Simulated Climate Warming Influenced Colony Microclimatic Conditions and Gut Bacterial Abundance of Honeybee Subspecies Apis mellifera ligustica and A. mellifera sinisxinyuan
Recently, symbiont bacteria are receiving attention as strong and effective immune-modulators of insects (Ryu et al., 2008; McFall-Ngai et al., 2012). Gut-harboring bacterial communities greatly influence the resistance of their hosts to biotic and abiotic stresses (Crotti et al., 2012; Engel & Moran, 2013), which suggests their crucial contribution to hosts’ adaptation and evolution (Rosenberg & Zilber-Rosenberg, 2011). Recent studies have shown that gut bacteria could change ectotherms’ population composition or metabolism to fight with low temperature stress by (Chevalier et al., 2015; Hylander & Repasky, 2019; Raza et al., 2020). However, during high temperature stress could reduce the gut bacterial abundance of reptile and amphibian ectotherms (Bestion et al., 2017; Li et al., 2020). A more comprehensive analysis of gut bacteria within a host under a simulated climate-warming pattern is of a great interest to predict the consequences of climate warming on the ecology of symbiotic microbes and their hosts.
An understanding of how abiotic factors affect host-microbiota interactions would improve our knowledge of honeybees’ response to climate warming. In this study, we investigated the impact of simulated climate-warming on the brood nest microclimate and gut bacterial populations of honeybees. We hypothesized that temperature increase may affect directly the incubation temperature inside the brood nest and indirectly mediate the gut bacterial abundance of Apis mellifera. To conduct this study, we used two subspecies of A. mellifera i.e. A. mellifera ligustica and A. mellifera sinisxinyuan to demonstrate how the response of gut bacteria might differ according to the evolution of a species. A. mellifera ligustica is a common honeybee dispersed in temperate and tropical climates, while A. mellifera sinisxinyuan is strictly an endemic subspecies in temperate climates particularly in north-east China.
MATERIAL AND METHODS
Honeybee colonies
The study was carried out from April to August 2017 at the Institute of Apicultural Research (40°00′12.8″N; 116°12′29.5″E), Beijing, China. The study population of each honeybee subspecies (A. mellifera ligustica and A. mellifera sinisxinyuan) was comprised of eight colonies. Each colony was comprised of approximately 6,000 worker bees at the beginning of the experiment and all queen bees were of the same age (~18 months) and descendants from different mother bees. There were eight frames in each bee hive.
Treatments
During the experiment, eight housed bee hives as the control were kept outdoors and shaded with ceramic tiles under ambient climatic conditions and eight hives for the heating treatment were maintained at 2–3°C higher than ambient temperature conditions. For each honeybee subspecies used in this study, there were four replications (four bee hives) in the control and four replications in the heating treatment. Under the heating treatment, heat was supplied to honeybee hives with a ceramic infrared radiator heater (FTE-L10-Y; 1000 W, 240 V; 60 mm long × 245 mm wide) the hives. Each hive was heated up with two such heaters set at a height of 190 cm at the front and behind of the hive to make the ambient climatic condition warmer. The heating treatment was designed according to the global warming prediction of IPCC (2014).
Monitoring of brood nest temperature and relative humidity
In order to monitor the brood nest microclimate conditions, one probe (Onset® HOBO® Pro V2 logger; Onset Computer Corporation, MA, USA) was introduced in each hive through the lateral holes (Ø=10 mm). The probe was installed in the brood area at 0.3 cm from the comb surface. The probes were wrapped in a plastic protective membrane to impede honeybee workers from covering the sensor with water, nectar, wax or propolis. Data regarding hive temperature and relative humidity were recorded and stored automatically by the logger in each hive at regular 10 min intervals throughout the duration of the experiment (24 hrs in a day) (Fig. 1). At the end of experiment, data collected by the loggers were downloaded to a laptop through an optic coupler (2-E) using the Onset Optical Base Station (BASE-U-4) software.
Fig. 1
Mean daily temperature (°C) recorded from April to August, 2017 under two temperature conditions. Blue and red lines indicate temperature fluctuations for the control and heating treatments, respectively.
