Low temperature exposure (20 °C) during the sealed brood stage induces abnormal venation of honey bee wings

<p>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 (<i>Apis mellifera</i>) 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.</p

Low-Temperature Stress during Capped Brood Stage Increases Pupal Mortality, Misorientation and Adult Mortality in Honey Bees

<div><p>Honey bees (<i>Apis mellifera</i>) 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.</p></div

Misorienting pupa and rates of misorientation after cold treatments.

<p>(A) Misorientation of a pupa, (B) ofan eclosed bee and (C) the same misorientated bee shown sideways). (D) Rate of misorientation of different aged brood (±SE) after they were exposed to 20°C for 2 h. (E) Rate of misorientation after 24 h old brood (L1) were exposed to different durations of 20°C. These misoriented bees died due to starvation, even though technically they survived until eclosion. Control group was indicated as “CK”.</p

Time (h ± SE) required to kill 50% of pupae at 20°C (LT₅₀) for different stages of sealed brood.

<p>Time (h ± SE) required to kill 50% of pupae at 20°C (LT₅₀) for different stages of sealed brood.</p

Mean longevity in days (±SE) of various aged bees (vertical) exposed at 20°C for different periods (horizontal).

<p>Mean longevity in days (±SE) of various aged bees (vertical) exposed at 20°C for different periods (horizontal).</p

Mean mortality (±SE) of capped brood.

<p>Each stage was treated with 20°C for different durations (with number of h indicated in parenthesis). Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154547#pone.0154547.g001" target="_blank">Fig 1</a> for age of different groups. Means were based on 3 colonies. Different letters on top of each bar indicate that they are statistically different from others by Student–Newman–Keuls tests after ANOVA showed a significant difference. Control group was indicated as “CK”. ND: not determined because this treatment at shorter durations (48–72 h) all died.</p

Morphology and color changes during the 12 days of capped brood.

<p>Larval (L) stage has 2 days (because we included the 0 h capped brood as the same day of being capped, L0), prepupae (PP) has 2 days and the rest are pupae (P4-P11). On day 12 the worker would eclose (emerge) as an adult.</p

Table1_The mushroom body development and learning ability of adult honeybees are influenced by cold exposure during their early pupal stage.DOCX

The honeybees are the most important pollinator in the production of crops and fresh produce. Temperature affects the survival of honeybees, and determines the quality of their development, which is of great significance for beekeeping production. Yet, little was known about how does low temperature stress during development stage cause bee death and any sub-lethal effect on subsequent. Early pupal stage is the most sensitive stage to low temperature in pupal stage. In this study, early pupal broods were exposed to 20°C for 12, 16, 24, and 48 h, followed by incubation at 35°C until emergence. We found that 48 h of low temperature duration cause 70% of individual bees to die. Although the mortality at 12 and 16 h seems not very high, the association learning ability of the surviving individuals was greatly affected. The brain slices of honeybees showed that low temperature treatment could cause the brain development of honeybees to almost stop. Gene expression profiles between low temperature treatment groups (T24, T48) and the control revealed that 1,267 and 1,174 genes were differentially expressed respectively. Functional enrichment analysis of differentially expressed genes showed that the differential expression of Map3k9, Dhrs4, and Sod-2 genes on MAPK and peroxisome signaling pathway caused oxidative damage to the honeybee head. On the FoxO signal pathway, InsR and FoxO were upregulated, and JNK, Akt, and Bsk were downregulated; and on the insect hormone synthesis signal pathway, Phm and Spo genes were downregulated. Therefore, we speculate that low temperature stress affects hormone regulation. It was detected that the pathways related to the nervous system were Cholinergic synapse, Dopaminergic synapse, GABAergic synapse, Glutamatergic synapse, Serotonergic synapse, Neurotrophin signaling pathway, and Synaptic vesicle cycle. This implies that the synaptic development of honeybees is quite possibly greatly affected by low temperature stress. Understanding how low temperature stress affects the physiology of bee brain development and how it affects bee behavior provide a theoretical foundation for a deeper comprehension of the temperature adaptation mechanism that underlies the “stenothermic” development of social insects, and help to improve honeybee management strategies to ensure the healthy of colony.</p

Morphological differentiation in Asian honey bee (<i>Apis cerana</i>) populations in the basin and highlands of southwestern China

<p>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 <i>Apis cerana</i> 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 <i>A. cerana</i> 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.</p