quality compared with those reared by larval grafting. The CC group showed significant improvements in egg length, egg weight, and number of capped brood cells (p < 0.05). Worker offspring from the CC group demonstrated significantly superior morphological traits—including forewing length, hindwing width, and lengths of the third and fourth tergites—as well as higher daily colony foraging activity, compared with those from the grafting larvae group (p < 0.05). Queen-rearing technology using CC effectively enhances the reproductive capacity and productive performance of colonies, promising high-quality queen rearing in A. cerana and sustainable beekeeping optimization.

1. Introduction

Honeybees are beneficial eusocial insects with considerable economic and ecological value. They play an irreplaceable role in the pollination of over 85% of global crop production [1,2]. Like Apis mellifera, Apis cerana also has outstanding pollination ability. However, the global diversity of bee species has been declining. Massive bee losses caused by Colony Collapse Disorder have led to a sharp decrease in colony populations, with some species facing extinction [3,4]. Consequently, nature is confronting the severe challenge of a “pollination crisis.” Therefore, maintaining honeybee colony populations is crucial for balancing agricultural development and ecological conservation. Research indicates that poor queen quality may be a key contributing factor to Colony Collapse Disorder [5]. The queen’s reproductive capacity is critical for colony development, as queen quality directly influences colony productivity, disease resistance, stress tolerance, and reproductive potential [6]. As queen bees senesce, their physiological functions naturally decline, resulting in a gradual annual decrease in egg-laying capacity [7]. This decline not only affects the colony population but also indirectly impairs the colony’s stress tolerance and disease resistance. Consequently, replacing old queens with new ones is necessary for beekeeping practices. In recent years, queen-rearing technology has emerged as a critical research focus in apiculture, with significant advancements. Its evolution has progressed from utilizing naturally formed queen cells to rearing queens by artificially grafting larvae (GL). This advancement has enabled beekeepers to rear queens on demand, based on production needs, a practice that continues today. Nevertheless, the traditional artificial queen-rearing method faces challenges, such as difficulties in larval grafting, particularly in Apis cerana. Compared with Apis mellifera, less royal jelly in the worker cells of Apis cerana colonies results in greater difficult in grafting larvae for queen rearing and mechanical damage to the eggs or larvae [8,9,10]. To address this issue, Pan et al. [11] developed a technology for rearing queen bees without transferring larvae, which is named “Queen Rearing without Grafting Larvae”. This method effectively resolved the challenges associated with artificial larval grafting. In addition, queens reared using this method outperformed those reared using traditional methods in terms of larval acceptance rate, weight at emerging, and ovarian development. The oviposition efficiency and queen cell acceptance rate of the queen rearing without grafting larvae group were higher than 91%. The weights at emergence of queens in the queen rearing without grafting larvae group and the artificial larval transfer queen-rearing group were 256.31 mg and 243.43 mg, respectively, and the number of ovarioles was 163.87 and 154.77, respectively [12]. Although these techniques have proven to be highly effective in colonies of Apis mellifera, their application in colonies of A. cerana has yielded suboptimal results. This is primarily because honeybees (A. cerana) have a highly sensitive olfactory system and show aversion to plastic materials used in these techniques [12,13].

A. cerana is a common bee species in Asia that plays a vital role in maintaining national biodiversity and supporting rural revitalization. The quality of its queens not only influences the quality of offspring worker bees and colony productivity but also impacts the contribution of the A. cerana industry to rural revitalization [14,15]. Therefore, it is imperative to explore novel techniques to rear high-quality queens. In 2024, Lu et al. [16] designed a cut-comb queen-rearing device specifically tailored to the biological characteristics of A. cerana. This technology has proven to be effective in improving the birth quality of reared queens. The queens in the CC (cutting comb) group outperformed those in the GL (grafting larvae) group in indicators including weight at emerging, queen morphological traits, number of ovarioles, and the expression levels of development-related genes. The diagram of this device and its operational steps are shown in Figure 1, which consists of three core steps: (1) assembling the specialized egg-laying frame, as shown in A, B, and C of Figure 1. The frame was placed in a honeybee colony, and the queen was allowed to lay eggs; (2) removal of the comb containing eggs, followed by using a knife to cut 3–4 queen-rearing strips in the 2-d-old larval comb, each 10–12 mm in width; and (3) fixation of the strips to a queen-rearing frame, as shown in Figure 1E.

