Isolation of Bacillus From the Gut of Bombus terrestris and Its Correlation in Queen Mating

The gut of bumblebees harbors bacteria that play a crucial role in physiology, nutrition, and health. The mating rate is important for the reproductive activity of a colony; however, few studies have investigated the relationship between mating and gut bacteria. In this study, bacterial functions were identified in the intestinal tract of bumblebees, and biochemical identification and screening were performed using genetic detection technology. By isolating and identifying bacteria, we obtained a single strain and fed it to queens. The results indicated that Bacillus cereus and Bacillus pumilus are present in the gut. The queen mating rates were 48.89% at the period of 4 days and higher than 28.89% mating rates of the control group (P < 0.05), and the latency time were 16.90 min (from entering the mating cage to mating success) and decreased than control (P < 0.05) which was 28.20 min. This finding confirmed that Bacillus was important in Bombus terrestris mating.


Introduction
Bumblebees are important pollinators in natural and agricultural ecosystems, as well as playing a significant role in maintaining plant diversity and ecological balance (Gallai, 2009;Williams, 2009). Additionally, their potential economic value has been recognized for a long time. Their tongues are generally longer than honeybees, so they are better at pollinating flowers with deep corollas. Furthermore, the dense hairs on their bodies allow efficient pollen transfer from flower to flower, and they can also sonicate ("buzz pollination") wildflowers and crops (including tomato) whose flowers shed pollen through apical pores. Thus, some bumblebees have become commercially bred species, notably for greenhouse pollination of many crops (Velthuis & van Doorn, 2006;Dasgan et al., 2004).
In 1912, the first monograph on artificial bumblebee rearing was published, thus starting a new process of artificial bumblebee breeding (Sladen, 1989). Chinese native bumblebees (post hibernating queens) have been reared in the laboratory for scientific studies of social behavior, biology and pollination since 1996, and remarkable progress of Bumblebees rearing techniques and pollination in greenhouses has been made . Additionally, mating behavior is significant to the development of colonies (Salvarrey, 2013). Therefore, for bumblebees rearing techniques, the successful mating of queens is a crucial step. Unmated queens cannot survive after hibernation; only the successfully mated queens can survive and reproduce colonies (Cao, 2014;Holland, 2013). Tasei et al. (1998) reported that the sperm vitality in the vas deferens and seminal vesicles of bumblebee adult males in Europe starts to decline 13 days after emergence, an essential time for male bees to mate successfully with queens. In artificially controlled mating cages, the optimal mating age of queen bees is 8-9 days, that of male bees is 11-12 days, and the ratio of the queen and male bees is 1:1.5 (An, 2004;Wu et al., 2005). The mating situation of artificial bumblebee breeding is mainly affected by the sexual maturity of bumblebees and the external environment, including light, temperature, humidity, and health (Thomas, 2005;Amin, 2012).
The bumblebee harbors a simple yet specialized gut microbiota (Kwong et al., 2013;Killer et al., 2014;Praet et al., 2017), which plays an essential role in host food digestion and absorption, enhancing the host's immunity, and resisting pathogen infection (Li et al., 2015;Kwong et al., 2017). Studies have shown that there is a close, however, very complicated relationship between the host and its intestinal flora. Many gut bacteria have a probiotic effect on the host. They can induce the host to produce antibacterial peptides and defensins (Evans, 2004;Cariveau, 2014). Bacillus subtilis can inhibit bee funga Ascosphaera apis and play an essential role in the prevention of Nosema sp. (Liu et al., 2016). Various Bacillus bacteria have inhibitory effects on Paenibacillus larvae that cause American foulbrood (Sabate, 2014). Additionally, studies have shown that bumblebee intestinal bacteria exert a defensive particular impact on the infection of Crithidia bombi (Koch & Schmid-Hempel, 2011. Moreover, the gut microbiota plays a vital role in the healthy reproduction of many insects, gut bacteria could influence the mating active through epidermal sex pheromone in Drosophila melanogaster. At the same time, in mouse intestinal symbiotic bacteria could impact sexual maturity time (Vanbelle, 1990;Benvacoub, 2003;Weger et al., 2018;Sharon et al., 2010). Our previous study showed a significant difference in the diversity and composition of the gut microbiota communities in queens of Bombus lantschouensis across different physiological states, and Bacillus is a relatively dominant bacterial genus in mated queens (Wang et al., 2019). However, few studies have examined how the gut microbiota responds to external changes, as well these changes affect the interaction between the host and the external environment (Kong et al., 2014).
In order to understand the complex interplay between the microbiome and queens that mated successfully. We isolated Bacillus cereus and Bacillus pumilus from the gut of queens that mated successfully and fed Bacillus cereus and Bacillus pumilus to newly emerging queen samples to observe the effect of Bacillus on the mating rates of queens. Our study is the first to explore the impact of the gut microbiome on the mating of bumblebee queens. It will provide useful information for future studies of the function of gut bacteria in queen mating and reproduction.

