著者

梶村 恒 KAJIMURA Hisashi

出版者

名古屋大学農学部附属演習林

雑誌

名古屋大学農学部演習林報告 (ISSN:04694708)

巻号頁・発行日

no.14, pp.89-171, 1995-12

被引用文献数

3

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養菌性キクイムシ(ambrosia beetle)は、樹木を介した昆虫と微生物との相互関係における進化の過程で、アンブロシア菌(ambrosia fungi)と総称される菌類を自分の食糧として積極的に利用する習性を獲得した昆虫群である。本研究では、養菌性キクイムシの一種であるクスノオオキクイムシとアンブロシア菌の共生機構とその適応的意義を解明することを目的として、野外調査と実験的手法を併用した総合的な研究を行った。具体的には、(1)名古屋大学農学部附属演習林に隣接する広葉樹林(愛知県北東部)における本種個体群の生体調査、(2)その坑道内および(3)胞子貯蔵器官(mycangia)内共生菌の走査型電子顕微鏡(SEM)観察と分離培養実験、(4)本種の人工飼育実験、(5)アンブロシア菌の化学的分類実験を行った。その結果、以下のことが明らかにされた。(1) クスノオオキクイムシ個体群の生態的特徴① 本種は、本調査地において1年で1世代を経過した。飛翔分散は雌成虫のみが行い、7月下旬をピークとして6月下旬から9月上旬まで続いた。性比は、約1:8(♂:♀)であった。② 寄生木の樹種別にみると、シロモジへは、本種を含む多種のキクイムシが穿孔した。コハウチワカエデにおいては、本種が優占的に穿孔した。これらの寄主木への単木(主樹幹)あたりの本種の穿孔数は、ウリハダカエデとリョウブよりも多かった。リョウブへは、他種のキクイムシ類の穿孔も少なかった。③ 本種の産卵坑道率は、ウリハダカエデ、リョウブ、シロモジ、コハウチワカエデの順に大きくなり、ウリハダカエデにおける繁殖成功率は他の樹種の約50%以下となった。また、ウリハダカエデにおける雌新成虫数が約2,3個体であったのに対し、他の樹種では平均5個体以上であった。④ 雌成虫は、水平坑道の完成後、垂直坑道を約1cm形成した時点(供試木伐倒後20日以内)で産卵を開始し、卵を数個ずつ塊状に産下した。また成虫は、穿入孔を蜘蛛糸状の膜で封鎖することが初めて明らかになった。⑤ 孵化幼虫は、産卵開始後20日間で蛹化、羽化した。この時期の坑道内には、卵から新成虫までの全成育ステージの個体が混在していた。子孫数は、供試木伐倒から60日後まで増加したが、その後は雄成虫の脱出と餌不足による幼虫の死亡により減少した。垂直坑道は幼虫期から蛹期にかけて約2倍に拡張され、幼虫の成育終了後(おそらく新成虫により)さらに延長された。最終的に、本種の坑道の全長は平均6cmとなった。⑥ 全成育ステージにおいて、坑道あたりの子孫数と坑道長との間には、正の相関が認められた。しかし、回帰直線の傾き(回帰係数)は、卵期(0.68)~蛹期から新成虫期~越冬期(0.16)にかけて大きく低下した。⑦ 各坑道あたりの雌蛹および雌新成虫の平均生重は、坑道長6cm以下の場合、極端に小さい場合があった。また、坑道長6cmで坑道を二分し、それぞれの次世代の平均生重と体サイズの頻度分布を比較した。その結果、6cm以上の坑道の平均生重は6cm以下のものより有意に大きかったが、体サイズの分布範囲はほぼ同一であった。⑧ 同一坑道内の各個体(5個体以上)の成育時期を、蛹は坑道内における新成虫の有無、新成虫は体色によりグループ分けし、これらの成育時期グループに含まれる蛹および新成虫の生重頻度分布を比較した。その結果、蛹、新成虫ともに早い時期に成育した個体の方が平均生重が有意に大きく、また生重の範囲もより大きい方へとシフトしていた。(2) 坑道内共生菌① シロモジ材の水平坑道から直接接種法により分離された共生菌は、主にAmbrosiella sp.,酵母類、Paecilomyces sp.であった。穿入・産卵期には、Ambrosiella sp.が優占種であったが、その後は酵母類、Paecilomyces sp.が次第に優占的となった。また、コハウチワカエデ、ウリハダカエデ、リョウブの穿入・産卵期の水平坑道からもAmbrosiella sp.が優占的に分離された。② 希釈平板法によっても、Ambrosiella sp. 酵母類、Paecilomyces sp. が分離され、坑道1cmあたりの生菌数で表された各分離菌の動態も直接接種法の結果とほぼ一致した。また、顕微鏡下で直接計測されたAmbrosiella sp.の全菌数は、1齢幼虫期までに急増し、終齢幼虫期に大きく減少した後、新成虫期までその数を維持した。③ 垂直坑道の共生菌相の遷移様式は、水平坑道とほぼ同様の傾向を示したが、幼虫~蛹期においてPaecilomyces sp.の優占度が同時期の水平坑道よりも大きく、またAmbrosiella sp.の生菌数が1齢幼虫期から終齢幼虫期にかけて水平坑道ほど大きく減少しない点が異なっていた。④ SEM観察によって明らかにされた実際の坑道内の共生菌繁殖状況は、①~③のようなキクイムシの成育に伴う共生菌相の動態をおおよそ反映するものであった。(3) mycangia内共生菌① 本種のmycangiaは、前胸背と中胸背の間の節間膜が陥没した一対のポーチ状をなし、その開口部は虫体背面に存在した。② SEM観察により、越冬期のmycangia内膜上には、平坦な板状の物質が付着していることが明らかになった。一方、飛翔成虫のmycangiaにおいては、開口部側の菌塊表面が粘液状物質により被膜され、貯蔵胞子自体からも粘液物が分泌されていた。また、開口部を閉鎖する節間膜の膜組織が隆起、肥大し、その表面では刺状突起物が多数形成されていることが初めて確認された。③ 未成熟新成虫および越冬成虫のmycangia内からは、Ambrosiella sp.以外に酵母類、Paecilomyces sp.などが分離された。しかし、その他の成育段階の成虫からは、Ambrosiella sp.のみが分離された。さらに、蛹を無菌的に脱蛹、成熟させた成虫のmycangiaからは共生菌が分離されなかったのに対し、未成熟成虫に同様の処理を加えた場合には、Ambrosiella sp.が100%分離された。また、飛翔成虫のmycangia内からは、飛翔分散した寄生樹種と関係なく、Ambrosiella sp.のみが分離された。④ mycangia内のAmbrosiella sp.生菌数は、飛翔期に最大量となり、その後産卵期までに急減し、幼虫期以降はさらに減少した。全菌数の変化もほぼ同様のパターンを示したが、産卵後の大きな減少傾向は認められなかった。また、飛翔成虫のmycangia内では胞子の割合が極めて大きくなった。⑤ 越冬成虫を5℃下に置いた場合、mycangia内の優占種は、常に酵母類とPaecilomyces sp.であった。これに対して、25℃処理区では、すべての処理期間においてAmbrosiella sp.のみが分離された。また、越冬成虫に5, 15, 20, 25℃の4段階の温度処理を加えた場合、20℃および25℃処理区のAmbrosiella sp. 生菌数が、処理日数の延長とともに増加した。しかし、どの処理日数においても、25℃処理区の方が20℃処理区より有意に生菌数が多く、また25℃処理区の胞子数のみが有意に増加した。(4) クスノオオキクイムシの人工飼育① 本種の羽化個体が得られたのは、本種のAmbrosiella sp.1とハネミジカキクイムシのAmbrosiella sp.3を与えて飼育した場合のみであった。