기형 날개 바이러스로 인하여 기억력 상실한 벌들을 부티르산나트륨이 회복시켜 준다(논문).

[꿀벌나라]

이 논문을 공개하지 않고 나만 알고 있을려고 했지만, 앞전 나의 글 "벌들은 어떻게 바이러스에

감염이 되는가?" 설명할 때, 나의 주관적인 생각이 아니라, 증거 논문에 근거하여 말한 것임을 보여

주기 위함입니다.

개미산으로 응애 구제만 확실히 하면, 필요가 없는 자료입니다.  유기양봉을 하지 않고 약제처리로 

해결할까봐, 또, 약품업자가 장사속으로 이용할까봐 공개를 안했습니다.

기형날개 바이러스(DWV)에 감염된 벌이 월동시 사라지는 이유는, DWV가 벌 머리의 뇌 버섯

모양체에서 복제를 하여 그로 인한 뇌 손상으로 방향 감각을 잃어 귀소를 못하기 때문입니다.

지구 온난화와는 관계가 없습니다.

이제, 증빙 논문을 수회, 순차적으로 공개합니다.

Real-time monitoring of deformed wing virus-infected bee foraging behavior

following histone deacetylase inhibitor treatment

히스톤 탈아세틸화 효소 억제제 처리 후 기형 날개 바이러스에 감염된 벌의 외역 행동의 실시간

모니터링

Cheng-KangTang1, Yu-HsienLin2, Joe-AirJiang34, Yun-HengLu1, Chih-HsuanTsai1, Yu-ChunLin1

Yun-RuChen1, Carol-PWu1, Yueh-LungWu

저자 : 청강탕, 유셴린, 조에어장, 윤형루, 지현차이, 유춘린, 윤루첸, 캐롤-퓨, 위룽우

Highlights

가장 중요한 점

* Sodium butyrate (NaB) reversed learning ability of bees infected by deformed wing virus

NaB(부티르산나트륨)이 기형 날개 바이러스에 감염된 벌들의 학습 능력을 완전히 바꾸어

놓았다

* RNA-Seq showed NaB restored the expression‎ of genes involved in glycolysis and memory

리보핵산 배열(순서)에 기반한 생물학적 기술로 NaB(부티르산나트륨)이 당분해와 기억에

관여하는 유전자의 발현을 회복시켰다는 것을 보여 주었다

* NaB improved the homing ability of deformed wing virus-infected bees

부티르산나트륨은 기형 날개 바이러스에 감염된 벌들의 귀환 능력을 향상시켰다.

Summary

요약

Impairment in the learning/memory behavior of bees is responsible for the massive disappearance of

bee populations and its consequent agricultural economic losses. Such impairment might be because of

both pesticide exposure and pathogen infection, with a key contributor deformed wing virus (DWV).

꿀벌의 학습/기억 행동의 손상은 꿀벌 개체군의 대량 소멸과 그에 따른 농업 경제적 손실의

원인이 된다. 이러한 손상은 살충제 노출과 병원균 감염으로 인한 것일 수 있으며, 주요 원인은

기형 날개 바이러스(DWV)이다.

The present study found that sodium butyrate (NaB) significantly increased survival and reversed the

learning/memory impairment of DWV-infected bees. A next-generation sequencing analysis showed that

NaB affected the expression‎ of genes involved in glycolytic processes and memory formation, which

were suppressed by DWV infection.

현재 연구에서는 부티르산나트륨(NaB)이 DWV에 감염된 벌들의 생존을 상당하게 증가시키고

학습/기억 손상을 되돌리는 놓는 것으로 밝혀졌다. 차세대 염기서열 분석에서는 부티르산나트륨이 

DWV 감염에 의해 억제되어진 당분해 과정과 기억 형성에 관여하는 유전자의 발현에 영향을

끼치는 것으로 밝혀졌다.

In addition, we performed a large-scale movement tracking experiment by using a wireless sensor

network-based automatic real-time monitoring system and confirmed that NaB could improve the

homing ability of DWV-infected bees. In short, we demonstrated the mechanism of how epigenetic 

regulation can resume the memory function of honeybees and suggest strategies for applying NaB

to reduce the incidence of colony losses.

또한, 무선 센서 네트워크 기반의 자동 실시간 모니터링 시스템을 이용하여 대규모 이동

추적 실험을 수행하여 부티르산나트륨(NaB)이 DWV에 감염된 꿀벌의 귀환 능력을

향상시킬 수 있다는 것을 확인했다. 간단히 말해, 우리는 후성 유전적 조절이 꿀벌의

기억 기능을 어떻게 회복할 수 있는지에 대한 메커니즘을 보여주고, 봉군 손실의 발생을

줄이기 위해 부티르산나트륨(NaB)을 적용하는 전략을 제의한다.

Graphical abstract

그래프한 개요

도해 설명 : DWV에 감염되어 부티르산  나트륨을 급이한 봉군은 나가는 벌량과 들어 오는 벌량은 거의 같다. 기억과 학습 유전자가 상향되고, 기억 상실을 회복하게 한다

Subject areas

Entomology; Ethology; Molecular biology; Omics; 

주제 분야

곤충학; 행동생물학; 분자 생물학; 유전체학 

Introduction

개론

* Apis mellifera are globally distributed, economically important insects, having particular value as pollinators 

(Hladun et al., 2012; Potts et al., 2010; Pardo and Borges, 2020). Through ever-improving bee rearing technology,

the number of bee farms has increased over the years (Zhou et al., 2017), as well as the variety of bee-derived

products. According to the Food and Agriculture Organization of the United Nations, the number of artificial

beehives reached 90 million in 2017 (Aizen and Harder, 2009).

서양종 꿀벌은 전 세계적으로 분포되어 있으며, 경제적으로 중요한 곤충으로, 수분 매개자로서

특별한 가치를 지니고 있다. 지속적으로 향상되는 벌 사육기술을 통해, 양봉장의 수와 다양한

양봉 제품이 수년에 걸쳐 증가했다. 유엔식량농업기구(FAO)에 따르면, 2017년 양봉 벌통의 수가

9천만 개에 달했다.

* However, over the last few decades the numbers of both wild and managed bee pollinators have been

declining, consequently decreasing their beneficial effects on ecosystems. Although the reasons for this

decline are under debate, it is highly likely that a combination of multiple stressors is to blame, in particular

deformed wing virus(DWV). This virus has been associated with winter mortality and colony losses, is globally

distributed, and has been shown to affect 50–75% of honeybee hives (Ryabov et al., 2014). DWV is an

RNA virus with a positive single-stranded RNA genome of 10,140 nt (GenBank accession NC_004830.2) and

belongs to the Picornaviridae family (Lanzi et al., 2006).

그러나 지난 수십 년 동안 야생 및 양봉벌 수분 매개체의 수가 감소하여, 결과적으로 생태계에

대한 이로운 효과가 감소했다. 이러한 감소에 대한 이유는 논란의 여지가 있지만, 여러 

스트레스 요인의 복합, 특히 기형 날개 바이러스(DWV)가 원인일 가능성이 높다. 

이 바이러스는 겨울철 사망률 및 봉군 감소와 관련이 있으며, 전 세계적으로 분포하며, 꿀벌

벌통의 50-75%에 영향을 주는 것으로 나타났다. DWV는 양성 단일 가닥 RNA 게놈이 10,140 

nt인 RNA 바이러스(GenBank 가입 NC_004830.2)이며 「피코르나바이러스」과 계통에 속한다.

