昆虫の感覚系（嗅覚系）

Sensory Systems of Insects

昆虫の嗅覚受容

昆虫は主に頭部に付属する一対の触角により環境の匂いを受容する。触角上には感覚子と呼ばれる多数の突起状の感覚器が存在しており、嗅覚感覚子はクチクラ上に多数の嗅孔をもつ多孔性感覚子である。嗅覚感覚子の内部には複数の嗅覚受容細胞があり、匂い受容部位である樹状突起を感覚子内へ、軸索を中大脳にある触角葉とよばれる嗅覚情報処理の一次中枢へ伸ばしている（触角の項参照）。匂い分子は触角上の嗅覚感覚子のクチクラへの吸着、拡散を経て、嗅孔を通り感覚子内部へと入る。感覚子内はリンパ液（感覚子リンパ）で満たされているため、揮発性で水に溶けにくいフェロモンや匂い分子は、感覚子リンパ中に高濃度で存在するフェロモン結合タンパク質（PBP）または匂い結合タンパク質（OBP）と結合することで可溶化され、樹状突起膜上に発現する嗅覚受容体へと移行すると考えられている。また最近PBPはフェロモンの可溶化・移行の機能だけでなく下記に示す嗅覚受容体の活性化にも必須であるという結果が報告されている。結合タンパク質により可溶化された匂い分子が受容細胞の樹状突起膜上に発現する嗅覚受容体と結合すると、受容細胞の脱分極が引き起こされ、活動電位が発生し匂い受容シグナルが触角葉へ伝えられる。本項では、フェロモンと一般臭の匂い受容の分子機構を中心に最近の知見を紹介する。

昆虫は花や食べ物の匂いなどの一般臭とフェロモンを受容する２種類の嗅覚受容器を備えている。一般臭の受容器は特異性が低く、さまざまな匂い物質に応答を示す「ジェネラリスト」の性質をもつ。また、匂い応答スペクトルは受容器どうしで一部重複する。一方、フェロモン受容器は特異性が高く、リガンドと受容細胞は一対一で対応する「スペシャリスト」である。一般臭の受容器はショウジョウバエやゴキブリ、ミツバチでよく調べられており、応答スペクトルによりいくつかのタイプに分類される1-3)。

カイコガ（Bombyx mori）は1959年にはじめてフェロモンの化学構造が同定された種であり16)、これまでにフェロモン受容系のモデルとして多くの知見が蓄積されてきた。フェロモンの特異的な受容器は雄の触角に密生する毛状感覚子である（リンク）。フェロモンは主成分であるボンビコール(bombykol) [(E、Z)-10、12-hexadecadien-1-ol]と副成分であるボンビカール(bombykal) [(E、Z)-10、12-hexadecadien-1-al]から構成される16,17)。カイコガの雄はボンビコールのみで完全な配偶行動を発現する。一方、ボンビカールはボンビコールによる雄の配偶行動の解発閾値をあげ、行動を抑制する効果がある17)。毛状感覚子には、ボンビコールとボンビカールに高感度かつ特異的に電気的応答を示すフェロモン受容細胞が対になって入っている。これらの細胞はわずか1分子のフェロモンに対し興奮するといわれ、他の化合物にはほとんど応答を示さない17,18,22,23)。

昆虫嗅覚受容体遺伝子

バックとアクセルにより、1991年に嗅覚受容体遺伝子がラットではじめて単離され、これがGタンパク質共役型受容体（GPCR）ファミリーに属することが示された19)。その後、魚類やセンチュウでもGPCRファミリーに属する嗅覚受容体遺伝子が単離された。

一方、昆虫においても、匂いやフェロモン受容後にGタンパク質を介した二次伝達物質であるイノシトール三燐酸（IP3）が急速かつ一過性の上昇を示すこと、嗅覚受容細胞の樹状突起膜上にIP3により開くイオンチャネルが存在すること、キイロショウジョウバエのホスホリパーゼC欠損変異体で匂い応答が減少することなどから、嗅覚受容体はGPCRであると推測された。そして1999年にフォッセルらによって、キイロショウジョウバエのゲノム解析から新規GPCRをコードする遺伝子の探索によって昆虫ではじめて嗅覚受容体候補遺伝子ファミリーDOr（Drosophila odorant receptor）が同定された20)。