The queen’s reproductive capacity is a key indicator of quality [17]. Queens with strong reproductive capacities can sustain robust colonies, and their offspring demonstrate enhanced vitality and disease resistance [18]. However, the reproductive performance and resulting colony production performance of queens reared using cutting comb (CC) technology require further investigation.

This study aimed to compare the differences between queen rearing by cutting comb and the larval grafting method. Referring to the experimental protocol described by Wang [19], the key investigated indicators included the queen reproductive capacity (length, width, and weight of queen-laid eggs, capped brood quantity), quality of worker bee offspring (worker offspring birth weight, worker bee morphological indicators), and overall colony production performance (honey sac weight in forager bees, daily foraging amount per colony). Furthermore, we explored the effect of queen rearing by CC on colony development and production performance. These findings will provide a theoretical basis for the widespread application of queen rearing using CC.

2. Materials and Methods

2.1. Experimental Honeybee Colonies

The experimental colonies consisted of the Eastern Honey Bee (A. cerana). All colonies were maintained at the Honeybee Research Institute of Jiangxi Agricultural University and managed according to standard apicultural practices between March and June 2023.

2.2. Experimental Methods

2.2.1. Queen Rearing

Virgin queens were reared using either queen rearing by CC (Cutting comb, following the methodology described by Lu [16]) or queen rearing by GL (Grafting larvae). The virgin queens were reared with the two methods from the same colony, and 3 colonies were used as three parallel experiments. Newly emerged virgin queens were collected for the subsequent experiments. Nucleus-mating colonies were established, and five virgin queens reared by each of the two methods (CC and GL) were introduced into these colonies to allow for natural mating. Ten days later, the mating status of the queens was checked, and the presence of normal fertilized eggs in the nucleus-mating colonies served as the criterion for confirming successful mating. After that, the queens were introduced into colonies with a population of 1.0 ± 0.050 kg of bees, allowing them to breed naturally. All colonies in the experiment were single-queen colonies. And then, the relevant indicators were measured.

2.2.2. Determination of Length, Width, and Weight of Queen-Laid Eggs

Queens in the two groups were each confined to empty combs (pretreated by worker bees) for a 4-h egg-laying period (from 10:00 to 14:00 daily). The queens were released after 4 h. Ten fertilized eggs were randomly picked from the cells using a larval transfer tool, and their weights were immediately measured using an electronic analytical balance (METTLER TOLEDO, ME204, Zürich, Switzerland). Subsequently, the eggs were transferred to glass slides, and their lengths and widths were measured using a stereomicroscope (CNOPTEC, OPTPro, Chongqing, China). Each measurement was repeated five times.

2.2.3. Determination of Capped Brood Quantity

After the queens in the two groups were introduced into their respective colonies, they began laying eggs. The sealed brood combs were photographed every 12 days, and the number of sealed worker bee cells of each colony in the photos was counted. This counting was repeated four times consecutively (total monitoring period: 48 d).

2.2.4. Determination of Worker Offspring Weight at Emerging

Following the completion of the aforementioned measurements, brood combs containing capped worker brood at the imminent emergence stage were transferred to an incubator (34 °C, relative humidity: 65%; AIKANE, DK250, Shanghai, China). The incubator was checked every 30 min to monitor worker bee emergence. Fifty newly emerged worker bees were randomly selected from each group, and the weight at emergence of each bee was measured individually using an analytical balance (METTLER TOLEDO, ME204, Zürich, Switzerland). To ensure data reliability, the sampling and measurement processes were replicated five times per group.

2.2.5. Determination of Honey Sac Weight in Forager Bees

Following the method described by Peng [20], 20 forager bees returning to the hive after foraging were captured and immediately frozen in liquid nitrogen. Before dissection, the bees were thawed at room temperature (about 25 ± 1 °C) for 5 min. Using sterile fine-tipped forceps and a dissecting microscope (CNOPTEC, OPTPro, Chongqing, China), the abdomen of each bee was carefully opened to isolate the honey sac. The weight of each isolated honey sac was immediately measured and recorded using an analytical balance. To ensure data reliability, measurements were repeated five times for each group.

