Bingfeng Zhou

Exposure to low temperatures during honeybee development has been shown to impede brain development and affect cognitive function in adult bees. On the other hand, neuronal damage due to oxidative stress has been reported in many cases. Hence, biochemical parameters related to oxidative stress in honeybee pupae brain were determined. The levels of GSH in the pupal brain decreased after 24 h and 48 h of exposure to low temperatures; there were also reduced activities of SOD and CAT enzymes following 48 h of low-temperature treatment compared to the control group. Furthermore, analysis of transcriptome data post-24 h and -48 h low-temperature stress revealed the suppression of the glutathione metabolism and peroxisome pathways in pupal brains. Additionally, expression pattern clustering analysis and KEGG enrichment showed that 10 differentially expressed genes with down-regulated expression trends post-low-temperature treatment were significantly enriched in the peroxisome pathway, including PEX10, highlighting their connection to peroxisome function. RT-qPCR validation was conducted on 11 core enriched genes in pathways identified via GSEA, and all these genes exhibited a downregulated expression pattern, confirming the inhibition of glutathione metabolism and peroxisome function under low-temperature stress. The present study showed that exposing honeybee pupae to low temperatures suppressed both the glutathione metabolism and peroxisome pathways, resulting in increased oxidative stress. This research enhances our understanding of how the pupal brain reacts to cold stress and illuminates the neural damage associated with low temperatures during honeybee capped brood development.

Thermal condition affects the development and growth of ectotherms. The stenothermic honeybee brood, particularly the prepupae, are sensitive to low rearing temperature. The fat body plays important roles in energy reserve and metabolism during the honeybee brood development. To date, the fat body metabolic changes in prepupae responding to cold stress have not been completely understood. In this study, the ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS)-based non-target metabolome was analyzed between the cold-treated (CT, 20 °C, 36 h) and control (CK, 35 °C) fat body in prepupal honeybees. The fat body metabolomic data showed that the levels of 1860 and 254 metabolites were significantly increased and decreased, respectively, in cold-stressed prepupae. These altered metabolites, glutamine, glutamic acid, pyroglutamic acid, and oxidized glutathione, were significantly enriched into glutamine metabolism and glutathione metabolism pathways. Furthermore, the expression levels of glutamine metabolism-related genes, glutaminase (GLS), glutamate dehydrogenase (GDH), and gamma-glutamyl transferase (GGT-1 and GGT-7), were significantly decreased in cold-exposed prepupae compared with the control groups. Meanwhile, the oxidized glutathione (GSSG), but not the reduced glutathione (GSH) content, was increased in the cold-exposed group compared with controls. Collectively, our data revealed the fat body metabolomic changes in larva-to-pupa transition when exposed to cold stress. Our data provided new insights into stenothermic honeybee sensitivity to cold, characterized by perturbation of glutamine metabolism and oxidative stress.

Many species of high-altitude plateaus tend to be narrowly distributed along river valleys at lower elevations due to a limitation of suitable habitats. The eastern honeybee (Apis cerana) is such a species and this study explored the effects of long and narrow geographic distributions on honeybee populations. Genetic differentiation and diversity were assessed across populations of the southeastern Qinghai-Tibet Plateau. A total of 492 honeybee samples from eight sampling sites in four valleys were analyzed for the genetic differentiation and diversity of 31 microsatellite loci and mitochondrial tRNA^leu-COII fragments. The following results were obtained: (1) Microsatellite genetic differentiation coefficients (F_ST) ranged from 0.06 to 0.16, and mitochondrial F_ST estimates ranged from 0.18 to 0.70 for different sampling sites in the same valley, indicating genetic differentiation. (2) Honeybees in adjacent valleys were also genetically differentiated. The F_ST of microsatellites and mitochondria were 0.04–0.29 and 0.06–0.76, respectively. (3) Likely a result of small population sizes, the observed genetic diversity was low. The observed impedance of honeybee gene flow among valleys increased both genetic differentiation and population numbers in the Qinghai-Tibet Plateau. This study contributes significantly to the current understanding of the mechanism underlying population genetic differentiation and highlights the potential effects of utilizing genetic resources that are subject to the ecological conditions of the long and narrow geographic distributions of plateau-valley landforms.

To better understand the morphological characteristics of Apis cerana after a long-time in isolation from the Chinese mainland, coupled with the impact of recent exotic gene flow, multivariate morphometric analyses of A. cerana in Hainan Island were undertaken. A total of 1,020 worker bees of A. cerana were collected from three localities in Guangdong Province, nine localities in Hainan province, and five apiaries that were introduced from the adjacent mainland over two years. In all, 33 morphometric characters were measured, and multiple analyses including analysis of significance of difference, principal component analysis, discriminant analysis and cluster analysis were conducted. The samples were divided into three groups: Guangdong group (M), the introduced honey bee group (I), and the Hainan group (H). The local population in Hainan Province was differentiated from the honey bees collected from Guangdong Province. The population in Hainan was adapted to island environments over a long time in isolation, and this resulted in unusual morphologies, such as the smallest body size, smallest leg, and smallest wing among the populations, as well as a large cubital index. The size of most morphometric characters of Group I were between those of the Group M and Group H. Therefore, we conclude that the honey bee in Hainan Island is still differentiated from the honey bee on the adjacent mainland, even though many exotic alleles have been introduced onto the island in recent decades.