さらに、2000年にショウジョウバエの全ゲノム配列が解読され、DOrファミリーは60遺伝子から構成され、62の受容体タンパク質をコードすることが示された。そのうち42遺伝子が成虫の嗅覚受容細胞で特異的に発現している。DOrの推定アミノ酸配列には膜貫通領域と推定される7つの疎水性領域があり、脊椎動物やセンチュウの嗅覚受容体と同様にGPCRファミリーに属すると考えられる。しかし、既知のGPCRのアミノ酸配列との間に保存性がないことから、昆虫は独自の嗅覚受容体ファミリーを形成しているようである。ショウジョウバエでは、個々の嗅覚受容細胞は一種類の嗅覚受容体と、例外的にほとんどすべての嗅覚受容細胞で発現するOr83の２種類の受容体を発現している21)。

一方、フェロモン受容体遺伝子は、櫻井らにより2004年にカイコガではじめて同定された22)。カイコガのフェロモン受容器は雄の触角にあり、雌は自らの放出するフェロモンに反応しない。そこで櫻井らは、雄触角cDNAライブラリーについてdifferential screeningを行い、雄触角で特異的もしくは多量に発現する遺伝子を単離することでフェロモン受容体遺伝子を得た。得られたcDNAクローンのひとつは、既知の昆虫嗅覚受容体遺伝子とアミノ酸配列に類似性を示した。このクローンはカイコガの学名にちなんでBmOR1（Bombyx mori olfactory receptor 1）と名付けられた22)。アフリカツメガエル卵母細胞にBmOR1を発現させた一連の研究から、BmOR1はボンビコールを特異的に受容し、BmGaqを介したシグナル伝達系を活性化することが示された。また、BmOR1と既知の昆虫嗅覚受容体配列をもとにカイコゲノム中に29個の嗅覚受容体様の配列が見出された。それらのうち4遺伝子が雄特異的もしくは優位に発現していたが、BmOR1以外に卵母細胞発現系でボンビコールに応答する受容体はなかった22)。
　さらに、カイコガ雌の触角に、組換えバキュロウイルス感染によりBmOR1を発現させると、ボンビコールに対してのみ電気的応答を示した22)。以上の櫻井らによる一連の研究を通して、カイコガのフェロモン受容体の実体が、ブテナントがフェロモンの化学構造を決定してから、実に半世紀を経てはじめて明らかになったのである22)。

一般臭受容機構

Transduction Mechanisms of General Odor in Insects

一般の匂い

匂い物質とは、ヒトや動物の嗅覚受容系を経て匂いとして認識される分子量約300以下の揮発性の化学物質である（Touhara and Vosshall, 2009）。これら匂い物質の中で、食物、火災、外敵の存在を知らせるような環境中に存在する匂い物質が一般臭と呼ばれている（フェロモンについては、匂いの受容機構フェロモン参照）。匂い物質の大多数は親油性であり水に溶けにくいが、哺乳類や昆虫はそれぞれ異なる匂い溶解、受容機構を発達させ、匂いを認識してきた。本項では、昆虫における一般臭の受容機構についての分子機構をカイコガでの知見を交えて紹介する。