Molecular analysis
For the characterization of the gut bacterial community of each honeybee subspecies, i.e. A. mellifera ligustica and A. mellifera sinisxinyuan, ten 5-days old larvae and ten teneral worker bee imagoes were randomly collected from each of the sixteen hives (including 4 heated and 4 control hives for each subspecies) once at the end of the field experiment. Insects collected from each bee hive were surface-sterilized with 70% ethanol, rinsed thoroughly with sterilized double-distilled water, dried over a lint-free laboratory wipe and weighed with a digital balance (accuracy 0.001 g, Shimadzu Corporation, Japan). All sampled specimens were preserved at −20°C in 95% ethanol for the dissection of their guts.
DNA extraction
Total genomic DNA was extracted from the ileums and rectums of individual honeybee larvae and adults with the use TIANamp® genomic DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. Purified DNA was eluted from each sample using 200 μl of AE buffer provided in the extraction kit. Extracted DNA quality was verified by gel electrophoresis through staining 1.5% (w/v) agarose gel with ethidium bromide and visualizing it under UV-transillumination system and then was quantified with NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Extracted DNA samples were preserved at −20°C until downstream molecular analysis. Final DNA concentrations in all samples were normalized to 10 ng/μl with nuclease-free water.
Abundance of total symbiotic bacterial community
Total bacterial symbionts harboured by the honeybee adults and larvae of A. mellifera ligustica and A. mellifera sinisxinyuan were estimated through the amplification and quantification of the 16S rRNA gene copy numbers with a set of universal primers i.e. 341F (5′-CCT ACG GGA GGC AGC AG-3′) and 515R (5′-ATT CCG CGG CTG GCA-3′) targeting 174 bp region of eubacterial gene.
Quantitative PCR assay
The real-time quantitative PCR assays were performed using a C1000TM thermo-cycler (CFX96®, BioRad, Hercules, California). A final reaction mixture of 20 μl contained 0.6 μl of each 10 μM 16S rRNA primer, 10 μl of SYBR Green PCR mix having HotStar TaqTM DNA Polymerase, SYBR Green PCR Buffer, dNTP mix, SYBR Green I, ROX and 5 mM MgCl2 (FastFire qPCR PreMix; Tiangen®, Beijing, P.R. China), 1 μl of template DNA corresponding to 10 ng of total DNA and 7.8 μl of DNase- and RNase-free distilled deionized water. Thermal protocol for 16S rRNA amplification reactions were comprised of 900 s at 95°C for enzyme activation followed by forty cycles of 15 s at 95°C for denaturation, 30 s at 60°C for annealing and 30 s at 72°C for extension steps. At the end of each reaction, melting curve analysis for data acquisition involved an additional step of heating from 65°C to 95°C (@ 0.2°C/s). Specificity and purity of the amplified qPCR products were confirmed by melt-curve analysis and by observing the band of expected fragment size in 1.5% (wt/v) agarose gel stained by GelRed® 10,000X (Coollab, Beijing, P.R. China). The standard curve for 16S rRNA gene quantification was worked out through the plotting of threshold cycle (Ct) values as a function of log10 of the copy numbers of target gene fragment. To this end, amplified target gene fragments were cloned in Top10® (Escherichia coli) Competent Cells (Tiangen®, Beijing, P.R. China) using pGM-T Blunt Cloning Kit (Tiangen®, Beijing, P.R. China) according to the manufacturer’s instructions. Cloned plasmids carrying a standard target sequence were linearized through incubation at 37°C for 1 hr along with the 10 U of SalI restriction enzyme. Standards were prepared by tenfold serial dilution of these linearized plasmids containing 102 to 109 16S rRNA gene copies, calculated from the concentration of extracted plasmids.