All insects are influenced by low temperature. However, as a typical stenothermic insect, the honey bee is perhaps the most severely affected. Low temperature exposure during the capped brood stages leads to high mortality and shortened worker longevity. The impact of low temperature stress on vein development has not, however, been investigated. In this study, the eight different developmental stages of capped brood (Apis mellifera) were exposed to 20 °C for 12, 24, 36, 48, 60, 72, 84, and 96 h, and then incubated at 35 °C (the optimum temperature for brood development) until emergence. We found a total of 21 abnormal vein types, consisting of 16 types of supernumerary veins and five types of lost veins. The abnormal phenomena in the brood occurred mainly 1–4 days after capping, when the metamorphosis process from larvae to pupa occurs. Our results suggest that this metamorphosis process is critical for the development of veins, indicating it as the most sensitive period to low-temperature. In addition, we found a cross vein “rs-m” between Rs and M in the hind wing of the honey bee, which has previously been ignored due to its very short length. This study on the effects of temperature on wing venation in stenothermic insects adds to our understanding of the thermal requirements for shaping the wing vein pattern, and for predicting wing venation deformation. This also adds a new research route for the investigation of the evolutionary relationship between honey bees and other hymenopteran insects.

In the evolutionary process, honey bee populations became divergent due to geographical barriers or ecological adaptation, but to date which specific barriers and ecological isolation conditions that blocked the honey bee gene flow remain uncertain. Here, the morphometric divergence of Apis cerana was studied across southwestern China, a region of diverse topography, featuring a low-elevation basin in the east and a high-elevation mountain and plateau in the west. A multivariate morphometric analysis was conducted on 1482 A. cerana individuals from 25 sampling sites across Sichuan province. The populations dwelling in high-elevation areas with larger body sizes and darker coloration were differentiated from the eastern low-elevation basin population. Moreover, the populations dwelling in the Jinshajiang, Yalongjiang, and DaduRiver Valleys showed significant divergence. Our data indicate that the morphometric variation between the high and low altitudes, probably due to the adaptation to the integrated effects of ecological factors such as air temperature, humidity, the nectar and pollen plants, and light intensity etc. Additionally, the mountains with an altitude of more than 3300 m and a honey bee-free zone probably served as a barrier to gene flow between the river valley populations.

We aimed to determine the optimal cryopreservation method for eradication of the greater wax moth (Galleria mellonella) from honey bee comb. To pursue this goal, mortality rates of G. mellonella eggs, larvae, pupae and adults were studied after treatment at −15 or −20 °C for varying durations. The results showed that larvae, pupae and adults all died after they were treated for one hour at either −15 or −20 °C. Moth eggs, on the other hand, had strong frost resistance. It was only when the treatment time increased to 10 h that all G. mellonella eggs were killed. Freeze tolerance of 7-day-old eggs was higher than that of 1, 3 and 5 day-old eggs. We propose two protocols for cryopreservation of comb to kill all stages of the greater wax moth.

To study the intraspecies taxonomy of the Western honeybee (Apis mellifera), geometric morphometrics analyses can now replace the standard morphometry method, yet its availability for microtaxonomy of the Asian honeybee (Apis cerana F.) has not been reported. In order to assess effectiveness of geometric morphometrics analyses using the forewing in the microtaxonomy of the Asian honeybee, 180 individual worker bees were sampled from three sample sites, Jilin, Hainan, and Shaanxi, representing different habitats for A. cerana within China. Bees were analyzed by discrimination analysis using three methods: (1) standard morphometry method with 33 traditional characters, (2) simplified standard morphometry method with only 11 wing vein angles, and (3) geometric morphometrics analyses with 18 wing vein junctions. The results revealed that geometric morphometrics analyses could reflect the variability information of the forewing vein structures of A. cerana. Morphological differentiation at the three ...

Honey bees (Apis mellifera) are key pollinators, playing a vital role in ecosystem maintenance and stability of crop yields. Recently, reduced honey bee survival has attracted intensive attention. Among all other honey bee stresses, temperature is a fundamental ecological factor that has been shown to affect honey bee survival. Yet, the impact of low temperature stress during capped brood on brood mortality has not been systematically investigated. In addition, little was known about how low temperature exposure during capped brood affects subsequent adult longevity. In this study, capped worker broods at 12 different developmental stages were exposed to 20°C for 12, 24, 36, 48, 60, 72, 84 and 96 hours, followed by incubation at 35°C until emergence. We found that longer durations of low temperature during capped brood led to higher mortality, higher incidences of misorientation inside cells and shorter worker longevity. Capped brood as prepupae and near emergence were more sensitive to low-temperature exposure, while capped larvae and mid-pupal stages showed the highest resistance to low-temperature stress. Our results suggest that prepupae and pupae prior to eclosion are the most sensitive stages to low temperature stress, as they are to other stresses, presumably due to many physiological changes related to metamorphosis happening during these two stages. Understanding how low-temperature stress affects honey bee physiology and longevity can improve honey bee management strategies.

The complete genome sequence of Chinese sacbrood virus (CSBV), isolated from diseased larvae of Apis cerana in Fujian province, China was analyzed. The viral genome consisted of 8,800 nucleotides, encoding 2,848 amino acids. A phylogenetic tree analysis showed the sacbrood virus (SBV) segregated into three distinct groups. The isolates originated from A. c. indica and were the first distinct evolutionary group. The AcSBV–SBM2 isolate, from A. c. cerana, belonged to the second distinct group. The remaining SBV isolates formed the third group. The phylogenetic relationships of SBV isolates suggest that they are derived from similar honeybee species or geographic origins. The 3C-like cysteine protease protein plays an important role in viral replication. The 3C-like cysteine protease protein of CSBV-FZ was predicted to contain a transmembrane domain. The subcellular localization of 3C-like cysteine protease was distributed as discrete punctate inclusions and co-localized with VP1 of CSBV. These results suggest that the non-structural protein 3C-like cysteine protease might be involved in viral replication. Insect cell cultures can further advance our understanding of picorna-like virus replication.