本種のPaecilomyces sp.およびCandida sp.,ミカドキクイムシおよびサクキクイムシのAmbrosiella spp.では成虫まで成育できなかった。しかし、羽化率はAmbrosiella sp.1を与えた処理区(68.8%)の方がAmbrosiella sp.3を投与した区(17.6%)よりも有意に高く、また雌蛹の平均生重も有意に大きかった。② 4樹種の鋸屑添加培地上で生育させたAmbrosiella sp.1によって飼育した結果、シロモジとコハウチワカエデの鋸屑を添加した場合のみ、羽化成虫が得られた。これらの平均雌蛹重間には、有意な差は認められなかった。③ クスノオオキクイムシの発育日数は、雌の場合、卵期が約3日、幼虫期は約10~14日、蛹期は約7日、卵から成虫羽化までは約22日前後であり、雄は約3日早く羽化した。また、同一培養基上において、早く孵化した個体は遅れて孵化してくる個体よりも確実に早く成育を完了した。④ 菌の培地上での面積を食物資源量の指標としたとき、孵化幼虫1個体あたりのAmbrosiella sp.1の菌面積が10c㎡までは、平均蛹生重、蛹化率ともに菌面積の増加につれてほぼ直線的に上昇した。しかし、この面積以上になると増加傾向は小さくなり、平均蛹重は9mg、蛹化率は80%の一定の上限値に到達した。⑤ Ambrosiella sp.1の1幼虫区では、各飼育個体の雌蛹重と蛹化するまでの発育速度との間に正の相関が認められた。(5)アンブロシア菌の化学的分類①SDS-ポリアクリルアミドゲル電気泳動法を用いて、クスノオオキクイムシ、ミカドキクイムシ、ハネミジカキクイムシ、サクキクイムシと共生する7種のアンブロシア菌のタンパク質分析を行った結果、各キクイムシのAmbrosiella spp.は本種のPaecilomyces sp.やCandida sp.より多数の明瞭なバンドを持ち、またキクイムシ種特異的な泳動パターンを示した。②ミカドキクイムシのAmbrosiella sp.は、他属の3種とかなり疎遠であり、一方、多くの共通する生活様式を持つクスノオオキクイムシとハネミジカキクイムシのAmbrosiella sp.はその類縁性がきわめて高かった。以上の結果を総合して、次のことが示唆された。① 本種の主要な食物資源(PAF)はAmbrosiella sp.であり、彼らはこの菌を様々な樹種の寄主木内に持ち込み、坑道全体をその繁殖場所としている。しかし、本種の繁殖成功は奇主木の樹木成分により大きな影響を受けるものと推察される。② 親成虫は、坑道サイズを拡大することによってAmbrosiella sp.の量を増大させ、この資源量に応じて産卵数を調節している。早く孵化した幼虫は、良質の食物資源を優占的に利用して体サイズの大きい個体となる。このような資源利用様式により、本種は限られた資源量下でより大きな個体を効率的に生産できるものと思われる。③ mycangia内への共生菌の獲得は脱蛹直後に行われ、坑道内に存在する菌を非選択的に取り込んでいる。その後、取り込まれた共生菌の中でPAFのみが選択的に培養される。このPAFの選択的培養の発現は温度により既定され、至近要因としてはmycangiaの附属腺からの分泌物やPAFの生産する抗生物質による影響が考えられる。また、坑道内への貯蔵胞子の接種様式が、坑道部位によるPAFの繁殖速度の違いに対応した、適応的なものであることが示唆された。④ 養菌性キクイムシは、同一樹木内で共存しながら種特異的なPAFと共生関係を成立させている。また、キクイムシ間の生態学的特徴を比較することによって、その類似性が高いほど共生するPAFはより近縁である可能性が示された。さらに、他種キクイムシの共生菌は自種のPAFの近縁種であれば潜在的に利用可能であるが、その場合にはキクイムシの適応度が低下することが初めて実証された。⑤ ④の結果に基づき、キクイムシとアンブロシア菌との種特異的な共生関係の進化ルートを説明する新しい仮説(PCM : pairwise coevolutionary mutualism, DCM : diffuse coevolutionary mutualism)を提案した。⑥ 本研究結果をもとに、キクイムシとアンブロシア菌の共生機構の特質を明らかにし、また両者の相互作用系の特徴とその成立過程を共生(symbiosis)の定義に従って詳細に検討した。Ambrosia beetles (Coleoptera : Scolytidae and Platypodidae) are xylomycetophagous insects which have evolved mutualistic associations with micro-oganisms in host trees. The present study has clarified symbiotic interactions between the ambrosia beetle, Xylosandrus mutilatus (BLANDFORD), and their associated fungi and its adaptive significance by various creative methods : (1) successive censuses of X. mutilatus field population, (2) scanning electron microscopy (SEM) observations and isolation experiments of fungal symbionts in gallery system, and those of fungal symbionts in mycangia of the beetle, (3) artificial rearing experiments of X. mutilatus on fungal diets and (4) protein analysis of fungi associated with several species of ambrosia beetles. The study was conducted in a mixed stand of deciduous trees and shrubs near the Nagoya University Forest, in the northeast of Aichi Prefecture, in central Japan. The major results are summarized as follows.(1) Biological features of X. mutilatus population in the field① X. mutilatus was univoltine in the study area. From late June to early September, with a peak in late July, overwintered female adults made their dispersal flights in search of host trees suitable for colonization. The sex ratio of their offspring (♂: ♀) was about 1:8.