* DWV infects bee species such as Apis mellifera, Apis cerana, and Bombus terrestris (Ai et al., 2012; 

Kajobe et al. Genersch et al., 2006), with all growth stages susceptible (di Prisco et al., 2016; 

Martin et al., 2012; de Miranda and Genersch, 2010). Previous studies have demonstrated that the

 Varroa mite can increase the frequency of DWVs from 10 to 100%, making it one of the biggest factors

causing the worldwide decimation of bee colonies. Varroa destructor harbors virus levels higher than

those seen in severely infected bees, and V. destructor beehive infestations may be related to severe

DWV infections (Bowen-Walker et al., 1999).

DWV는 서양종, 동양종 봉군 및 서양 뒤영벌과 같은 벌 종을 감염시키며, 모든 성장 단계가

영향을 받기 쉽다. 이전 연구에 바로아 응애는 DWV의 빈도를 10%에서 100%로 증가시켜,

전 세계적으로 벌 봉군을 대량 파괴하는 가장 큰 요인 중 하나가 된다. 바로아 응애는 심하게

감염된 벌들에서 볼 수 있는 것보다 높은 수준의 바이러스를 보유하고 있으며, 바로아 응애

벌통 만연은 심각한 기형날개 바이러스(DWV) 감염과 관련이 있을 수 있다.

*Thus, V. destructor may serve as both a concentrating vector and replicating incubator for the virus as

it can magnify and increase the effects of the virus on bees and beehives. Even though most infected bees

show no obvious symptoms before metamorphosis, seriously infected bees will have wrinkled and

deformed wings after metamorphosis, resulting from the replication and accumulation of DWV

in the brain and in the three/four segments of the abdomen. Infection leads to deprivation of nutrients

which may cause further impairment of the nervous system and therefore impact on survival

(Brettell et al., 2017).

따라서 바로아 응애는 벌과 봉군에 대한 바이러스의 영향을 확대 및 증가시킬 수 있으므로,

바이러스의 농축 매개 곤충 및 복제 배양기로서 역할을 할 수 있다. 대부분의 감염된 벌들은

변태 전에는 뚜렷한 증상을 보이지 않지만, 심각하게 감염된 벌들은 변태 후에 주름이 생기고

날개가 변형되어, 뇌와 복부의 3/4 환절에서 DWV가 복제되고 축적된다. 감염은 신경계의

추가 손상을 유발할 수 있는 영양소 결핍으로 이어져 생존에 영향을 미칠 수 있다(브레텔 외,

2017).

The DWV infection rate reaches a peak in winter, leading to the massive death of the worker bees

(Genersch et al., 2010; Berthoud et al., 2010).In addition to lethality, it has also been reported that

replication of DWV in the mushroom body of the bee brain results in decreased neural signal transduction 

(Shah et al., 2009). The learning/memory ability of worker bees infected with DWV was found to be

significantly impaired as compared to uninfected worker bees, based on learning behavioral studies

(Traniello et al., 2020; de Miranda and Genersch, 2010). This may explain the massive disappearance

of worker bees in hives positive for DWV infection, because impairment of the brain would prevent them

from successfully navigating back to their hives.

DWV(기형날개 바이러스) 감염률은 겨울철에 최고조에 달하여, 일벌의 대량 사망으로 이어진다.

치명적인 것 외에도, 벌 뇌의 버섯 모양체에서 DWV의 복제는 신경 신호 전달을 줄인다고 또한

보고되어 왔다. 학습 행동 연구에 따르면, DWV에 감염된 일벌의 학습/기억 능력은 감염되지

않은 일벌에 비해 현저히 손상된 것으로 나타났다. 이것은 DWV 감염에 양성인 일벌이 벌통에서

대량으로 사라지는 것을 설명할 수 있는데, 왜냐하면, 뇌의 손상으로 벌들이 벌통으로 성공적으로

돌아갈 수 없기 때문이다.

Whether if it is by direct death or reduced homing ability, the loss of worker bees in hives results in

insufficient food (pollen and honey) for the developing larvae. The hives become poorly maintained,

leading to the eventual collapse of the colony. DWV infections were first reported in Japan, and have

since been reported in Europe, North America, and Africa. At least three main strains, including

DWV-A, DWV-B, and DWV-C, have been reported. DWV-A is classified into two subtypes: DWV and

Kakugo virus (KV) (Lanzi et al., 2006; Fujiyuki et al., 2005).

DWV-B, which can replicate in V. destructor and honeybees (Ongus et al., 2004), has a nucleotide identity,

which is 84% similar to that of DWV-A. A third variant, DWV-C, reported by Mordecai et al. (2016),

is an established variant rather than a recently emerged strain (Mordecai et al., 2016).

직접적인 죽음에 의한 것이든 귀환 능력의 감소에 의한 것이든, 벌통에 있는 일벌의 손실은 

발육하는 유충에게 음식(화분 및 꿀)의 부족을 초래한다. 벌통(봉군)은 제대로 관리되지 않아,

결국에는 봉군은 붕괴가 된다. DWV 감염은 일본에서 처음 보고되었으며, 이후 유럽, 북미 및

아프리카에서도 보고되어 왔다. DWV-A, DWV-B 및 DWV-C를 포함한, 적어도 세 가지 주요

균주가 보고되었다. DWV-A는 DWV와 카쿠고 바이러스(KV)의 두 가지 하위 유형으로 분류된다.

바로아 응애와 꿀벌에서 복제할 수 있는, DWV-B는, DWV-A와 84% 유사하게 뉴클레오티드가

일치한다. 2016년 모르데카이 등에 의해 보고된, 세 번째 변종인 DWV-C는, 최근에 출현한

균주가 아닌 확립된 변종이다.

주)뉴클레오티드 ((핵산의 구성 성분))

Among them, DWV-B and DWV-A-VDV1 recombinant are known to be the most virulent variants

(Moore et al., 2011; Ryabov et al., 2014).The transmission modes of DWV have been studied, and

show that the virus can be vertically or orally transmitted through nursing bees to larvae and

horizontally transmitted through predators feeding on infected bees (Amiri et al., 2018; Yanez et al., 2012; 

Chen et al., 2006). However, an efficient strategy to prevent and manage viral infection in bee rearing

has yet to be developed. Histone deacetylase inhibitors (HDACi) are a class of compounds which prevent

 deacetylation of histones and therefore increase gene expression‎ (Marks et al., 2000; Fuchikami et al., 2016).

그 중에서, DWV-B와 DWV-A-VDV1 재조합형이 가장 치명적인 변종으로 알려져 있다.

DWV의 전염 방식이 연구되어 왔으며, 바이러스는 내역봉을 통해 유충에 수직 또는 구강으로

전염될 수 있으며 감염된 벌을 잡아먹는 포식자를 통해 수평으로 전염될 수 있다. 그러나,

꿀벌 사육에서 바이러스 감염을 예방하고 관리하기 위한 효율적인 전략은 아직 개발되지

않고 있다. 히스톤 탈아세틸화효소 억제제(HDACi)는 히스톤의 탈아세틸화를 방지하여 유전자

발현을 증가시키는 화합물의 한 종류이다.

주) 히스톤(histone) : 단순 단백질의 하나

These epigenetic regulators have been eval‎uated for their application in medical treatments, including

anti-inflammatory response, anti-neural degeneration, anti-cancer, and anti-Alzheimer’s disease

(Shein and Shohami, 2011; Mckinsey, 2011; Consalvi et al., 2011; Wu et al., 2007). Feeding aged mice with

 sodium butyrate (NaB; Formula name: NaC4H8O2), a HDACi, can significantly improve their memory

(Garcez et al., 2018; Blank et al., 2015). NaB is found in plants and also in human guts as a fermentation

product. NaB could work on histone deacetylase (HDAC) class I, including HDAC1, HDAC2, HDAC3, and HDAC8.

