Insects have great difficulty in utilizing wood as food resource. Wood is composed of mostly of polymers that insects are difficult to digest, and almost lacking in essential nutrients. Therefore, most insects that reside in wood are known t o have an association with microbes that help digestion of wood. Some wood-boring insects have a mutualistic association with fungi. For example, ambrosia beetles carry a specific ambrosia fungus into the wood where they deposit eggs, cultivate it in the wood, and feed on it. Wood wasps also have symbiotic fungi that may enhance the nutrient value of wood or help digestion of wood. For these insects the association with microbes is clear. However, there are other insect groups that do not have a definite symbiotic association unlike ambrosia beetles or wood wasps, but do utilize the fungi that have already existed in wood. The typical insects are stag beetles.

Adult females of the stag beetle Dorcus rectus (Mo t s chulsky) (Coleoptera:Lucanidae) deposit eggs in dead trunks and boughs of broad-leaved trees that have been infested with white-rotting fungi. The mothers place granulated wood besides the eggs at oviposition. Hatched larvae initially feed on the granulated wood and then move to feed on the decaying wood. The granulated wood have been thought to harbor some microbes beneficial to the larval nutrition. In Chapter 1, I examined the effect of wood decay by different wood-rotting fungi on larval growth, and the effect of initial ingestion of the granulated wood on larval growth. To prepare decaying wood, autoclaved Japanese beech (Fagus crenata) wood sawdust were inoculated with each two white-rotting fungus Bjerkandera adusta and Trametes versicolor, and incubated at 25°C for 3 or 10 months. Newly hatched larvae of D. rectus were initially supplied with small amount of the granulated wood, and then reared on the decaying w ood or non-decaying wood for 28 days. For a control, newly hatched larvae were soon placed on the decaying wood or non-decaying wood and reared in the same manner. Consequently, I demonstrated that the larvae cannot grow on non-decaying wood, and that initial ingestion of the granulated wood positively affected the larval growth although the larvae which were not supplied with the granulated wood also exhibits a substantial growth on decaying wood.

Insects that feed on decay i n g wood are usually classified as xylophagy although decaying wood contains substantial amount of fungal mycelia. In Chapter 2, I examined whether D. rectus larvae can utilize only fungal mycelia as food. For this purpose, newly hatched larvae of D. rectus were reared for 14 days on artificial diets containing a fixed amount of freeze-dried mycelia of the following fungi: Bjerkandera adusta, Trametes versicolor, Pleurotus ostreatus and Fomitopsis pinicola. The mean incremental gain in larval body mass was greatest on diets containing B. adusta, followed by T. versicolor, P. ostreatus, and F. pinicola. The growth rate of body mass correlated positively with mycelial nitrogen content of the different fungi. It also correlated positively with the mycelial content of B. adusta in the diet. Addition of antibiotics to diets with mycelia nearly halved larval growth, indicating that larvae were able to use fungal mycelia as food without the assistance of associated microbes although the microbes positively affected larval growth. Four newly hatched larvae reared on artificial diets containing B. adusta mycelia developed to the second instar in 21 to 34 d; and one developed to the third (= final) instar. These results provide evidence that fungi may constitute the bulk of the diet of D. rectus larvae.