② Many species of ambrosia beetles were found in freshly-felled trees of Lindera triloba, whereas few beetles invaded the trees of Clethra barbinervis. The trees of Acer sieboldianum were dominantly attacked by X. mutilatus adults, and the number of attacks by this insect per main trunk of A. sieboldianum was larger than that of Acer rufinerve and that of C. barbinervis.③ The proportion of galleries in which female adults of X. mutilatus laid eggs to all galleries monitored (the proportion of oviposition) was lower in the order, A. sieboldianum, L. triloba, C. barbinervis and A. rufinerve. In A. rufinerve, the proportion of galleries in which adult eclosion occurred to the galleries oviposited (the proportion of reproduction ) was less than half of that in A. sieboldianum, in L. triloba and in C. barbinervis. From a gallery system constructed in A. rufinerve, it was observed that 2 or 3 female adults emerged newly, but more than 5 female adults were observed in other three species of host trees.④ A female adult began to lay clumped eggs in the gallery system when she excavated a side gallery (length of approx. 1 cm) soon after construction of a main gallery and within 20 days after tree-felling. It was found that the female adult spreads a thin membrane completely over the entrance hole before oviposition.⑤ Twenty days after oviposition, pupation and adult eclosion occurred, and all developmental stages (eggs, larvae, pupae and new adults) were present in the gallery systems. The brood size increased until the 60th day after tree-felling, but thereafter decreased because the new male adults disappeared from the galleries and the larvae died due to shortage of the fungal food. The total length of the side galleries at the end of the larva-pupa period was about twice that during the egg period. After brood development, further extension of the side galleries was most likely due to browsing by new adults, and resulted in an average main-side gallery of 6 cm in length.⑥ There was a positive correlation between the total length of gallery system and the number of offspring per gallery system at every growing stage of the brood population. However, the slope of the linear regression during the egg (0.68) to pupa period was less than that during the new adult to overwintering adult (0.16) period.⑦ In gallery systems less than 6 cm long, some of offspring (female pupae and new female adults) had an extremely light mean body weight, compared with other individuals that had developed in the gallery systems. The mean body weights of the offspring in the ≧ 6 cm long gallery systems were significantly greater than those in the < 6 cm long gallery systems. However, there were large size variations, within the same range, in offsprings from both ≧ 6 cm and < 6 cm long gallery systems.⑧ Frequency distributions of female body weight were compared among the groups of pupae and new adults with different rates of development, which were classified according to the time of pupation (whether pupae alone were present or both pupae and eclosed adults coexisted together ) and the time of eclosion determined by adult body colors in the same brood, respectively. The mean body weights of pupae and new adults were heavier in individuals that pupated and eclosed earlier, and the weight ranges also shifted from light towards heavy.