2.2.6. Determination of Daily Foraging Amount per Colony

Sixty-five days after the queens were introduced into the colonies, the total weight of each beehive (including hive bodies, frames, adult bees, and stored resources—honey, pollen, and nectar) was measured at 08:00 daily for 11 consecutive days. Weighing was conducted using a digital platform scale (precision: ±5 g; Ex-E650, Wolant, Shenzhen, China). Hives were checked for external moisture (such as dew or rain) before weighing, and any surface water was gently wiped dry to avoid overestimating weight.

The daily foraging amount was calculated using the formula: Daily Foraging Amount (g) = Hive Weight on Day (n) (g) − Hive Weight on Day (n − 1) (g).

2.2.7. Determination of Worker Bee Morphological Indicators

Worker bee morphological traits were measured following a modified protocol described by Wang, the anatomical diagrams of worker bees as shown in Figure 2 [19]. Fifty worker bees were randomly captured from each of the two groups of colonies, preserved in 75% analytical pure (AR)-grade ethanol (Xilong Scientific Co., Ltd., Shantou, China), and labeled. From each group, 15 worker bees with fully extended proboscises were selected for analysis. Each bee was transferred to a wax dish containing 75% AR-grade ethanol to maintain hydration during dissection.

Following the morphological indicators proposed by Ruttner [21] in 1988, and using sterile fine-tipped dissecting forceps and a stereomicroscope, the following body parts were dissected in sequence: proboscis, head, forewings, hindwings, third sternum, sixth sternum, third tergite, fourth tergite, and hind legs. The third and sixth sternum were stained with 0.5% (w/v) eosin Y solution (dissolved in distilled water) for 3 min to enhance anatomical feature visibility and then rinsed with distilled water for 1 min to remove excess stain. The dissected body parts were individually mounted on glass slides (with a drop of 75% ethanol to prevent drying) and photographed using a digital camera attached to the stereomicroscope, controlled with OptPro software V4.0. Morphological indices were measured from the digital images using a calibrated measurement tool (calibrated with a stage micrometer before analysis). Each index was measured four times per body part, and the mean value was used for statistical analysis to reduce measurement errors.

2.3. Data Statistics and Analysis

The daily foraging amount of colonies was statistically analyzed using a generalized linear mixed model in SPSS Statistics 27.0. For datasets with independent observations (no repeated measurements per experimental unit), statistical analyses were performed using ANOVA and t-tests in StatView 5.0, with test selection based on the number of comparison groups. All statistical analyses were conducted using a significance threshold of α = 0.05 (p < 0.05 was considered statistically significant, whereas p > 0.05 indicated no significant difference). All experimental data are presented as mean ± standard deviation (Mean ± SD).

3. Results

3.1. Effects of Queen Rearing Methods on Queen-Laid Eggs

As shown in Figure 3, there were significant differences in egg weight and length between the two groups (p < 0.05). The CC group had an average egg weight of 0.1909 mg, which was 10.4% higher than that of the GL group (0.1729 mg). The CC group also produced longer eggs (1.817 mm) than those of the GL group (1.772 mm), representing a 2.5% increase in egg length. By contrast, no significant difference was observed in egg width between the groups: the CC group had an average egg width of 0.463 mm, while the GL group had 0.444 mm (p > 0.05).

3.2. Effects of Queen Rearing Methods on the Number of Capped Brood Cells in Colonies

As shown in Figure 4, the number of capped brood cells in the CC group was significantly higher than that in the GL group (p < 0.05).

3.3. Effects of Queen Rearing Methods on the Weight at Emergence of Worker Offspring

As shown in Figure 5, the average weight at emergence of worker bee offspring in the CC group was 94.96 mg, whereas that in the GL group was 92.01 mg. The CC group produced worker offspring with significantly higher weights at emergence than those of the GL group (p < 0.05).

3.4. Effects of Queen Rearing Methods on the Honey Sac Weight of Forager Offspring

As shown in Figure 6, the CC group exhibited a slightly higher average honey sac weight than that of the GL group, although the difference between the two groups was not significant (p > 0.05). Specifically, the average honey sac weight of forager offspring in the CC group was 24.51 mg, whereas that in the GL group was 23.29 mg.