一般臭の可溶化と輸送

昆虫は、一般臭を触角に存在する嗅覚感覚子によって受容する。嗅覚感覚子は主に触角に分布しているが、キイロショウジョウバエでは少数ではあるが頭部の小顎髭（maxillary palp）や唇弁（labial palp）にも存在することが報告されている（Stange, 1992、de Bruyne et al., 1999、Kwon et al., 2006）。嗅覚感覚子は特徴的な構造をしており、表面に嗅孔（pore）と呼ばれる多数の孔が開いた構造をしている（触角参照）。感覚子の内部は感覚子リンパで満たされており、嗅覚受容ニューロン（olfactory receptor neuron; ORN）の樹状突起が存在する。ORNの細胞体は、匂い結合タンパク質（odorant binding protein; OBP、Vogt et al., 1991）やフェロモン結合タンパク質（pheromone binding protein; PBP、Vogt and Riddiford, 1981）を分泌する補助細胞（鞘生細胞、毛生細胞、窩生細胞）によって囲まれている（Steinbrecht et al., 1995、Shanbhag et al., 1999、Shanbhag et al., 2000）。ORNは双極細胞であり（Keil, 1997）、一方は感覚子リンパ中に樹状突起を伸ばし、もう一方は脳へ投射している（神経経路参照）。ORNは樹状突起上に存在する受容体が匂い分子と結合することによって興奮し、脳へと刺激を伝える。このような嗅覚感覚子はその形態と大きさから複数のタイプに分類されており（Shanbhag et al., 1999、触角参照）、各タイプごとに感覚子の匂い応答は単一感覚子記録法により観察できる（de Bruyne et al., 2001）。キイロショウジョウバエでは、ほとんどのタイプの感覚子について匂い応答スペクトルが決定されており、感覚子はタイプごとに異なる匂い応答スペクトルを示す（de Bruyne et al., 1999, 2001、Hallem et al., 2004、Yao et al., 2005、van der Goes van Naters and Carlson, 2007）。各感覚子には2-4個の異なる受容体をもつORNが存在し、感覚子の匂い応答スペクトルはこれらのORNの匂い応答スペクトルを表している。たいていの場合、一般臭を受容する感覚子は異なる匂いに様々な強度で応答するスペクトルを示す（de Bruyne et al., 1999, 2001、Hallem et al., 2004）。これらORNの応答スペクトルは、嗅覚受容体のリガンド親和性によるものであることが示されている（以下参照）。

嗅覚感覚子に吸着された匂い物質は感覚子リンパ中に高濃度で存在するOBPによって可溶化され、受容体へと輸送される。昆虫のOBPはポリフェムス蚕（Antheraea polyphemus）で初めて発見された（Vogt and Raddiford, 1981）。それ以降、40種類以上の昆虫種で単離されており、ゲノム解析により、カイコガでは44種類、キイロショウジョウバエでは51種類、ハマダラカでは57種類あると推定されている（Maida et al., 1993、Krieger et al., 1996、Pelosi et al., 2006）。これまでに単離されているOBPはアミノ酸配列の類似性に基づいて4つのタイプ（PBP、General odorant binding protein；GOBP1、GOBP2、Antennal binding protein X；ABPX）に分類される（Vogt et al., 1991、1999、Pelosi et al., 2006）。OBPは約15kDaの可溶性のタンパク質で、6つのシステイン残基が保存されており3つのジスルフィド結合をもつ（Scaloni et al., 1999、Leal et al., 1999）。立体構造については、カイコガのPBPを用いて初めてX線結晶構造解析が行われて以降、キイロショウジョウバエのOBP（LUSH）など6つのOBPについて結晶構造解析が行われている（Sandler et al., 2000、Pelosi et al., 2006）。これら結晶構造解析による結果を元に、匂い分子を結合したOBPは樹状突起膜付近の低pH領域に入ると、pH依存的にコンフォーメーションを変化させ、匂い分子をリリースし受容体へと受け渡されると考えられている（Leal et al., 2005）。

一般臭の嗅覚受容体

嗅覚受容体遺伝子は、ラットで初めて同定され（Buck and Axel, 1991）、Gタンパク質共役型受容体であることが示された（Firestein, 2001）。その後、脊椎動物ではヒト（Ben-Arie et al., 1994）、魚類（Ngai et al., 1993）や鳥類（Nef et al., 1996）の嗅覚受容体遺伝子が同定された。無脊椎動物では、ゲノム解析により線虫から7回膜貫通部位を持つ嗅覚受容体遺伝子が同定された（Troemel et al., 1995、Sengupta et al., 1996）。昆虫においてもゲノム解析が進み、現在までにキイロショウジョウバエ（Drosophila melanogaster）から62種類、ミツバチ（Apis mellifera）から170種類、ハマダラカ（Anopheles gambiae）から79種類、ネッタイシマカ（Aedes aegypti）から131種類、コクヌストモドキ（Tribolium castaneum）から341種類、カイコガ（Bombyx mori）から66種類の嗅覚受容体遺伝子が推定されている（Clyne et al., 1999、Vosshall et al., 1999、Gao and Chess, 1999、Fox et al., 2001, 2002、Hill et al., 2002、Krieger et al., 2002, 2004、Robertson and Wanner, 2006、Wanner et al., 2007、Bohbot et al., 2007、Engsontia et al., 2008、Tanaka et al., 2009）。昆虫の嗅覚受容体は疎水性領域の解析から、7回膜貫通型であることが示されている。しかし、哺乳類の嗅覚受容体と比較して、昆虫の嗅覚受容体は配列の類似性が低く、DRYアミノ酸モチーフ構造のような類似配列が存在しない。また、C末端が細胞内、N末端が細胞外に位置するトポロジーをとるなど、脊椎動物の嗅覚受容体や他のGタンパク質共役型受容体とは異なり、Gタンパク質共役型受容体に見られる多くの特徴を欠くことが指摘されている（Wistrand et al., 2006、Benton et al., 2006、Lundin et al., 2007）。