(2) Dynamics of fungal symbionts in the gallery system① By the direct inoculation technique, three fungal groups, Ambrosiella sp., yeasts and Paecilomyces sp., were isolated from main galleries excavated in L. triloba, where Ambrosiella sp. was predominant during the boring-oviposition period, but then yeasts and Paecilomyces sp. gradually became co-dominant. From the galleries in A. sieboldianum, in A. rufinerve and in C. barbinervis Ambrosiella sp. was also isolated dominantly during the boring-oviposition period.② The same three fungal groups were also found in galleries by the viable plate count technique. The number of colony-forming units of each fungal species per 1 cm long galleries changed with the developmental stages of the beetle in the same manner, as the relative dominance of fungal symbionts isolated by the direct inoculation technique also changed. Direct microscopic obsevations using a counting chamber revealed that the total number of spores and hyphae of Ambrosiella sp. in the 1 cm long galleries rose to a peak during the period from boring to 1st-instar larva, steeply declined during the last instar larva period and then reached a plateau until the new adult period.③ The dynamics of the fungal flora in side galleries showed a trend similar to that in main galleries, except that Paecilomyces sp. was isolated more frequently during the larva-pupa period and that there was a slither decrease in the viable count of Ambrosiella sp. during the period from 1st-instar larva to last instar larva.④ These results were supported by SEM observations which revealed that growing phase of fungal symbionts in the gallery system at various developmental stages of the beetle.(3) Dynamics of fungal symbionts in the mycangia ① It was shown that female adults of X. mutilatus have a pair of dorsal pouched formed by the intersegmental membrane between the pronotum and the mesonotum for carrying fungal spores.② Under a SEM, it was observed that (1) there are only lamellar substances attached to the membrane in the mycangia of overwintering adults, (2) in contrast, during the dispersal period, every mycangial sac is tightly filled with fungal spores, the surfaces of which a are covered with a sticky liquid and which also secretes viscous substances themselves and (3) the sacs are sealed by a swollen membrane that is equipped with many needle-like projectrions.③ Only a single fungal species, Ambrosiella sp., was consistently stored in the mycangia in all adult stages, except for the periods of teneral and overwintering adults during which yeasts and Paecilomyces sp. predominated over Ambrosiella sp. No fungal spores occurred in the mycangia of the adult beetles reared under aseptic conditions from the pupal stage, whilst only Ambrosiella sp. was stored in those from the teneral adult stage. Regardless of species of host trees attacked, Ambrosiella sp. was the dominant fungus in the mycangia of the disperising adults.