The molecular mechanism of hot defensive bee ball in Apis cerana japonica

概要

Honeybees of Apis mellifera and A. cerana inhabit in a wide temperature range from tropical to temperate, which is exceptional in insects. This is because honeybees have the ability to control body and nest temperatures by metabolic heat production regulation. Therefore, studying thermoregulation of honeybees is essential for understanding insect temperature adaptation.

A. c. japonica, which is a subspecies of the A. cerana and distributes from Honsyu to Kyusyu in Japan, exhibits specific collective defensive behavior by utilizing thermoregulation against the giant hornet, Vespa mandarinia. This behavior is called a “hot defensive bee ball”, and is performed when the bee nest is attacked by V. mandarinia (Ono et al., 1995). A hot defensive bee ball is formed by hundreds of bee workers catching a hornet. The balling state is maintained for ~ 30 minutes, and its core temperature reaches ~46 °C, which is higher than the lethal temperature of hornets. This temperature is produced by the flight muscles of honeybees. Recently reported that the survival rate of honeybee workers participated in bee ball decreased few days after the hot defensive bee ball (Yamaguchi et al., 2018). Thus, the hot defensive bee ball is an behavior that utilizes temperature regulation near the lethal temperature by applying the heat-generating ability of flight muscles.

To clarify the molecular mechanism related to the thermoregulation during the hot defensive bee ball leads to understanding the thermoregulation in insects. However, only a few researches have been done to elucidate the molecular mechanism of this behavior. Therefore, I conducted two studies to elucidate the molecular mechanism of the hot defensive bee ball. In the chapter 1 of this thesis, RNA-seq was used to analyze gene expression level in the brain, fat body, and flight muscle to detect candidate genes associated with balling behavior. In the chapter 2 of this thesis, in order to clarify the detailed molecular mechanism involved in temperature sensitivity in A. c. japonica, I conducted the heat avoidance experiment using honeybee workers that inhibited the temperature-sensitive channel AmHsTRPA in honeybees.

1. Genes associated with hot defensive bee ball in the Japanese honeybee, A. c. japonica
Materials and methods
In chapter 1, in order to detect candidate genes related to the hot defensive bee ball in A. c. japonica, we measured the gene expression level using RNA-seq. Honeybee workers 15 days after emergence were prepared and sampled under the following three conditions; 30 minutes after the formation of the hot defensive bee ball (“balling”), 30 minutes incubation of standard temperature 31°C (“control”), 30 minutes high temperature 46°C (“heat”). RNA-seq was used to measure gene expression levels in the brain, fat body, and flight muscle. The gene expression levels of “balling” and “control” (“comparison 1”) or “heat” and “control” (“comparison 2”) were compared, and differentially expressed genes (DEG) were calculated. The DEGs included in “comparison 1” from which the DEGs overlapping with “comparison 1” and “comparison 2” was removed was defined as the DEG only in the bee ball (“ball-only”).

Results and discussion
We observed that expression level of genes related to rhodopsin signaling (i.e., rhodopsin long wavelength and arrestin 2) were increased in all three tissues during the hot defensive bee ball in the group of “ball-only” (Figure 1). The expression of other genes involved in rhodopsin signaling (i.e., PLC and arrestin 1) was also increased in the brain.

Rhodopsin is mostly known as a photo-receptor protein that is involved in the downstream GPCR cascade and the light sensitive transient receptor potential channels (Katz & Minke, 2009; Montell, 2012). In Drosophila phototransduction cascade, PLC genes (norpA) involved in the amplification of rhodopsin GPCR signaling (Katz & Minke, 2009). Arrestin ptoteins are known to interact with and inactivate the rhodopsin in Drosophila (Dolph et al., 1993). These "turn-on" and "turn-off" mechanisms in this signaling are important in both sensitivity and temporal resolution of the visual system (Katz & Minke, 2009).

In Drosophila, rhodopsin signaling affects thermotaxis via the temperature-sensitive channel TRPA1 (Shen et al., 2011; Sokabe et al., 2016; Leung & Montel, 2017). In Drosophila, three TRPA group channels of TRPA1, painless, pyrexia act as thermosensor, and deletion of these channels reduces the avoidance from noxious high temperatures (Tracey et al., 2003; Lee et al., 2005; Rosenzweig et al., 2005; Neely et al., 2011). TRPA1 channel is also involved in thermotaxis in third-instar larva (Rosenzweig et al., 2005; Kwon et al., 2008; Shen et al., 2011; Sokabe et al., 2016). Mid- or late-third-instar larvae of Drosophila prefer 18°C (Shen et al., 2011; Sokabe et al., 2016). However. the TRPA1, Rh1, and PLC deleted mutant lose the 18°C preference (Kwon et al., 2008; Shen et al., 2011). Furthermore, mutant of Rh5 and Rh6 lose the 18°C preference in the late-third-instar larvae (Sokabe et al., 2016). These results suggest that rhodopsin signaling regulates the activity of TRPA1 and influences temperature preference in these developmental stage of larvae.

Thus, the results of chapter 1 suggested that the rhodopsin signaling could be associated with temperature sensitivity, presumably high temperatures during hot defensive bee ball. In honeybee of A. mellifera, AmHsTRPA, a hymenoptera specific TRPA channel, is involved in temperature sensing (Kohno et al., 2010). However, it has not been clarified whether AmHsTRPA is involved in high temperature sensitivity in A. c. japonica.

In chapter 1, I also revealed differentially expressed genes in each tissue. In the brain, genes related to behavior, inflammation, and glucose metabolism involved; in the fat body, genes related to energy metabolism, stress tolerance, and immunity involved; in the flight muscle, genes related to the exoskeleton involved in DEGs of “ball-only”.

2. Thermal sensitivity in the Japanese honeybee, Apis cerana japonica
Materials and methods
In chapter 2, in order to clarify whether AmHsTRPA is involved in high temperature sensitivity in A. c. japonica, I performed heat avoidance experiments using honeybee workers which inhibited the AmHsTRPA. Forager bees were captured in front of the nest. The honeybee workers captured were incubated overnight. These honeybee workers were anesthetized, and 1 µl of 10 mM of menthol, which is an AmHsTRPA inhibitor (Kohno et al., 2010), or water wese injected into the head. A honeybee awakened from anesthesia was placed in a case. A honeybee could walk around in the case and drink sugar water freely. The case was placed in a heat block heated at high temperature (55℃) or standard temperature (31℃). The time the bee workers stayed on the heated surface of the case was calculated.

Results and discussion
In honeybee workers injected with water into the brain, the avoidance from high temperature (55℃) was lower than those from low temperature (31℃). I also demonstrated that honeybee workers injected with menthol showed lower avoidance from high temperature (55℃) than those injected with water. These results suggested that inhibition of AmHsTRPA reduced heat avoidance in A. c. japonica.

My results demonstrated that AmHsTRPA was involved in heat avoidance in A. c. japonica. In chapter 1, it was suggested that the temperature sensitivity might be changed by the rhodopsin signaling during the hot defensive bee ball. From these results, I propose the following hypothesis: rhodopsin signaling regulated AmHsTRPA activity, presumably by inhibiting it to reduce the heat avoidance, which results in honeybee workers maintaining bee ball without escaping from the high temperature. However, my thesis did not show a relationship between the rhodopsin signaling and AmHsTRPA. In the future, it is necessary to investigate the relationship between the rhodopsin signaling and AmHsTRPA by functional analysis.