Statistical analysis
Using mean daily temperature and relative humidity in the brood nest of each hive, we calculated the mean brood nest temperature and relative humidity under the two temperature treatments. For statistical analyses, samples were grouped by treatment categories for each subspecies. Data regarding brood temperature and relative humidity were analysed with the use of one-way analysis of variance (ANOVA), followed by Fisher’s Least Significant Difference (LSD) test to assess the difference between the means of treatment groups and honeybees subspecies at standard probability level (p ≤ 0.05). Gut bacterial gene abundances for both treatments and subspecies were analysed using two-sample Student’s t test at α=0.05. Data analysis was carried out with IBM® SPSS Statistics V. 20 (IBM Corp., NY, USA).
RESULTS
Effects of simulated climate warming on the brood nest microclimate
For both A. mellifera subspecies, the brood nest mean temperature under heating treatment significantly differed (p≤0.001) from that under the control treatment (Fig. 2 and Fig. S1). The mean brood nest temperatures of A. mellifera ligustica and A. mellifera sinisxinyuan during the heating treatment was recorded as 34.90 and 34.54°C, respectively, and both were significantly higher than the brood temperatures of 33.51 and 34.14°C, recorded respectively for the control treatments. Moreover, the brood nest temperature during heating treatment did not significantly differ between both honeybee subspecies (p≥0.05), while for the control treatment, the brood nest temperature of A. mellifera sinisxinyuan was significantly higher than that of A. mellifera ligustica (p≤0.05).
Fig. 2
Brood nest temperature (mean±SD; n=8) of A. mellifera ligustica and A. mellifera sinisxinyuan recorded during the control and heating treatments from April to August 2017. Cumulative data of eight bee hives is presented for each treatment. * indicates significant difference between treatments for each subspecies (two-sample Student’s t-test; p≤0.05).
Similarly, the mean relative humidity in both A. mellifera subspecies brood nests during the control and heating treatments was significantly (p≤0.001) different (Fig. 3 and Fig. S2). Mean brood nest relative humidity values of A. mellifera ligustica (60.14%) and A. mellifera sinisxinyuan (55.63%) during the heating treatment were significantly lower than those recorded during the control treatment i.e. 61.71 and 61.21%, respectively (Fig. 3). Under the control treatment, mean brood nest relative humidity for both subspecies was statistically similar (p≥0.05). However, the brood nest relative humidity of A. mellifera ligustica was significantly higher than that of A. mellifera sinisxinyuan (p≤0.05) during the heating treatment.
Fig. 3
Brood nest relative humidity (mean±SD; n=8) of A. mellifera ligustica and A. mellifera sinisxinyuan recorded during control and heating treatments from April to August 2017. Cumulative data of eight bee hives is presented for each treatment. * indicates significant difference between treatments for each subspecies (two-sample Student’s t-test; p≤0.05).
Impact of simulated climate warming on the honeybee gut bacterial population
Regarding the abundance of total gut bacteria, qPCR data revealed that 16S rRNA gene copy numbers in the larval guts of both honeybee subspecies were higher during the heating treatment than those under the control condition (Fig. 4), although the difference was statistically significant only for A. mellifera sinisxinyuan (p≤0.05). The bacterial abundance of A. mellifera sinisxinyuan larvae increased significantly (p≤0.05) from 1.73 × 107 copies g−1 fw under the control condition to 9.21 × 107 copies g−1 fw under the heating treatment. On the other hand, bacterial population of A. mellifera ligustica larval gut increased from 1.67 × 107 copies g−1 fw under the control to 2.89 × 107 copies g−1 fw under heating treatment (Fig. 4). Moreover, no significant difference (p≥0.05) was found between the gut bacterial abundance of A. mellifera ligustica (1.67 × 107 copies g−1 fw) and A. mellifera sinisxinyuan (1.7 × 107 copies g−1 fw) under the control. In contrast, under heating treatment, A. mellifera sinisxinyuan gut bacterial abundance (9.21 × 107 copies g−1 fw) was three times higher than that of A. mellifera ligustica (2.89 × 107 copies g−1 fw) (p≥0.05).
Fig. 4
Bacterial 16S ribosomal RNA gene abundance (mean±SD; n=40) in larval guts of A. mellifera lingustica and A. mellifera sinisxinyuan during control and heating treatments. * indicates significant difference between treatments for each subspecies (two-sample Student’s t-test; p≤0.05).