④ The viable count of Ambrosiella sp. per mycangia showed a maximum during the dispersal flight period and an abrupt decrease during the boring-oviposition period, followed by a gradual fall. The change in the total number of spores and hyphae of the fungus in relation to life history of the beetle was exactly similar to observed with the viable count, except for a slower decline after the oviposition period. Highest percentages of spores in Ambrosiella sp. were strored in the mycangia of the disperaing adults.⑤ In the mycangia of the overwintering adults placed at 5℃, yeasts and Paecilomyces sp. were consistently co-dominant, whereas only Ambrosiella sp. was isolated from the mycangia at 25℃ throughout the experimental period. In case of controlled-temperature treatments at 5,15,20 and 25℃, the number of colony-forming units of Ambrosiella sp. per mycangia increased greatly with time after treatment at 20 and 25℃, where the viable fungal number at 25℃ was significantly larger than that at 20℃. It was the number of fungal spores at 25℃ that increased significantly with treatment time.(4) Artificial rearing experiments of X. mutilatus on fungal diets① On diet plates of Ambrosiella sp. (sp. 1) associated with X. mutilatus and Ambrosiella sp. (sp. 3) associated with Xylosandrus brevis, hatched larvae of X. mutilatus successfully grew into adults, but no larvae survived on those of Paecilomyces sp. and yeast (Candida sp.) associated with X. mutilatus Ambrosiella sp. (sp. 2) associated with Scolytoplatypus mikado and Ambrosiella sp. (sp. 4) associated with Xylosandrus crasiussculus. The percentage of emerged adults reared on Ambrosiella sp.1 (68.8%) was significantly higher than that on Ambrosiella sp.3 (17.6%), and the mean body weight of female pupae on Ambrosiella sp.1 was also significantly heavier than that on Ambrosiella sp.3.② Of the four species of host trees, hatched larvae of X. mutilatus pupated and eclosed on Ambrosiella sp. 1 that was propagated on potato-dextrose agar mixed with saw dust of L. triloba and of A. sieboldianum. However, there was no significant difference in mean body weight of female pupae fed on dietary fungus of L. triloba or A. sieboldianum.③ The developmental periods of X. mutilatus females from eggs to 1st-instar larvae, pupae and teneral adults were about 3,15(13-17) and 22 days, respectively, and males emerged about 3 days earlier than females. In each diet plate, however, earlier-hatched larvae completed their development before later-hatched larvae.④ The mean pupal weight and the proportion of successful pupation were both positively correlated with increasing fungal area per individual larva, but both were likely to approach upper limits (9 mg and 80%, respectively ) above a fungal area of about 10 c㎡.⑤ There was a positive correlation between the pupal weight and the developmental rate during the period from 1st-instar larva to pupa in solitary reared females (one-larva per plate of Ambrosiella sp. 1).⑤ Protein analysis of fungi associated with several species of ambrosia beetles① Protein variations among seven ambrosia fungi associated with X. mutilatus, S. mikado, X. brevis and X. crasiussculus was examined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The genus Ambrosiella associated with the four different species of ambrosia beetles contained more visible proteins than Paecilomyces sp. and Candida sp. associated with X. mutilatus, showing the beetle species-specific patterns of protein bands.