In our previous study (Hu et al., 2018), we found that including NaB in the diet of honeybees resulted in

a significant increase in the expression‎ of acetyl histone H3 and H4 and the genes involved in immune

response, detoxification, and learning/memory.

이러한 후성 조절 인자는 항염증 반응, 항신경 변성, 항암 및 항알츠하이머병을 포함한, 의학적 

치료에서의 적용에 대해 평가를 해 왔다. 나이든 쥐에게 HDACi (히스톤 탈아세틸화 효소 억제제)인

부티르산나트륨(NaB, 화학식명: NaC4H8O2)을 먹이면 기억력이 크게 향상될 수 있다.

부티르산나트륨(NaB)은 발효 산물로 식물에서 발견되며 인간의 내장에서도 발견된다. NaB는

HDAC1, HDAC2, HDAC3 및 HDAC8을 포함하는, 히스톤 탈아세틸화효소(HDAC) 1등급에 작용한다.

우리의 이전 연구에서, 꿀벌의 식단에 NaB를 포함하면 아세틸 히스톤 H3 및 H4 및 면역 반응, 해독 및

학습/기억에 관여하는 유전자의 발현을 크게 증가시킨다는 사실을 발견했다.

Furthermore, the addition of NaB restored learning ability in neonicotinoid-treated bees, proving that

the functionality of NaB could enhance the ability of bees to resist outer stress (Hu et al., 2017, 2018).

As a valuable economic insect, the loss of honeybees, particularly through Colony Collapse Disorder (CCD),

has attracted much attention; yet, much remains unknown about the causes, mechanisms, and potential

treatment or prevention of CCD. Because of the previously found anti-stress effects of NaB on bees,

the present study set out to eval‎uate the potential of NaB in preventing DWV-induced colony collapse.

또한, NaB를 추가하면 「네오니코티노이드」로 처리한 벌들의 학습 능력이 회복되어, NaB의 기능이 

외부 스트레스에 저항하는 벌들의 능력을 향상시킬 수 있음을 증명했다(후 外 2017, 2018).

귀중한 경제 곤충으로서, 꿀벌의 손실은 특히 봉군붕괴현상(CCD)을 겪으면서 많은 관심을 끌었다.

그러나 CCD의 원인, 메카니즘(구조) 및 잠재적인 처리방법 또는 예방에 대해서는 아직 많은 것이

알려져 있지 않다. 이전에 발견한 NaB의 벌들에 대한 항스트레스 효과 때문에, 본 연구는 DWV에

의해 유발된 봉군 붕괴를 예방하는 NaB의 잠재력을 평가하기 시작했다.

주) 네오니코티노이드(neonicotinoid) : 니코틴과 유사한 방식으로 중추신경계에 작용하는 특정 종류의 곤충살충제.

We determined the survival rate of DWV-infected bees after feeding them with NaB and investigated the

learning/memory ability of bees by a proboscis extension response (PER) assay. Next-generation sequencing (NGS)

was performed to comprehensively assess genes that underwent significant changes in expression‎ as a result of

DWV infection and dietary sodium butyrate. As NGS revealed that NaB increased the expression‎ of genes

involved in carbohydrate metabolism and learning/memory, we examined the detailed physiological changes

in bee brains upon DWV infection and NaB addition.

우리는 벌들에게 NaB를 급이한 후 DWV에 감염된 벌들의 생존율을 알아내었고 주둥이 확장 반응(PER)

분석을 통해 벌들의 학습/기억 능력을 조사했다. 차세대 염기서열분석(NGS)은 DWV 감염과 식이성

부티르산 나트륨의 효과에 따라 발현하는데 상당한 변화를 겪은 유전자를 종합적으로 평가하기 위해

수행되었다. NGS가 NaB가 탄수화물 대사 및 학습/기억에 관여하는 유전자의 발현을 증가시키는 것으로

밝혀짐에 따라, 우리는 DWV 감염 및 NaB 첨가에 대하여 꿀벌 뇌의 상세한 생리학적 변화를 조사했다.

주) 차세대 염기 서열 분석(NGS) : 다량의 DNA를 병렬로 처리하여 기존 방법보다 시간과 비용을 줄인 염기 서열 분석 방법

Finally, to validate the feasibility of applying NaB in hive maintenance, a real-time monitoring system,

the “wireless sensor network (WSN)-based automatic monitoring system (Jiang et al., 2016)”, was utilized

to track the movement of DWV-infected bees with or without a NaB diet on a large scale that simulated

real field conditions. This study uncovered gene regulations under DWV infection and proved that

the epigenetic regulator NaB could be used to rescue the impaired physiological functions after virus infection.

A detailed mechanism of the NaB effects is discussed and feasible in-field treatment strategies are provided.

마지막으로, 벌통을 유지 관리하는데 NaB 적용 가능성을 검증하기 위해 실시간 모니터링 시스템인,

"무선 센서 네트워크(WSN) 기반 자동 모니터링 시스템을" 활용하여 실제 현장 조건을 시뮬레이션한

대규모 NaB 식단을 급이하였거나 하지 않는 DWV 감염된 벌들의 이동을 추적했다.

이 연구는 DWV 감염에 따른 유전자 조절을 밝혀냈고, 후성유전 조절자인 NaB가 바이러스 감염 후에

손상된 생리적 기능을 구조하는데 사용될 수 있음을 입증했다.

NaB 효과의 자세한 메커니즘이 논의되고 실행 가능한 현장 치료 전략이 제공된다.

Results 

성과들

Sodium butyrate (NaB) significantly reversed the low survival rate and learning ability of DWV-infected

bees. In the present study, we first examined whether the intake of NaB can decrease the negative impacts of

DWV infection in bees.

부티르산 나트륨(NaB)은 DWV에 감염된 벌들의 낮은 생존율과 학습 능력을 크게 반전시켰다.

본 연구에서는, NaB 섭취가 벌들에서 DWV 감염의 부정적 영향을 줄일 수 있는지 먼저 조사했다.

It has been suggested that severe infection by DWV may lead to a high mortality rate in worker bees

(Schroeder and Martin, 2012). Therefore, we determined the survival rate of DWV-infected bees with and

without the addition of NaB to their diet. First, in order to confirm‎ that DWV can infect bees through feeding,

we measured the virus copy number in the hemolymph and brain of the bees after five days of feeding.

The results showed that the virus successfully infected bees through this method. In addition, we also

analyzed the effect of dietary NaB on virus replication; the results showed that NaB could inhibit DWV

replication (Figure S1).

DWV에 의한 심각한 감염은 일벌의 높은 사망률로 이어질 수 있다고 제안되었다. 따라서, 우리는

식단에 NaB를 첨가하거나 첨가하지 않은 DWV에 감염된 벌들의 생존율을 밝혀 내었다. 먼저, DWV가

먹이를 통해 꿀벌을 감염시킬 수 있음을 확인하기 위해 급이한지 5일 후 벌들의 혈림프(체액) 및 뇌에서

바이러스 카피 수를 측정했다. 결과는 바이러스가 이 방법을 통해 벌들을 성공적으로 감염시킨 것으로

나타냈다. 또한, 우리는 식이성 NaB가 복제에 영향을 주는지도 분석했다; 결과는 NaB가 DWV 복제를

억제시킬 수 있음을 보여주었다(그림 S1).

