Studies on Lignocellulose Decomposition and Structure of Gut Microbiota of Death Watch Beetle, Nicobium hirtum (Coleoptera: Anobiidae)

概要

（ 続紙 １ ）
京都大学

博士（

農

学

）

氏名 NI PUTU RATNA AYU KRISHANTI

Studies on Lignocellulose Decomposition and Structure of Gut Microbiota of Death

論文題目

Watch Beetle, Nicobium hirtum (Coleoptera: Anobiidae)
（ケブカシバンムシのリグノセルロース分解と腸内微生物叢に関する研究）

（論文内容の要旨）
Wood-destroying insects, mainly classified in the two major orders of Insecta,
i.e., Coleoptera (beetles) and Blattodea (cockroaches and termites) which cause
great economic damage to wood industries. Wood-feeding beetles are recognized as
wood pests because some species can inflict severe damage to economically
important forest trees or dried timbers used in buildings. There is much to learn
about the mechanisms underlying lignocellulose digestion by wood-feeding insects,
i.e., how they achieve efficient hydrolysis and subsequent assimilation of
polysaccharides (cellulose and hemicelluloses) and their conversion to energy, and
also how they circumvent recalcitrant lignin barriers to access embedded
polysaccharides. These topics are of great interest not only because they will lead
to better understanding of the physiology and evolution of herbivorous insects, but
also to the development of biotechnological strategies for wood pest management
and biochemical production methods from lignocellulosic biomass.
Among the wood-feeding beetles, anobiid beetles have a broad range of
feeding substrates and are responsible for many attacks on historic wooden
structures and artifacts in Japan. Nicobium hirtum is a serious pest of wooden
furniture, cultural heritage, and architectural structures. Although many
members of Anobiidae family have been observed to utilize plant/wood cell walls,
little is yet known about the ability to degrade lignocellulose on N. hirtum. This
beetle is likely to have developed a unique and diverse digestive system, often
associated with intestinal microbial symbionts, which can produce the enzymes
necessary to decompose lignocellulose, a recalcitrant biocomposite of cellulose,
hemicelluloses, and lignin that constitutes the woody cell walls. Therefore, the aim
of this thesis was to investigate in detail the properties of lignocellulose
degradability by N. hirtum larvae.
Chapter 1 reviewed the biology of N. hirtum. In Chapter 2, a laboratory
culture of N. hirtum was developed to assess the basic physiological and feeding
characteristics of N. hirtum larvae fed different types of wood and lignocellulosic
artificial diet. In-depth structural characterizations of lignocellulose in their feces
found that N. hirtum larvae are able to deconstruct lignocellulose in their gut
digestive system. The larvae can subsist on plain softwood and hardwood
lignocelluloses, utilizing cellulose and hemicelluloses, especially mannans, for
assimilation and energy. Such efficient polysaccharide digestion could be achieved
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by overcoming lignin barriers through structural modifications of lignin polymers
via reductive and/or oxidative degradation pathways.
The effects of artificial diets with different contents of starch and hardwood
lignocellulose compounds were compared with the survival and growth of N.
hirtum larvae in Chapter 3. Larvae fed diet with 80% starch content grew faster
and developed larger bodies than larvae fed diets with lower starch concentrations
(20% and 50%), although survival rates were significantly lower. Thus, while
starch is partially important for larval growth, lignocellulose is also important for
maintaining physiological activity and improving survival of N. hirtum larvae.
Studying the microbial communities of N. hirtum may provide insight into
their role and capability in digesting wood components and broaden the sources of
biocatalysts that may impact biotechnology. To shed light on how gut microbiota of
N. hirtum is affected by various dietary patterns, amplicon sequencing of bacterial
16S-rRNA and fungal ITS1 were used to determine how communities vary among
these populations, and along different regions of an individual insect’s digestive
system in Chapter 4. The feeding habits and different sources of diet may
determine microorganisms’ abundance, which in turn affects the cellulolytic
activities of N. hirtum’s digestive system. The amplicon sequencing data revealed
that both bacterial and fungal community profiles of the N. hirtum larvae gut are
related to the dietary variation, in particular, lignocellulose and starch ratio in the
diet. Microorganisms in the host guts are nutritionally endogenous or
interdependent relationships with the host and are co-evolutionary adapted.
Therefore, the predominant symbionts in the gut of N. hirtum larvae may be
highly related to host fitness. Further studies will be required to confirm the
abilities of gut bacteria and fungi to metabolize wood components and defend
against toxic compounds ingested by the hosts, N. hirtum.
In conclusion, this thesis confirmed that starch is an essential carbon source
for N. hirtum larval growth, but lignocellulose also plays an important role as a
nutrient that maintains N. hirtum larval physiological activity and enhances
survival. The microbial community and abundance of N. hirtum larvae varies with
the diet types, which in turn influences wood composition and carbohydrate
degradation. At the same time, the study showed that it is also critical for N.
hirtum larvae to maintain a healthy gut microbiota in the digestive tract in order
to efficiently degrade lignocellulose.