② The protein pattern of Ambrosiella sp. 2 associated with S. mikado differed greatly from that of other Ambrosiella spp. associated with Xylosandrus spp. On the contrary, there was a remarkable similarity in protein patterns of Ambrosiella spp. between X. mutilatus and X. brevis, both of which also presented and an almost complete similarity in biological features.All the present results suggest that : ① X. mutilatus utilizes Ambrosiella sp. as the most important food resource (primary ambrosia fungi : PAF) for its development, and uses the whole gallery system in various host trees as the space available for cultivating the fungus. The reproductive success of X. mutilatus, however, is most likely to be affected mainly by chemical ingredients contained in each species of the trees.② A mother beetle may be capable of manipulating her fecundity according to the expected quantities of food resource by expanding the gallery system for cultivating Ambrosiella sp. Earlier-hatched larvae in a brood can grow into large-sized progeny adults by predominantly utilizing fungal food resources of the best quality. The combination of resource-dependent oviposition by a mother beetle and dominant resource utilization by earlier-hatched individuals in a brood can contribute to the efficient production of larger females under limited resource conditions.③ Immediately after eclosion, new female adults may take at least four associated fungal species, without any selection, into their mycangia from the walls of the galleries, and conditions may well be produced in the mycangia of both matured and dispersing beetles whereby only the spores of Ambrosiella sp. can proliferate. Temperature surrounding the adult beetles is probably a key factor that influences the activity of the gland cells and/or the viability of the fungal spores in the mycangia. The cells of mycangia may secrete some chemicals favoring the propagation of the PAF and/or inhibiting the growth of other contaminating fungi, and antibiotics produced by the PAF may also limit contamination by other microbes. It would be adaptive that a mother beetle can disseminate the PAF over the walls of both the main gallery and side galleries, allocating the amount of fungal spores according to growth rate of the PAF in each gallery.④ Different species of ambrosia beetles living in the same tree have different symbiotic associations with their own specific fungi (PAF). As similarity of biological features among scolytid beetles increases, degrees of relatedness among PAF associated with the beetles also increases. Each PAF is most likely to have a nutritional potential as a food resource for scolytid larvae of other related species, but consequently giving lower "fitness" to the beetles.⑤ From the results mentioned above (④), two new hypotheses, PCM (pairwise coevolutionary mutualism ) and DCM (diffuse coevolutionary mutualism), are proposed for coevolutionary pathways between scolytid beetles and their species-specific PAF.⑥ Finally, based on all results of the present study, symbiotic interactions between the ambrosia beetles and their associated fungi are clarified, and attributes of the interactions are discussed according to the definition of symbiosis.農林水産研究情報センターで作成したPDFファイルを使用している。