주)혈림프(hemolymph) : 절지〔연체〕동물의 혈강을 흐르는 체액

When bees were infected with DWV, 50% of the bees died by the end of day 2 post-infection and only 10%

survived by the end of day 5 post-infection (Figure 1A) (p < 0.05). When NaB was added to the diet prior to

DWV infection, the survival rate of DWV-infected bees (N/D group) remained >90% after 5 days (Figure 1A).

It is interesting to note that under these laboratory rearing conditions, around 30% of the control bees

died over a period of 5 days (Control group); however, when NaB was included in the diet of the uninfected bees,

less than 15% of the bees died. These results indicate that feeding bees with NaB could significantly increase

their survival with or without DWV infection

벌들이 DWV에 감염되었을 때, 꿀벌의 50%는 감염 후 2일이 끝날 무렵에 죽고 10%만이 감염 후

5일째가 끝날 무렵까지 생존했다(그림 1A)(p <0.05).

DWV 감염 전에 NaB를 음식(식단)에 첨가했을 때, DWV에 감염된 벌들(N/D 그룹)의 생존율은

5일 후에 90% 이상으로 유지되었다(그림 1A).

이러한 실험실 사육 조건 아래서, 대조군 벌들의 약 30%가 5일 동안 죽었다는 점은 흥미롭다(대조군);

그러나, 감염되지 않은 벌들의 식단에 NaB가 포함되었을 때, 벌들의 15% 미만이 죽었다. 

이러한 결과는 벌들에게 NaB를 먹이는 것이 DWV 감염 여부에 관계없이 생존을 상당히 증가시킬 수 

있음을 나타낸다.

Figure 1. Sodium butyrate increases the survival rate of DWV-infected bees by reversing memory loss

그림 1. 부티르산나트륨은 기억 상실을 반전시켜 DWV에 감염된 벌의 생존율을 증가시킨다(A)

(A) Survival rate of DWV-infected (DWV), sodium butyrate-fed (NaB), DWV-infected and sodium butyrate-fed

(N/D) bees and bees without any treatment (Control). For the DWV group, bees were fed with a solution

containing 106 virus copy number/bee. For the NaB group, bees were fed with 10 mM of sodium butyrate.

For the N/D group, bees were fed with 10 mM sodium butyrate for 7 days, followed by infection with DWV.

Three independent experiments were performed and 35 bees were included in each group for every experiment.

The survival rate after DWV infection was recorded for 5 days. Statistical analysis was performed using

Student’s t test; “∗” indicates p < 0.05 as compared to the Control group. The data are presented as mean ± SD.

 DWV 감염(DWV)된 벌들, 부티르산나트륨(NaB)을 급이한 벌들, DWV 감염되어 부티르산나트륨을

급이한(N/ D) 벌들 및 어떤 처리도 하지 않은 벌들(대조군).

DWV 그룹의 경우, 벌들은 106개 바이러스 복제본 수/벌이 들어 있는 용액을 먹었다. NaB 그룹의 경우,

벌들에게 10mM의 부티르산나트륨을 먹였다. N/D 그룹의 경우, 꿀벌에게 7일 동안 10mM

부티르산나트륨을 급이한 후에, DWV에 감염되었다. 3개의 독립적인 실험이 수행되었으며 모든

실험에 대해 각 그룹에 35마리의 벌들이 포함되어 있다.

DWV 감염 후 생존율을 5일 동안 기록되었다. 통계 분석은 스튜던트 t 테스트를 사용하여 수행되었다.

 "*"는 대조군과 비교하여 p < 0.05를 나타낸다. 데이터는 평균 ± SD로 표시된다.

(B) PER assay results from days 1–5 post-DWV infection with or without 10 mM sodium butyrate treatment.

The memory formation was expressed as the percentage of PER in each group (n = 35 bees) to antennal

stimulation. The data are presented as mean ± SD from 3 independent experiments and analyzed using

a Mann–Whitney U test. An asterisk indicates a statistically significant difference between the treatment

group and the Control group (∗p < 0.05).

(B) PER 분석은 10mM 부티르산 나트륨 처리 유무에 관계없이 DWV 감염 후 1-5일째 나온 결과이다. 

기억 형성은 안테나 자극에 대한 각 그룹(n = 35마리 벌들)의 PER의 백분율로 표현하였다. 

데이터는 3개의 독립적인 실험에서 평균 ± SD로 표시되고, 만-휘트니 U 검증을 사용하여 분석된다.

별표는 처리군과 대조군 사이의 통계적으로 상당한 차이를 가리킨다(*p < 0.05).

(C) Western blotting analysis of the expression‎ of histone proteins in the brain. Bees were treated with DWV

or sodium butyrate and brain lysates were prepared after treatment to analyze the expression‎ of acetylated

histone protein H3 and H4. Lysate prepared from bees without any treatment served as the control.

The expression‎ level of actin in each sample was also detected and used as a loading control.

Signals of acetyl-histone H3 and H4 were first normalized to that of actin and the normalized acetyl-histone H3

and H4 signals of the control group were set to 1. Signals from treatment groups were adjusted accordingly

and expressed as relative protein intensity. Statistical analysis was performed using Student’s t test;

an asterisk indicates a statistically significant difference between the treatment and control groups (∗p < 0.05).

The data are presented as mean ± SD.

(C) 뇌에서 히스톤 단백질의 발현에 대한 웨스턴 블로팅 분석. 

벌들에게 DWV 또는 부티르산나트륨을 처리하고 처리 후 뇌 용해물을 준비하여 아세틸화 히스톤 

단백질 H3 및 H4의 발현을 분석하였다. 

어떠한 처리도 하지 않은 벌로부터 준비된 용해물을 대조군으로 사용하였다. 각 샘플에서 액틴의 

발현 수치도 검출하여 부하 제어로 사용하였다. 아세틸-히스톤 H3 및 H4의 신호를 먼저 

액틴의 신호로 정규화하고 대조군의 정규화된 아세틸-히스톤 H3 및 H4 신호를 1로 설정하였다. 

처리군의 신호를 그에 따라 조정하고 상대 단백질 강도로 표현하였다. 통계 분석은 스튜던트 t 

검정을 사용하여 수행하였다. 별표는 처리군과 대조군 사이의 통계적으로 상당한 차이를 

나타낸다(*p < 0.05). 데이터는 평균 ± SD로 표시된다.

주) 웨스턴 블롯팅 분석(western blotting analysis) : 단백질 발현 분석법

NaB has been shown to reverse memory impairment in bees caused by exposure to pesticides (Hu et al., 2017).

The results of our proboscis extension reflex (PER) experiment showed that DWV infection significantly impaired

the learning/memory ability of bees (Figure 1B). While bees were fed with NaB prior to DWV infection,

the infected bees exhibited a similar level of learning/memory ability as those uninfected (p < 0.05).

This suggests that DWV infection could cause learning and memory impairment in bees, and this could be

reversed or prevented by NaB supplement.

NaB는 살충제 노출로 인한 벌들의 기억력 손상을 되돌려 놓는 것으로 밝혀졌다(후 외, 2017). 

PER 실험의 결과는 DWV 감염이 벌들의 학습/기억 능력을 현저하게 손상시키는 것으로 나타났다

(그림 1B).  DWV 감염되기 전에 벌들에게 NaB를 급이하였지만, 감염된 벌들은 감염되지 않은

벌들과 유사한 수준의 학습/기억 능력을 나타내었다(p < 0.05). 이것은 DWV 감염이 벌들의 학습

및 기억 장애를 유발할 수 있으며, 이는 NaB 보충제를 줌으로써 반전되거나 예방될 수 있음을

시사한다.