3.5. Effects of Queen Rearing Methods on the Daily Foraging Amount of Colonies

As shown in Figure 7, while the two groups exhibited comparable daily foraging amounts during the initial 3 d of monitoring, a significant difference emerged starting on the fourth day: colonies with offspring reared by the CC method had significantly higher daily foraging amounts than those reared by the GL method (p < 0.05).

3.6. Effects of Queen Rearing Methods on the Morphological Indicators of Workers

As summarized in

Table 1. Effects of queen rearing by cutting comb and grafting larvae on the morphological indicators of the worker (n = 75).

Morphological Indicators	Cutting Comb (CC)	Grafting Larvae (GL)
Proboscis length/mm	4.770 ± 0.140 a	4.750 ± 0.170 a
Forewing length/mm	8.640 ± 0.180 a	8.690 ± 0.200 a
Forewing width/mm	2.990 ± 0.048 a	2.900 ± 0.110 b
Cubital vein a/mm	0.880 ± 0.130 a	1.100 ± 0.058 b
Cubital vein b/mm	0.310 ± 0.031 a	0.270 ± 0.024 b
Length of wax mirror on 3rd sternum/mm	3.720 ± 0.150 a	3.120 ± 0.470 b
Distance between wax mirrors on 3rd sternum/mm	0.270 ± 0.025 a	0.250 ± 0.020 b
Oblique distance between wax mirrors/mm	3.320 ± 0.079 a	3.260 ± 0.060 a
3rd tergite Length/mm	2.090 ± 0.053 a	1.990 ± 0.039 b
4th tergite Length/mm	2.013 ± 0.050 a	1.940 ± 0.062 b
Hind leg femur length/mm	2.430 ± 0.046 a	2.390 ± 0.065 a
Tibia length/mm	2.958 ± 0.077 a	2.800 ± 0.190 b
Tarsus length/mm	2.240 ± 0.061 a	2.019 ± 0.060 b
Tarsus width/mm	1.110 ± 0.087 a	1.055 ± 0.096 a
Hindwing length/mm	6.460 ± 0.200 a	6.070 ± 0.150 b
Hindwing width/mm	1.850 ± 0.064 a	1.790 ± 0.110 a
Length of the 6th sternum/mm	2.360 ± 0.083 a	2.314 ± 0.090 a
Width of the 6th sternum/mm	2.890 ± 0.070 a	2.770 ± 0.160 b

Note: The table indicates that the difference is not significant (p > 0.05), and the different letters indicate that the difference is significant (p < 0.05).

, the two queen-rearing methods had different effects on worker morphology, with most traits showing significant advantages in the CC group. The worker offspring of the CC group had significantly higher values than those of the GL group for the following indicators (p < 0.05): forewing width, cubital vein b, length of wax mirror on the third sternum, distance between wax mirrors on the third sternum, third tergite length, fourth tergite length, tibia length, tarsus length, hindwing length, and width of the sixth sternum. However, the cubital vein value in the GL group was significantly higher than that in the CC group (p < 0.05). There were no significant differences between the two groups in proboscis length, forewing length, oblique distance between wax mirrors, hind leg femur length, tarsus width, hindwing width, or length of the sixth sternum (p > 0.05).

4. Discussion

With the annual decline in the egg-laying capacity of queens, the reproductive capacity of colonies progressively decreases; therefore, the periodic replacement of queens with newly reared individuals is necessary to sustain colony health and productivity. Over a century ago, Doolittle [22] and others developed the technology of queen rearing by artificial grafting of larvae, which has since become a foundational and widely adopted practice in apiculture worldwide. However, because of operational difficulties, cumbersome procedures, and visual limitations associated with artificial larval-grafted queen rearing, beekeepers often prefer natural queen rearing within the colony. This practice, however, frequently results in queens of suboptimal quality, which fail to ensure adequate colony reproductive capacity and productive performance. Therefore, there is an urgent need to develop a simple and practically feasible high-quality queen-rearing technique. In recent years, in-depth studies of queen-rearing technology have demonstrated that queens reared from eggs or younger larvae exhibit significantly superior physiological characteristics compared with those of queens reared from older larvae [23]. The rearing of queen bees without GL has been validated as an effective method to improve queen quality because it allows the direct use of eggs or younger larvae for queen rearing [10]. However, A. cerana shows relatively low acceptance of the plastic materials used in the queen rearing without grafting larvae devices, with the larval acceptance rate and queen emergence rate being 40.87% and 38.52%, respectively [24]. For artificial larval grafting queen-rearing using wax queen cells, the larval acceptance rate is as high as 74.38%, and the queen emergence rate reaches 69.13% [24]. Zhang et al. [25] found that the larval acceptance rates of the queen rearing without grafting larvae method and the artificial larval-grafting queen-rearing method for Apis mellifera were 90.13% and 79.01%, respectively. Lu et al. [16] developed a comb-cutting queen-rearing device tailored to the biological characteristics of A. cerana, which effectively enhanced the quality of reared queens. This study investigated the effects of comb-cutting queen-rearing technology on queen reproductive performance, foraging capacity, multiple morphological traits of worker bee offspring, and other relevant factors to further validate its feasibility.