Gut bacterial abundance as quantified from imago (adult) honeybees showed that 16S rRNA gene copy numbers of A. mellifera sinisxinyuan recorded during the control and heating treatments significantly differed from each other and from those of A. mellifera ligustica (p≤0.05). However, the population of gut bacteria in A. mellifera ligustica adults did not change significantly between the two treatments (p≥005) (Fig. 5).
Fig. 5
Bacterial 16S ribosomal RNA gene abundance (mean±SD; n=40) in guts of newly emerged adults of A. mellifera lingustica and A. mellifera sinisxinyuan under control and heating treatments. * indicates significant difference between treatments for each subspecies (two-sample Student’s t-test; p≤0.05).
Results revealed that simulated climate warming significantly impacted the brood nest microclimate (temperature and relative humidity) and the gut bacterial population of both larval and adult bees. Simulated climate warming increased the mean brood nest temperature by about 1.38±1.17 and 0.39±0.14°C of A. mellifera ligustica and A. mellifera sinisxinyuan, respectively, and reduced the relative humidity inside the brood nest by about 1.62±3.09 and 5.57±1.14%, respectively. Despite the augmentation of ambient temperature under heating treatment, honeybee workers maintained their brood nest temperature in the optimum range of 33–36°C (Silva et al., 2009; Kaftanoglu et al., 2011), most probably due to their fanning and water collection activities (Human et al., 2006). However, the relative humidity decreased from the optimum range suggesting that climate warming directly affected the brood nest microclimatee by decreasing the relative humidity inside the brood nest. Honeybee workers might not be able to regulate the relative humidity inside their nest during climate warming. Several studies have shown that low humidity inside the honeybee nest impacts negatively egg-hatching and brood-survival rates through desiccation of the broods (Ellis et al., 2008; Al-Ghamdi et al., 2016) and increases the rate of infestation of honeybees by Varroa mites (Kraus & Velthuis, 1997). Among high temperature and low humidity occurring during the summer in temperate climate, low relative humidity may be one of the key factors leading to the collapse of honeybee colonies (Cox-Foster et al., 2007; Switanek et al., 2017). Our results showed that the adaptation of a species to environmental change might be linked to the thermal plasticity of its gut microbiota population (Hylander & Repasky, 2019; Raza et al., 2020; Sepulveda & Moeller, 2020).
A relatively higher number of gut bacteria (16S rRNA gene copy numbers) in the indigenous honeybee subspecies (A. mellifera sinisxinyuan) than in the cosmopolitan (A. mellifera ligustica) one could mean that indigenous honeybee subspecies from temperate climate are more sensitive to climate warming than cosmopolitan ones (Chen et al., 2016). Therefore, this subspecies harbours more bacteria to sustain the environmental stress. Many studies have documented that an increased number of gut bacteria usually helps their host insects to better tolerate the abiotic stress (Rosenberg & Zilber-Rosenberg, 2011; Crotti et al., 2012; Engel & Moran, 2013). Our results corroborate the findings of previous studies demonstrating that elevated temperatures usually increase the gut bacterial community of ectotherms (Lokmer & Wegner, 2015; Kohl & Yahn, 2016) but are not in line with the effect of climate warming on vertebrate ectotherms reported by Bestion et al. (2017).
This preliminary study cannot answer completely how climate warming impacts honeybees. Although there is increasing evidence that there may be connection between the gut microbiota balance and the tolerance of honeybees to high temperature and low humidity stresses, the underlying mechanisms are still not clear. The employment and exploitation of microorganisms in a defined environment could solve such practical problems as colony collapse disorders (CCDs) and abiotic stress under climate warming (Crotti et al., 2012). Thus, symbiotic microorganisms can exert their beneficial contribution to the host to sustain its health and to tolerate biotic and abiotic stress in different ways, i.e. by activation or stimulation of the host innate immune system. However, further in-vitro and in-vivo research is needed to clarify the molecular mechanisms of insect gut symbiosis and to better understand how the honeybee gut microbial community would impact the learning, physiological and behavioural mechanisms of the host bees under temperature extremes as manifested in Sepulveda & Moeller (2020).