NaB is an HDACi that inhibits the deacetylation of the histone core, resulting in increased gene expression‎.

The expression‎ of acetylated histones, specifically H3 and H4, in the brains of NaB-fed bees was analyzed;

it was found that NaB significantly increased acetylated H3 and H4 expression‎ (acetyl-H3 and acetyl-H4)

(Figure 1C) (p < 0.05). Based on these results, we postulate that NaB helped to improve learning and

memory by up-regulating histone modification and subsequently the expression‎ of genes involved in

learning and memory.

NaB는 핵심 히스톤 단백질의 탈아세틸화를 억제하여, 유전자 발현을 증가시키는 HDACi(히스톤

탈아세틸화효소 억제제) 이다.

NaB를 먹인 꿀벌의 뇌에서, 아세틸화된 히스톤, 특히 H3와 H4의 발현을 분석하였다.

NaB는 아세틸화된 H3 및 H4 발현(아세틸-H3 및 아세틸-H4)을 상당하게 증가시키는 것으로

밝혀졌다(그림 1C)(p < 0.05). 이러한 결과를 바탕으로, 우리는 NaB가 히스톤 변형을

상향 조절하여 학습과 기억에 관여하는 유전자의 발현을 조절함으로써 학습과 기억을

향상시키는데 도움이 된다고 가정한다.

주) histone core =core histone protein (핵심 히스톤 단백질)

NaB enhances the expression‎ of genes involved in diverse biological processes in bees

To identify the possible gene regulation that rescues the negative effects of DWV infection on memory

in NaB treatment, NGS was used to elucidate gene expression‎ profiles in untreated bees (Control),

DWV-infected bees (DWV), NaB-fed bees (NaB), and DWV-infected bees fed with NaB (N/D).

After aligning and quantifying the NGS reads with the genes in the honeybee genome, an expression‎ library

for each treatment group was established. In the principal component analysis (PCA), each treatment group

had a distinct gene expression‎ profile and did not cluster with one another (Figure 2A).

NaB는 벌들의 다양한 생물학적 과정에 관여하는 유전자의 발현을 향상시킨다. NaB 치료에서, 

DWV 감염이 기억력에 미치는 부정적인 영향을 구제하는데 가능한 유전자 조절을 확인하기 위해,

NGS를 사용하여, 처리되지 않은 벌들(대조군), DWV에 감염된 벌들, NaB를 급이한 벌들, 그리고

NaB를 먹인 DWV에 감염된 벌들의 유전자 발현 프로파일(개요) 설명하였다

꿀벌 게놈의 유전자와 NGS 판독값을 정렬하고 정량화한 후, 각 처리 그룹에 대한 발현 라이브러리를 

확립하였다. 주성분 분석(PCA)에서, 각 처리 그룹은 고유한 유전자 발현 프로파일을 갖고 서로 

무리를 지어 이루지 않았다(그림 2A).

A total of 4,320 genes showed differential expression‎ in the treatment groups compared to the control group:

2,344 genes in the DWV group, 2,367 genes in the NaB group, and 2,226 genes in the N/D group

(Figure 2B and Data S1). Among these genes, 673 were found in all 3 groups and the DWV and NaB groups

had the most uniquely expressed genes (868 genes), which were not found in the other 2 groups.

DWV 그룹에서 2,344개의 유전자, NaB 그룹에서 2,367개의 유전자, N/D 그룹에서 2,226개의 

유전자(그림 2B 및 데이터 S1)의 총 4,320개의 유전자가 대조군과 비교하여 처리 그룹에서

차등 발현을 보였다.

이들 유전자들 중에서, 3개 군 모두에서 673개가 발견되었고, DWV 및 NaB 군이 다른 2개 군에서

발견되지 않는, 가장 독특하게 발현된 유전자(868개 유전자)를 가졌다.

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Figure 2. Analysis of gene expression‎ profile in experimental groups by using next-generation sequencing (NGS)

그림 2. 차세대 염기 서열 분석(NGS)을 이용한 실험군의 유전자 발현 프로파일(개요) 분석

(A) Genes were differentially expressed between the treatment groups. A scatterplot of the principal

component analysis of the gene expression‎ profile of the 3 experiment groups and 1 control group

is shown on the left. The maximum variance is 37.99% for PC1 and 32.68% for PC2. A correlation heatmap

was constructed and shown on the right. The number indicates the degree of correlation in the gene

expression‎ profile between 2 indicated groups and the strongest correlation was 1 by comparing between

the same group. Red and blue color indicate a positive and negative correlation, respectively. Control:

no treatment; DWV: DWV-infected; NaB: sodium butyrate-treated; N/B: DWV-infected with sodium butyrate 

treatment.

(A) 유전자는 처리군 간에 차등적으로 발현되었다. 3개의 실험군과 1개의 대조군의 유전자 발현 

프로파일의 주성분 분석 산점도를 왼쪽에 나타내었다. 최대 분산은 PC1의 경우 37.99%, PC2의 

경우 32.68% 이다. 상관 열지도가 구성되어 오른쪽에 표시된다. 숫자는 표시된 2개의 그룹 간의 

유전자 발현 프로파일의 상관 정도를 나타내며, 동일한 그룹 간의 비교에서 가장 강한 상관 관계는 

1이었다. 빨간색과 파란색은 각각 양의 상관관계와 음의 상관관계를 나타낸다. 대조군: 

처리하지 않음; DWV 감염; NaB: 부티르산나트륨으로 처리함; N/B: 부티르산나트륨 처리로 DWV에 

감염됨.

(B) Venn diagram showing the concordance of differentially expressed genes in treatment groups.

(C) Hierarchical clustering of gene expression‎ in the brain of bees in treatment groups. Each column

represents the gene expression‎ profile from a treatment group and each row represents the logarithm-transformed

expression‎ values of a particular gene in 3 samples. Red and blue color indicate up- and down-regulation,

respectively. The functions of genes in the clustering with the most diverse expression‎ profiles in 3 groups

are listed on the lower-right side of the heatmap.

(B) 처리군에서 차등적으로 발현된 유전자의 일치를 보여주는 벤 다이어그램.
(C) 처리군에서 벌들의 뇌에서 유전자 발현의 계층적 클러스터링. 각 열은 처리 그룹의 유전자 

발현 프로필을 나타내고 각 행은 3개의 샘플에서 특정 유전자의 대수 변환된 발현 값을 나타낸다. 

빨간색과 파란색은 각각 상향 및 하향 조절을 나타낸다. 3개 그룹에서 가장 다양한 발현 

프로파일을 가진 클러스터링에서 유전자의 기능은 적외선 열지도의 오른쪽 하단에 나열된다.

(D) Semantic space scatterplot of gene ontology annotations associated with the most differentially-expressed

genes in the heads harvested from DWV-infected bees with or without sodium butyrate treatment. Clusters of

highly similar GO terms were formed using REVIGO with enrichment p value ≤ 0.05. Two-dimensional spatial

organization of the enriched annotations was based on semantic similarity. The color of the bubble indicates

the logarithm of the p value used for each annotation with yellow representing p values closer to 0.05 and

purple colors representing p values much smaller than 0.05. The size of the bubble represents the number of

genes included in each cluster.