Dead wood is decomposed by wood rot fungi, resulting i n decaying wood. Because wood has a solid structure that remains for a long time, the chemical components of wood are mostly kept within wood. Some water-soluble components are released from decaying wood by reaching, however, most of the water-insoluble components remain in decaying wood. These water-insoluble components may be released by insects and incorporated into the nutrient circulation in forest. In Chapter 3, three independent experiments were conducted to clarify whether D. rectus larvae can utilize the water-insoluble fraction of decaying wood and fungal mycelia. In the first experiment, third instar larvae of D. rectus were reared on decaying willow (Salix sp.) wood and the decaying wood and larval feces were analyzed for the carbon and nitrogen contents in each fraction of hot-water- and hot-alkaline-extraction. Nitrogen of the water-soluble and alkaline-soluble-water-insoluble fraction exhibited 39% and 38% decrease, respectively, from decaying wood to larval feces, although nitrogen of the alkaline-insoluble fraction shoed almost no change. Carbon contents showed the same tendency, however, the decrease was only 21% and 26% for water-soluble and alkaline-soluble-water-insoluble fraction, respectively. In the second experiment, larvae were reared on the same decaying wood and the absorption ratio was determined as the decrease in dry mass through the larval ingestion and excretion. The absorption ratio was calculated as 26%, and using this value, the net utilization rate of carbon and nitrogen of each extractive fraction were recalculated. The third experiments was conducted to determine whether the larvae can grow merely on either water-soluble or water-insoluble fraction of fungal mycelia. To make artificial diets, freeze-dried mycelia of B. adusta was extracted by distilled water at 100°C for 3 hours and the extract and residue that were equivalent to 100 mg dry mycelia were suspended in agar. Newly hatched larvae did not grow either on the diet containing mycelial extract or on the diet containing residue, although the larval growth on the mixed diet containing both extract and residue was as much as that on the diets containing the equivalent amount of the non-extracted mycelia.

Fungal decom p osing activity can enhance the nutrient value of wood, however, the nitrogen and other nutrient levels in decaying wood are still much lower than those of insect bodies. Therefore, cannibalism usually plays an important role in nutrient ecology of wood-feeding insects. In Chapter 4, I examined the nutritional significance of cannibalism in D. rectus larvae, with reference to their interference competition in laboratory and field. A laboratory experiment, in which two larvae were placed on milled decaying wood in test tubes for two weeks, showed that cannibalism occurred in the first and second instar larvae. Cannibals tended to have larger head capsules than their victims. Cannibalizing larvae gained more body mass than non-cannibals. The carbon/nitrogen (C/N) ratio of decaying wood was much higher than that of larvae, explaining an increased body mass following cannibalism. Sixty-two percent of surviving second-instar larvae were wounded by their opponents. When cannibalism did not occur at the second larval instar, large-headed larvae grew but the growth of small-headed larvae was restricted, suggesting strong interference. However, a field study suggested low rates of interference competition between larvae.

Wood-boring insects that associate with mutualistic m i c robes usually possess special organs to store and convey the microbes. In Chapter 5, I newly discovered the microbe-storage organ (mycangium) in the stag beetles. The mycangium being located under the tergum was present in all adult females of 22 lucanid species examined, but absent in adult males. On the contrary, adult insects of both sexes of Passalidae and Scarabaeidae, which are closely related to Lucanidae, had no mycangium unlike lucanids. Yeast-like microbes were isolated from the mycangium of five lucanid species. Base sequence of ITS/5.8S and Dl/D2 rDNA indicated that the microbes are closely related to the xylose-fermenting yeasts Pichia stipitis, P segobiensis, or Pichia sp.

This study showed two microbial associations of stag beetles in reference to their nutrition: one is the one-sided utilization of wood-rotting fungi by insects, and another is the symbiotic association with xylose-fermenting yeasts. Stag beetles are utilizing wood-rotting fungi within decaying wood as well as having a symbiotic association with xylose-fermenting yeasts to help digestion and/or detoxification of wood xylose. There are many ecologically and evolutionally interesting phenomena left in the association of stag beetles and microbes, therefore, further studies are needed for fully understanding of nutrient ecology of stag beetles.

材穿孔性昆虫は樹木の材に入り,材を物理的に破砕しながら栄養を摂取する。材はその大部分が難消化性の高分子化合物から構成され,窒素化合物やステロール類,ビタミン類といった栄養をほとんど含まず,昆虫にとって利用困難な餌資源である。枯死直後の樹木の材に穿孔する昆虫の一部では,微生物と密接な関係を持って材から栄養を得ることが知られている。野外では枯死後時間が経つと,材は木材腐朽菌によって変質して腐朽材となる。シロアリを除くと腐朽材穿孔性昆虫では微生物との明確な共生関係があまり知られていない。その代表的なグループがクワガタムシ科の昆虫であり,幼虫は腐朽材を食べて成長する。その中でも,コクワガタの幼虫は,森林の腐朽材においてかなり大きいバイオマスを占める。この研究では,コクワガタを材料にして,微生物との相互関係を明らかにしたものである。