注)論文内容の要旨と論文審査の結果の要旨は１頁を３８字×３６行で作成し、合わせ
て、３,０００字を標準とすること。
論文内容の要旨を英語で記入する場合は、４００～１,１００wordsで作成し
審査結果の要旨は日本語５００～２,０００字程度で作成すること。
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（続紙 ２ ）
（論文審査の結果の要旨）
ケブカシバンムシは日本各地の木造文化財で最も高頻度で発見される木材害虫で
あるが、そのリグノセルロース分解機構については未解明な点が多い。本論文はケ
ブカシバンムシのリグノセルロース分解特性を明らかにすることを目的として、ケ
ブカシバンムシ幼虫にデンプン／木粉含有比率の異なる飼料の投与実験を行い、リ
グノセルロース分解特性と腸内共生微生物の機能解明を目指したものであり、評価
すべき点として以下の３点を挙げることができる。
１．幼虫の消化前後の飼料の成分比較により、幼虫がリグノセルロースを分解でき
ることを明らかにした。すなわち、デンプン含量0.3%のアカマツ辺材飼料の場合、
デンプンに加えてセルロースとマンナンを優先的に分解すること、デンプン含量50%
の人工飼料を与えた場合、デンプンとマンナンをセルロースより優先的に分解する
こと、デンプン量の多寡にかかわらず、リグニンを部分的に変性すること、を明ら
かにした。このことは、ケブカシバンムシが多様な餌に順応できるポテンシャルを
有する昆虫であることを示唆している。
２．幼虫の成長、生存率を検討した結果、デンプン含量の増加に伴い幼虫の体長や
体重が増加するが、生存率にマイナスの影響を及ぼすことを明らかにした。これに
よってデンプンは幼虫の成長に重要であるが、リグノセルロースは幼虫の生理活性
を維持し、生存率を高める効果があることが示唆された。
３. 幼虫の腸内微生物叢を解析した結果、腸内細菌および真菌の群集は、飼料中の
デンプン／木粉含有比率により大きく異なることを明らかにした。具体的にはデン
プン含量の減少（リグノセルロース含量の増加）に伴い α-ProteobacteriaやBacilli
などの特定の細菌種と、他のシバンムシ科でも見られる 真菌 Symbiotaphrina
buchneri が優先種になることを明らかにした。
以上のように、本論文は古材の害虫として知られるケブカシバンムシについて、
リグノセルロースの分解特性とその分解における腸内共生微生物の寄与について明
らかにしたものであり、木材保存学、昆虫生理学、害虫管理学の発展に寄与すると
ころが大きい。
よって、本論文は博士（農学）の学位論文として価値あるものと認める。
なお、令和 5 年 7 月 20 日、論文並びにそれに関連した分野にわたり試問し
た結果、博士（農学）の学位を授与される学力が十分あるものと認めた。
また、本論文は、京都大学学位規程第１４条第２項に該当するものと判断し、公
表に際しては、当該論文の全文に代えてその内容を要約したものとすることを認め
る。
注）論文内容の要旨、審査の結果の要旨及び学位論文は、本学学術情報リポジトリに
掲載し、公表とする。
ただし、特許申請、雑誌掲載等の関係により、要旨を学位授与後即日公表するこ
とに支障がある場合は、以下に公表可能とする日付を記入すること。 ...