(D) 부티르산나트륨 처리 여부에 관계없이 DWV에 감염된 벌들에서 수확한 머리에서 가장 다르게

 발현된 유전자와 관련된 유전자 존재론 주석의 의미 공간 산점도. 매우 유사한 GO 용어의 

클러스터는 농축 p 값이 ≤ 0.05인 REVIGO를 사용하여 형성되었다. 풍부한 주석의 평면 

공간 구성은 의미론적 유사성을 기반으로 한다. 비눗방울 모양의 색상은 각 주석에 사용된 p 값의

로그를 나타내며 노란색은 0.05에 가까운 p 값을 나타내고 보라색은 0.05보다 훨씬 작은 p 값을

나타낸다. 비눗방울 모양의 크기는 각 클러스터에 포함된 유전자의 수를 나타낸다.

<이하 번역은 생략함, 실험 내용이므로 어렵고 알 필요는 없어 생략합니다>

Gene ontology of these differentially expressed genes was constructed with their relative respective expression‎ in each group. There were 219 genes whose expressions were highly up-regulated in the N/D group but significantly suppressed in the DWV group. These genes were involved in signal transduction, the G protein-coupled receptor signaling pathway, regulation of biological and cellular processes, biological regulation, integral component of membrane, cellular component, and intrinsic component of membrane (Figure 2C). Of these 219 genes, 53 exhibited Log rER >0.05 between the N/D group and the DWV group (Table S1) and many were involved in memory behavior.

A semantic scatterplot of these 53 genes yielded clusters with high levels of relatedness (Log p value < −2.5) including RNA processing, the glycolytic process, regulation of gene expression‎ and epigenetic, as well as learning and memory (Figure 2D). The NGS results thus indicate that NaB might help repair the impairments caused by DWV infection through several different mechanisms, including directly inducing the expression‎ of memory and learning-related genes or modulating the cellular processes.

Gene Ontology - an overview | ScienceDirect Topics

NaB restores neurotransmitters in the bee brain via the glycolytic process

In NGS analysis, genes involved in the glycolytic process were among the genes whose expression‎ levels were significantly different in DWV-infected bees with or without NaB treatment. Glycolysis is important for energy generation and involves a sequence of enzymatic reactions that convert a glucose molecule into two pyruvate molecules and generates adenosine triphosphate as a product. Previous research found that mushroom body glycolysis is required for olfactory memory in Drosophila (Wu et al., 2018).

Disruption of glycolysis in the brain may be one pathogenesis of memory impairment caused by DWV infection, and NaB might antagonize the disruption to restore memory functions. To verify this assumption, we analyzed the expression‎ of glycolytic enzyme genes in the brain of honeybees after exposure to DWV, NaB, or a combination of both. In order to confirm‎ that DWV can successfully infect bees via feeding, we measured the virus copy number in the brain of bees after five days post infection. After confirm‎ing virus infection, the same samples were used for viral gene expression‎ assay (Figure S2). The expressions of glycolytic enzymes glucokinase (gk), phosphofructokinase (pfk), and pyruvate kinase (pyk) were determined by qPCR. The results showed that expression‎ levels decreased significantly after bees were infected with DWV, whereas the decrease was not as significant in the N/D group, suggesting that NaB might help prevent the negative effect on gene expression‎ induced by DWV (Figure 3A). At the same time, we also analyzed the ATP level, which yielded a similar result (Figure 3B). ATP provides energy for neurotransmission and is also involved in regulating neurotransmitters (Gold et al., 1986). The main neurotransmitter in bees is glutamate, which is normally synthesized via the Glu-Gln cycle. The concentration of glutamate and the expression‎ level of glutaminase mRNA in the brain was measured; the result showed that glutamate concentration in the brain decreased significantly after bees were infected with DWV (Figure 3C) (p < 0.05) and that glutaminase mRNA expression‎ decreased (Figure 3D). When bees were administered NaB prior to DWV infection (N/D group), the decrease in glutaminase gene expression‎ was not as significant. This suggests that DWV infection negatively influences glutamate synthesis (via a decrease in glutaminase) and can be partially recovered by NaB as it mediates glutaminase gene expression‎ (Figure 3D). N-methyl-D-aspartate receptor (NMDAR) is a major subunit of the glutamate receptor and is involved in the plasticity of dendrites and memory function. The expression‎ levels of NMDA receptor subunits NR1 and NR2 were measured to assess whether their expression‎ was affected by different levels of glutamate. Similarly, to the results for glutaminase, the expression‎ levels of nr1 and nr2 decreased in DWV-infected bees compared to the control, and the pre-treatment of bees with NaB rescued this decrease (Figure 3E). In a previous study, it was shown that the activation of NMDAR was accompanied by calpain activation (Wu et al., 2005), and it was subsequently demonstrated that calpain is involved in learning and memory functions (Chakrabarti et al., 2015). In the present study, Calpain 1 expression‎ was found to be significantly lower in the brains of bees from the DWV group than that of the N/D group, indicating that NaB could help restore its expression‎ in DWV-infected bees (Figure 3F).

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Figure 3. Sodium butyrate restores ATP and neurotransmitter glutamate in bee brains via glycolysis

(A) RT-qPCR analysis of glycolytic genes glucokinase (gk), phosphofructokinase (pfk), and pyruvate kinase (pyk) in honeybee brains 48 hr after DWV infection with or without sodium butyrate treatment. All results were normalized to the expression‎ of the 18S rRNA gene and non-infected control (ΔΔCt).

(B) ATP levels in honeybee brains were measured 48 hr after DWV infection with or without sodium butyrate treatment. All results were normalized to those of the control group.

(C) Glutamate levels in honeybee brains were measured 48 hr after DWV infection with or without sodium butyrate treatment.

(D) Expression‎ of glutaminase (gls).

(E) Expression‎ of NMDA receptor subunits nr1 and nr2, respectively. For the NaB group, bees were fed with 10 mM of sodium butyrate. For the N/D group, bees were fed with 10 mM sodium butyrate for 7 days, followed by infection with DWV. Three independent experiments were performed and 35 bees were included in each group for every experiment.

(F) Western blotting analysis of the expression‎ of Calpain-1 in the brain. Bees were treated with DWV or sodium butyrate and brain lysates were prepared after treatment to analyze the expression‎ of Calpain-1. Lysate prepared from bees without any treatment served as the control. The expression‎ level of actin in each sample was also detected and used as a loading control. All the bar graphs are presented as mean ± SD. p value were calculated using Student’s t-test (∗, p<0.05) as compared to the control group.

Overall, the above results suggest that NaBcould help rescue the negative effects on memory caused by DWV infection by resuming the normal glycolysis and the synthesis of factors involved in neural transmission in honeybees.

Short treatment of NaB reduces the negative physiological effect of DWV infection in bees and increases their field activity

The results so far indicated that NaB could help reverse the damage induced by DWV infection in bees, especially in learning/memory behavior. To validate this further, a field trial was devised. Hives were divided into 4 groups with different treatments as described above. As the queen of each hive came from the same source, thus each colony shared identical genetic traits, generating a comparable result. We also confirmed the successful DWV inoculation in DWV-infected hives by monitoring the viral loads with qPCR (Figure S3). Each hive was wired to a WSN-based automatic monitoring system, which recorded outgoing and incoming activity in addition to the ambient environmental parameters (Figure S4). Bees in the N/D groups were fed with NaB (10 mM/L) prepared in 40% sucrose water for 7 days; those in the infection groups (DWV and N/D) were infected with DWV after following the 7-day NaB diet (Figure 4A). The activity of these bees was monitored and recorded for 37 days post-DWV infection, during which bees were fed with sucrose water only. The activity of DWV-infected bees deviated significantly from other groups on the second day of recording, with an in/out ratio of 0.4971, indicating that at least 70% of the outgoing bees did not return to the hive, whereas the other 2 groups maintained an in/out ratio around 0.8250 (Figure 4B). This suggests that the bees from the DWV-infected hive may have lost their homing ability. A statistical analysis of the in/out ratio showed that DWV infection decreased the in/out ratio, and the DWV-infected colony supplied with NaB had a significantly higher in/out ratio (Figure 4C). This suggests that NaB treatment prior to DWV infection was sufficient to suppress the negative effect induced by DWV throughout the course of the infection. The DWV group lost the greatest number of bees during the course of the 30-day experiment (Figure 4C), which was 1.53-fold greater than that of the N/D group.