本論文は6章からなる。第1章は序論であり,材食性昆虫と微生物の関係,クワガタムシにおける栄養生態学,および森林生態系における材食性昆虫の位置づけについて概観している。

第2章では,まず幼虫の齢査定法を確立した。次に,幼虫間の共食いの可能性を明らかにするために,実験室で一定量の腐朽材粉末を2頭の1齢または2齢幼虫に与えた。その結果,共食いが観察され,他個体を食べた個体は被食個体よりも頭幅が大きい傾向があり,食べた個体は共食いをしなかった個体より大きな体重増加を示した。この結果から,腐朽材の窒素含有率は昆虫の体に比べて極めて低いことと共食いの関係について考察している。

第3章では,木材腐朽菌の種類と雌成虫が孵化幼虫のために用意する食物(木屑)が幼虫の成長に及ぼす影響を明らかにするために,オートクレーブしたブナおが屑と無菌的に孵化した幼虫を用いて実験を行っている。その結果,白色腐朽菌ヤケイロタケまたはカワラタケを接種した滅菌おが屑(腐朽材)で幼虫は成長したが,菌を接種しなかったおが屑(未腐朽材)では成長は起こらなかった。孵化幼虫に与えられた食物(木屑)を上述の餌に添加した場合,未腐朽材では効果がなかったが,腐朽材では幼虫の成長を有意に高めた。実験後の検査では,木屑を与えた幼虫はそうでない幼虫に比べて有意に高い頻度で微生物が検出された。これらの結果から,コクワガタの栄養摂取には木材腐朽菌による材の腐朽が必須であること,および雌成虫が準備した木屑には幼虫の栄養摂取を助ける微生物が含まれることが示唆されたと結論付けている。

第4章では,木材腐朽菌そのものが幼虫にとって十分な栄養源になるかどうかを明らかにするために,4種の木材腐朽菌ヤケイロタケ,カワラタケ,ヒラタケ,およびツガサルノコシカケの凍結乾燥菌糸粉末の一定量を寒天中に懸濁して人工飼料とし,孵化幼虫を14日間飼育している。その結果,幼虫の体重増加はこの順で大きかった。幼虫の成長率と菌糸の窒素含有率との間には有意な正の相関がみられた。また,人工飼料中のヤケイロタケ菌糸の濃度を変えると,幼虫の成長率は菌糸濃度と正の相関を示した。人工飼料に抗生物質を加えた場合,幼虫の成長は約半分になったことから,腸内バクテリアは幼虫の栄養摂取に正の影響を与えるが,幼虫は腸内バクテリアがなくても菌糸を栄養源として利用できることが示唆された。さらに,ヤケイロタケ菌糸を含む人工飼料によって幼虫が3齢(終齢)まで発育することが示された。これらの結果から,菌糸が幼虫の主な栄養源であることが示されている。

第5章では,クワガタムシの雌成虫から共生微生物の保持器官と思われる特異的な器官(菌嚢)を初めて発見したこと,および菌嚢が日本産クワガタムシ科のほぼ全ての13属にわたって広く存在することが示されている。この器官からはキシロースの代謝能力を持つ菌類(酵母)が高頻度で分離された。キシロースは広葉樹材の白色腐朽材に多く含まれ,昆虫や微生物を含め多くの生物にとっては利用が困難な物質である。クワガタムシはキシロースの代謝能力を持つ酵母と共生することによって,自然界に豊富に存在する白色腐朽材を利用できるように進化した可能性が示されている。

第6章では,コクワガタの栄養生態学とそれに関係する微生物について,昆虫による微生物の「利用」と「共生」の二つの観点から議論しており,それらが森林の物質循環の中で果たす役割についても言及している。

このように,本論文では材食性昆虫であるコクワガタの栄養生態とそれに関係する微生物の関係を明らかにしたものである。特に,クワガタムシ科での菌嚢の存在とその中のキシロース分解性の酵母の存在を初めて明らかにしたことは,この分野の発展に大きく寄与することになると考えられ,審査委員一同は,本論文が学術的にも応用的にも価値が高く,博士(農学)の学位論文に値すると判断した。