Sample Collection
Queens of Bombus terrestris (Linnaeus) (Hymenoptera: Apidae) were collected from the Institute of Apicultural Research, CAAS, China. The colonies were reared in the dark room at a temperature of 27±1 °C and relative humidity of 50-60%. Sugar water (1:1 v/v) and apricot (Prunus armeniaca L) pollen were provided ad libitum to Paenibacillus larvae he colonies subsequently produced 100 colonies until males and gynes (new queens) emerged.

Isolation and Culture of Bacteria
Three healthy mated queen bees from the bumblebee breeding room were collected and pretreated in the refrigerator at 4 °C for 5 minutes. After that, the queen was moved to an ultra-clean bench for intestinal extraction (Wang et al., 2019). The whole gut was placed in a 1.5-mL centrifuge tube, and then 800 μL of solution (Krebs-Ringer Solution) was added. Thereafter, the sample was fully ground into a homogenate, and then 100 μL and 900 μL of ddH 2 O were added and mixed with a pipette. The sample was diluted 10 times, 100 times and 1000 times, and finally coated with 1000 times diluted bacterial solution (Tryptic Soy Broth-TSB solid culture medium). The culture medium was placed in a constant-temperature incubator for 48-72 h, and the temperature was 37±1 °C. After the strain spot appeared, strain spot was streaked and cultured purely 2-3 times. The obtained strain was subjected to Gram staining, and then the morphology of the bacteria was observed under a microscope.
We selected representative and single strains grown on the culture medium. After repeated purification for 2-3 times, we placed them in an incubator room for 2-3 days. We observed and recorded the morphological characteristics of bacterial colonies on the plate.

Identification of Bacteria
Total genomic DNA was extracted using a DNA purification kit (WizardR @ SV 96 Genomic DNA Purification System; (Promega) from 18 hour cultures of each isolate. DNA samples were stored at -20 °C for PCR and further analysis. The bacterial 16S rRNA gene was amplified by PCR from total DNA using primers 27F and 1492R (Engel et al., 2013). The PCR products were electrophoresed in 1.2% agarose gels containing 0.5 µg/ml GoldView (GV) and visualized under UV light. Some of the PCR-amplified bands were purified and sequenced to verify their identities. The PCR products were bi-directionally sequenced, and the sequencing work was completed by Sino Geno Max (Beijing, China). The sequences obtained were BLAST analyzed and matched with valid reference sequences in the NCBI (National Center for Biotechnology Information) to determine the bacterial species types. Individual sequences of isolates that were ≥ 98% identical to each other were considered the same phylotype and were combined for analysis.
At the same time, the physiological and biochemical identification of Bacillus were performed according to Dong et al. (2001), including starch hydrolysis, VP, gelatin, nitrate, and lecithin test.

Feeding Bacillus
In total, 270 newly emerging queen samples were collected from the colonies of 3 Bacillus-fed groups (Bacillus + Sugar water, 5 × 107 copies/ml (Wang et al., 2019) mixed with 50% (1:1 v/v) sugar) and 3 control groups without Bacillus cereus and Bacillus pumilus feeding, each group comprising 15 queens randomly distributed into 6 experimental groups (N = 15 queens/group): 3 groups with Bacillus supplement and 3 without Bacillus supplement. Bacillus was collected from the guts of healthy mated queens and isolated on TSB medium. Then the queens were fed individually with the Bacillus liquid each day, for a period of 3, 4, and 5 days before undergoing a mating test. The concentration of the Bacillus cereus and Bacillus pumilus mixture (1:1) was 5 × 10 7 copies/ml (Wang et al., 2019) mixed with 50% (1:1 v/v) sugar solution. All the tests were repeated three times.