Queen fecundity (egg laying) and egg morphological indicators are key parameters for evaluating the reproductive performance of queens [26]. This study found that queens in the CC group had significantly superior performance across multiple reproductive indices compared with those in the GL group. Among these indicators, the length and weight of eggs laid by queens in the CC group were significantly higher than those in the GL group. This is mainly because larger queens have more ovarioles and exhibit stronger egg-laying capacity, thereby producing larger eggs [27]. Queens reared by CC exhibit significantly higher weights at emerging than those of queens reared by the artificial larval-grafting method, along with larger body size and a greater number of ovarioles [12]. Furthermore, this result may be attributed to the fact that comb-cutting technology effectively avoids the damage caused by larval grafting operations, and this technical advantage directly leads to better physical fitness of the queens in the CC group.

In addition, compared with the GL group, the CC group exhibited a significant increase in the number of capped brood cells. This is primarily attributed to the superior egg-laying capacity of queens reared using the comb-cutting method [16]. These results further confirm that comb-cutting queen-rearing technology can improve the reproductive performance of queens. This study also found that the weights at the emergence of offspring in the CC group were significantly higher than those in the GL group. This phenomenon is primarily due to a positive correlation among queen weight, egg weight, and the weight at emergence of worker bee offspring [28].

There was a significant correlation between the morphological traits of worker bees and their foraging capacity. The third and fourth tergites on the abdomen of worker bees not only indicate body size but also reflect the volume of their honey sacs and their capacity to store honey [29]. The tarsus length and width of the hind legs of worker bees indicate their ability to forage and carry pollen [30]. In addition, the length and width of the wings of worker bees reflect their foraging range and capacity [31]. This study found that the worker offspring of the CC group exhibited significantly superior performance across multiple morphological indices compared with those of the GL group, demonstrating a stronger foraging capacity. Consistent with this result, the daily foraging amount of colonies with offspring from the CC group was significantly higher than that of the GL group, which corresponds with the findings regarding worker bee morphological indicators and the number of capped brood cells. Furthermore, the honey sac weight of foragers is a direct indicator of individual foraging capacity; larger honey sacs allow bees to carry more nectar per foraging trip, which contributes to overall colony honey production [32]. However, there was no significant difference in honey sac weight between the two groups. Potential reasons for this include the experimental period not coinciding with the main nectar flow period or factors related to the foragers themselves. Nevertheless, the specific causes require further investigation.

5. Conclusions

The experimental results clearly demonstrated the advantages of queen rearing by CC: compared with the GL group, queens in the CC group produced eggs of significantly superior morphological quality (longer length and heavier weight) and a higher number of capped brood cells (p < 0.05), directly reflecting enhanced colony reproductive potential. Additionally, worker offspring from the CC group exhibited better morphological traits (including longer forewings, wider hindwings, and longer tergites). They contributed to a significantly higher daily foraging amount in the colonies (p < 0.05), thereby improving colony productivity. Although no significant difference was observed in the honey sac weight of foragers between the two groups (p > 0.05), this does not negate the comprehensive advantages of comb-cutting technology in key indicators of reproduction and production.

In summary, queen rearing using CC technology effectively enhanced the reproductive and productive performance of A. cerana colonies. As a practical and efficient method for rearing high-quality queens, this study provides a reliable theoretical basis and technical support for optimizing traditional beekeeping practices, facilitating the application of high-efficiency queen-rearing technology, and ensuring the sustainable development of apiculture.