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Figure 4. Short-term supply of sodium butyrate could restore the bee activity and the ratio of lost bees after DWV infection

(A) Real-time monitoring of the incoming and outgoing activity of DWV-infected bees with or without sodium butyrate treatment for 30 days. For DWV infection groups (DWV and N/D), bees were infected with the DWV solution containing 106 virus copy numbers in a time frame of 48 hr before their activities were monitored. Bees without any treatment were assigned to the Control group. Bee activity is expressed as an activity ratio, defined as the ratio of incoming to outgoing activities on the indicated day post-infection. The activity ratio on days 2, 6, 11, 22, and 26 post-infection was recorded. The highest activity ratio on an indicated day was set to 1 and the ratios from other groups were adjusted accordingly.

(B) The activity ratio on days 2, 6, 11, 22, and 26 post-infection was recorded. Significance was determined by one-way ANOVA with Post-hoc analysis (Tukey test); different letters for the treatment group indicate significant differences at p < 0.05. The data are presented as mean ± SD.

(C) Sodium butyrate treatment decreased the ratio of lost bees after DWV infection. The lost bee ratio was calculated by subtracting the number of incoming bees from the number of outgoing bees on an indicated day and then dividing that number by the number of lost bees in the Control group on the same day.

(D) Expression‎ analysis of genes involved in learning and memory. DWV-infected bees with (N/D) or without (DWV) sodium butyrate treatment were collected on days 0, 10, 20, and 30 post-infection. The expression‎ of 17 genes involved in learning and memory was analyzed by real-time qPCR; the expression‎ level of 18S rRNA was also measured and used as an internal control. These 17 genes included Synt, Syntaxin 1A; Sec61Beta, Sec61 b subunit FBgn0010638; RPN9, regulatory particle non-ATPase 9; Tet, tetracycline resistance protein; SesB, stress-sensitive B; Rpb8, subunit 8 of RNA polymerase II; PPO, prophenol oxidase; Irp 1, iron regulatory protein 1; RPL32, ribosomal protein L32; NPL, N-acetylneuraminate pyruvate lyase; Mosc1, mitochondrial mosc domain-containing protein 1; Mal, myelin and lymphocyte protein; HSP90, heat shock protein 90; and HDC, histidine decarboxylase. The expression‎ levels of these 17 genes were normalized with that of 18S rRNA. Normalized expressions of these genes are presented with red and green color denoting up-and down-regulation, respectively. Triplicate samples were prepared from each group and each sample contained brains from 5 bees in each group

Worker bees from the 4 hives were collected on day 0, 10, 20, and 30 for gene expression‎ analysis. Genes that had been previously reported to be involved in learning/memory in bees were investigated, including synt, secllbrtata, rpn9, tet, sesB, bpb8, ppo, irp1, histone 1B, gb-18684, rpl32, npl, mosc1, mal, hsp90, histone1.2, and hdl. The gene expressions were mostly suppressed in DWV-infected bees from day 0 to day 30, except for irp and tet on day 0 and mosc1 from day 10 to day 30 (Figure 4D). For the N/D group bees, even though the expression‎ of some genes was still suppressed (irp1, gb-18684, histone 1B, rpn9, mosc1, and rpl32), a greater number showed active expression‎, in particular histone 1.2, ppo, ses-b, tet, and secllbrtata, which were up-regulated throughout the experiment (Figure 4D; Table S2).

Steady dietary supply of NaB significantly improves homing ability of DWV-infected worker bees by upregulating the expression‎ of genes involved in learning and memory

In the first WSN monitoring experiment, it was found that a relatively short period of NaB treatment (7 days prior to DWV infection) was able to minimize the number of bees lost from a colony. To confirm‎ whether a long-term supply of NaB had a negative effect on bee homing behavior, we performed another experiment by constantly supplying the NaB for more than 30 days and monitoring the number of bees entering and exiting the hive (Figure 5A). The in/out ratio of DWV group bees declined from day 2 and started to increase from day 17 (Figure 5B). The activity ratio of the N/D group bees, however, resembled that of the control and NaB only group bees (Figure 5B). Figure 5B shows the in/out ratio for Figure 5A at the time of drawing out for statistics [i.e., Figure 5B shows 7 time points (Figure 5A), and each treatment group has 7 data points]. The results highlighted in Figure 5C show that the continuous feeding of NaB has the ability to restore, and that there was no difference between the NaB treatment group and the control. This outcome is also reflected in lost bee ratio during the course of experiment, with the DWV group exhibiting the highest ratio (Figure 5C). There was no significant difference in the ratio of losing bees between the N/D and the NaB groups, which were both significantly lower than that of the DWV group. In addition, an analysis of the gene expression‎ in experimental bees showed that those involved in learning/memory were highly up-regulated in NaB-fed bees despite being infected with DWV (Figure 5D; Table S3). These results demonstrate that a continuous supply of NaB helped bees counteract the negative effects of DWV infection on homing ability by up-regulating the expression‎ of genes involved in learning/memory processes. In addition, we did not observe a significant impact of the NaB treatment on homing behavior.

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Figure 5. Routine supply of sodium butyrate in the diet significantly improves the activity ratio and minimizes the ratio of losing bees after DWV infection

(A) Real-time monitoring of the incoming and outgoing activity of DWV-infected bees with or without sodium butyrate treatment for 37 days. For the NaB group, bees were fed with sodium butyrate for the duration of the experiment. For the DWV and N/D groups, bees were infected with the DWV solution containing 106 virus copy numbers in a time frame of 48 hr before their activities were monitored. Bees without any treatment were assigned to the Control group. The activities of bees are expressed as activity ratios, i.e. the ratio of incoming to outgoing movement on the indicated day post-infection. The activity ratio on days 2, 7, 12, 17, 22, 27, and 37 post-infection was recorded. The highest activity ratio on an indicated day was set to 1 and the ratios from other groups were adjusted accordingly.

(B) The ratio of in and out of Figure 5A at the time of drawing out for statistics. Figure 5A records 7 time points, and each treatment group has five data points. Significance was determined by one-way ANOVA with Post-hoc analysis (Tukey test); different letters for the treatment group indicate significant differences at p < 0.05. The data are presented as mean ± SD.

(C) Sodium butyrate treatment significantly decreased the ratio of losing bees after DWV infection. The lost bee ratio was calculated by subtracting the number of incoming bees from the number of outgoing bees on an indicated day and then divided that number by the number of lost bees in the Control group on the same day.