Effect of Bacillus on the Mating Success Rate of Queen Bees
The queens and males of each group were placed together in 100 × 100 × 100 cm stainless steel cages with mesh and transparent walls to observe the mating success rate. During mating, the cages were illuminated with 200 W mercury bulbs positioned 1 m above the mating cages. The experiment was performed in rooms maintained at 26±1 °C with a queen to male ratio of 1:2 in the mating cages to determine the impact of Bacillus feeding on queen mating success. (Wu et al., 2005;An, 2004). Fifteen queens (each treatment group) and 30 sexually mature male bees of each group were placed in each mating cage. The mating experiment time was from 8:00 to 12:00 every day for one month (Peng, 2003). The latency (the time elapsed between the introduction of males and queens into the mating cage until the initiation of copulation) (Amin, 2012), mating time, and mating rate of the queen bees were recorded in the mating experiment. As the queens emerged from the cage, the sexual maturation increased until the full maturity and mating with the male bees completed the mating process. In this process, the queen maturity was proportional to the queen mating rate (Wu et al., 2005;An, 2004). We started to record the numbers of successful mated queens within 1 hour when queens and males entered the cage.

Statistical Analysis
SPSS software (version 23) was used to analyze the latency, mating time, and mating rate. The significant differences in the mating rate, mating time, and latency between the Bacillus cereus plus Bacillus pumilus mixture-fed queen group and control group (no feeding) were compared using analysis of GLMMs . The model used a binary logistic regression distribution and included mating status (success or failure) as the response variable, the days, and groups as fixed factors, and the number of bumble queens as a random factor. The latency and mating time were analyzed in the same way.

Morphological Identification of Bacillus
By morphological identification, we obtained the two pure bacterial cultures named TSB-01 and TSB-11. The growth habits of the strains were all aerobic, and the morphological characteristics were regular round, showing a milky-opaque shape, wet surface, and no apparent gloss. After gram staining, the bacteria were Gram-positive, rod-shaped, medium-sized, most of them existed individually, and a few were connected to each other, similar to Bacillus spp. under the microscope (Dong, 2001;Sha, 2019). jas.ccsenet.

Molecu
The 16rRN is 880bp, w similar wit accession physicoche chemical p 2001). For identical t lecithin an

Effect
The latenc without Ba Figure 2) (Figure 1). erature (Chen, nd lecithin wa sitive whereas acillus cereus ( s cereus (positi f than the cont as found at 3 t groups. The d e 2). roup. At 4 and p (4 days: P = as higher than for the feeding feeding group ly significant Vol. 13,No.  Bumblebee mating behavior is affected by many factors, including their sexual maturity and environmental conditions (An, 2004), The addition of Bacillus cereus to the feed can significantly increase the growth rate of shrimp and fish, as well as promote host body development (Tang, 2017). By promoting growth, the queen can enter sexual maturity, thereby increasing the mating rate. However, how Bacillus affects queen mating is unclear and needs further research.
The diversity of symbiotic flora in the intestinal tract of European bumblebees is very rich. Although some strains were isolated in vitro in this study, some symbiotic bacteria that could not be isolated persisted in the intestine of bumblebees. Bacillus was isolated in this study. The cause may be related to the bacteria culture conditions and choice of culture medium. The other two strains fully comprehend the physiological role and function of symbiotic bacteria in the bumblebee intestine. To isolate and culture Lactococcus and Pseudomonadaceae, selecting the appropriate culture medium and culture conditions according to their growth characteristics is necessary. In specific cases, specific nutrients must be added to promote bacterial growth. Thus, we isolated and cultured the bacteria and then analyzed the physiological and biochemical characteristics of the isolates.
In Drosophila melanogaster intestinal symbiotic bacteria could through epidermal sex pheromone influence their mating (Sharon et al., 2010). In mouse, intestinal symbiotic bacteria could impact sexual maturity time (Weger et al., 2018). This study showed that mixing B. cereus and Bacillus pumilus to feed bumblebees could increase their mating rate. A mixed preparation of Bacillus can be added to the artificial breeding system of bumblebees to increase their mating rate. However, it is not clear in which ways do Bacillus increase the mating rate on bumblebees.
Therefore, mating behavior represents an essential vital system to understand the link between bee reproduction and host-associated microbiota. The bumblebee gut bacteria likely play a role in shaping the ability of the bee to survive environmental extremes and reproduce due to long-established coevolutionary relationships between the host and microbiome members.
The results showed a significant difference in the diversity and composition of Bombus terrestris gut-isolated Bacillus, which plays a significant role in bumblebee copulation. This study provides insight into the relationship between the bacterial community and the mating of bumblebee queens. It also lays the foundation for further studies of the function of gut microbiota in the reproductive success of bumblebee queens.