(D) Expression‎ analysis of genes involved in learning and memory. DWV-infected bees with (N/D) or without (DWV) sodium butyrate treatment were collected on days 0, 10, 20, and 30 post-infection. The expression‎ of 17 genes involved in learning and memory was analyzed by real-time qPCR and the expression‎ level of 18S rRNA was also measured as an internal control. The expression‎ levels of 17 genes were normalized with that of 18S rRNA. Normalized expressions of these genes are presented with red and green color denoting up- and down-regulation, respectively. Triplicate samples were prepared from each group and each sample contained heads from 5 bees in each group

Discussion

In the present study, a comprehensive elucidation of DWV infection and the effect of sodium butyrate (NaB) treatment on bees is presented. We found that NaB could increase the survival rate of bees infected with DWV (Figure 1A). Furthermore, NaB was able to reverse the negative effects on learning/memory ability caused by DWV infection (Figure 1B). Genes that underwent a significant alteration in expression‎ level by NaB and DWV were identified through NGS. The identified genes were found to be involved in the following physiological functions: epigenetics, signal transduction, regulation of biological process, regulation of cellular process, biological process, and biological regulation (Figures 2C and 2D). By functionally clustering genes which exhibited significantly different expression‎ levels between DWV-infected bees with and without NaB treatment, it was found that many were involved in metabolic processes and the learning/memory pathway (Table S1). Previous studies have demonstrated that NaB could counteract the negative effects of imidacloprid (a nicotine-like pesticide) in bees, in addition to enhancing their immune responses and detoxification processes, and restore learning behavior (Hu et al., 2017, 2018). This suggests that NaB might have a use in preventing virus-induced CCD.

As an epigenetic regulator, the treatment of NaB might affect the expression‎ of genes associated with diverse biological processes. A gene cluster identified by NGS analysis with significantly different expression‎ levels between the DWV and N/D groups was the glycolytic process cluster. Detailed gene expression‎ analysis showed that DWV infection down-regulated the expression‎ of enzymes involved in the glycolysis pathway (Figure 3A) and disrupted the energy supply in the brain of bees (Figure 3B). The shortage in energy further affected neurotransmission. In honeybees, glutamate is one of the most abundant neurotransmitters. The majority of glutamate is produced through the conversion of glutamine to glutamate, which is mediated by glutaminase. Glutamine, one of the few amino acids which can cross blood-brain barriers, is primarily stored in muscles and is transported to the brain, where it is converted to glutamate. When the glutamate concentration reaches a certain level, it binds to glutamate receptors, e.g., NMDA receptors, which subsequently activate downstream calpain. This eventually forms long-term potentiation, which aids in memory formation and the consolidation of long-term memory (Chakrabarti et al., 2015). The present study showed that infection with DWV decreased the expression‎ of glutaminase, calpain and the subunits of NMDA receptors, and resulted in impairments to both memory and homing behavior in bees (Figure 3). This correlates to studies in humans, where patients with age-related brain/neuronal diseases have been found to display significantly lower levels of both glutamate and NMDA receptors (Newcomer et al., 2000). We further demonstrated that NaB treatment is able to reduce the impairments caused by DWV infection by stimulating the expression‎ of both glutamate and NMDA receptor subunits, which could therefore decrease colony loss (Figure 3). A similar phenomenon has been observed in mammals, where histone deacetylase inhibitors have been shown to enhance learning ability in mice (Malvaez et al., 2010).

The ability of NaB to reverse memory loss or impaired learning ability in bees infected with DWV was further demonstrated in field studies, which showed that pre-treating bees with NaB significantly restored the homing behavior of infected bees (Figure 4). Furthermore, DWV-infected bees provided with a continuous supply of NaB exhibited activity similar to uninfected bees (Figure 5). Gene expression‎ profile analysis showed that genes involved in learning/memory ability exhibited up-regulated expression‎ in DWV-infected bees on a NaB diet (Figure 5D). This suggests that NaB helps bees recover from the memory loss effects of DWV by up-regulating learning/memory gene expression‎, either by direct regulation or by in-direct regulation through resumed neurotransmission activity. These results further demonstrate that NaB could counteract colony collapse by restoring the homing behavior upon DWV infection. Our monitoring system was able to continuously track the in-out frequency of bees. Although only one hive was used for each treatment, each hive contained more than ten thousand bees. In addition, two separate experiments were performed, with either shorter or continuous NaB treatment, and they were conducted during different dates with different hives. These two independent experiments showed a similar trend that NaB could decrease the replication of DWV in bees.

Many countries have now banned farmers from using nicotine-like pesticides (Nicholls et al., 2018; Dewar, 2017; Gross, 2013); however, despite this measure the incidence of reported colony collapse has not decreased significantly. Therefore, more attention has been directed toward pathogens as a cause, since the reduced use of pesticides has resulted in an increased number of pathogens present in the environment. Varroa destructor, an external parasitic mite that was found cohabiting in hives, is the main carrier of DWV (Rosenkranz et al., 2010; Le Conte et al., 2010; Yang and Cox-Foster, 2005). At present, removal of the infected hives from the farms is used to manage the DWV infection, with chemical-based pesticides to eliminate V. destructor as an alternative (Locke et al., 2017). However, it has been noted that V. destructor-targeted pesticide is toxic to bees. Although some products have been developed to boost bees’ immunity against bacteria, fungi, and microbes by promoting digestive health, or fast expansion and restoration of the colony by providing sufficient nutrients (Tauber et al., 2019), no product or regimen has been developed to promote bees’ ability to fight against viral pathogens thus far. The results of the present study demonstrate that routinely including NaB in the bee diet can prevent memory loss and colony decline by DWV infection without significant adverse effects (Figures 1B and 5).

Previous studies have found that histone deacetylase inhibitors increase long-term memory in mice (Davie, 2003; Vecsey et al., 2007; Dokmanovic et al., 2007). Experiments in the present study also present a similar conclusion, because bees receiving a NaB-containing diet exhibited a similar or better memory ability in the PER assays (Figure 1B) and real-time activity monitoring study, even in DWV-infected bees (Figures 4 and 5). The present study provides evidence that NaB could help bees fight against DWV infection and reverse the adverse effects of the infection by up-regulating learning/memory gene expression‎. Our investigation provides valuable information on HDACi gene regulation associated with memory mechanisms at the epigenetic level (Figure 6). Combining this with the results of our previous studies (Hu et al., 2017) (i.e., that NaB could enhance immunity and detoxification), a diet incorporating histone deacetylase inhibitors could be used to maintain the overall wellbeing of the bees and integrity of the colony.

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Figure 6. Schematic diagram of how epigenetics is involved in regulating the memory function of honeybees.

그림 6. 꿀벌의 기억 기능 조절에 후성유전학이 관여하는 방법의 개략도.

HDACi restored the memory impariment of DWV-infected bees. The WSN-based automatic monitoring system

confirmed that sodium butyrate could improve the ability of DWV-infected bees to return to their hives.

Limitations of the study

In the present study, we showed the beneficial effects by short- and long-term treatment of NaB in

DWV-infected bees. Although we conducted two experiments independently in different periods and hives,

we could not ignore the fact that we only used one hive for each treatment because of the limited amount

of our WSN monitor systems. Future work is required to expand our WSN systems for surveying higher number

of colonies at the same time.

STAR★MethodsKey resources table

HDACi(히스톤 탈아세틸화효소 억제제)는 DWV에 감염된 벌들의 기억 손상을 회복하였다.

WSN 기반 자동 모니터링 시스템은 부티르산나트륨이 DWV에 감염된 벌들이 벌통으로

돌아가는 능력을 향상시킬 수 있음을 확인했다.

연구의 한계
현재 연구에서, 우리는 DWV에 감염된 벌들에서 NaB의 장단기 처리에 의한 유익한 효과를

보여주었다. 각기 다른 기간과 벌통에서 독립적으로 두 가지 실험을 수행했지만, WSN 모니터

시스템의 제한된 양으로 인해 각 처리에 하나의 벌통만 사용했다는 사실을 무시할 수 없었다.

동시에 더 많은 수의 봉군을 조사하기 위해 WSN 시스템을 확장을 위해서는 향후 작업이 필요하다.