昆虫の嗅覚受容体の機能同定は、アフリカツメガエル卵母細胞発現系やキイロショウジョウバエの形質転換体を用いた電気生理学実験と、ヨトウガ卵巣細胞由来のSf9細胞発現系を用いたカルシウムイメージング法により進められてきた（Wetzel et al, 2001、Stortkuhl and Kettler, 2001、Hallem et al., 2004、Lu et al., 2007、Anderson et al., 2009、Tanaka et al., 2009、Jordan et al., 2009）。昆虫の嗅覚受容体では、キイロショウジョウバエのOr43aを用いて初めて機能同定が行われた。アフリカツメガエル卵母細胞を用いた機能解析によりOr43aがベンズアルデヒドやシクロヘキサノンに応答することが示されている（Wetzel et al., 2001）。同時に報告された論文では、Or43aを異所発現させたキイロショウジョウバエ触角を用いて触角電位図EAG（electroantennogram）を行い、in vivoにおけるOr43aの応答特性を調べており、卵母細胞における応答と同様に、ベンズアルデヒドやシクロヘキサノンに応答することが示された（Stortkuhl and Kettler, 2001）。これら二つの研究により、in vivoで嗅覚受容体により匂いの受容と識別が行われていることが明らかとなった。

その後、Carlsonのグループにより、キイロショウジョウバエの形質転換体のempty neuronによる手法を用いて嗅覚受容体の大規模な機能解析が進められている（de Bruyne et al., 1999, 2001、Hallem et al., 2006）。本方法を用いて、キイロショウジョウバエの24種類の嗅覚受容体について110種類の匂い成分に対する応答測定が実施され、24種類の嗅覚受容体について機能同定が行われた（Hallem et al., 2004、2006）。また、最近では、ハマダラカの嗅覚受容体についても本方法の適用により、50種類の嗅覚受容体について応答特性が明らかにされている（Carey et al., 2010）。これら嗅覚受容体の応答特性はORNでの応答特性と同じであることが示されており、ORNの応答が嗅覚受容体によるものであることが示された。

カイコガにおいても一般臭に対する嗅覚受容体の同定が進められている。Andersonらは、Sf9細胞を用いたカルシウムイメージング法を用いて、3種類の雌に特異的に発現する嗅覚受容体（BmOR19、BmOR45、BmOR47）の機能を同定した（Anderson et al., 2009）。BmOR19はリナロールに、BmOR45、BmOR47は安息香酸に応答することが示され、これらの成分が植物由来の匂い成分であることから、産卵場所や雄の放出するフェロモンの認識に関わっている可能性が示唆されている。また、Tanakaらは23種類のカイコガ幼虫で発現する嗅覚受容体について機能解析を行った（Tanaka et al., 2009）。これらのうち、BmOR59がカイコガの食草である桑の葉に含まれるシスジャスモンに特異的な嗅覚受容体であることを示し、シスジャスモンによるBmOR59の活性化がカイコガの幼虫の食草の探索に関わることを示唆している。

昆虫では、最近、嗅覚受容体の特殊な分子メカニズムが明らかになってきた。これまで、昆虫において触角にGタンパク質が存在すること（Laue et al., 1997）、三量体Gタンパク質を介した二次伝達物質であるIP3（inositol-trisphosphate）が匂い応答時に一過性で急速に上昇すること（Boekhoff et al., 1993）、そしてIP3によって開閉されるイオンチャネルが存在すること（Stengel, 1994）などの観察結果から、昆虫の嗅覚応答はGタンパク質共役型受容体（GPCR）を介したシグナル伝達経路によって化学的なシグナルを電気的なシグナルに変換し、匂いの情報を伝えると考えられてきた（Krieger and Breer, 1999）。しかし、キイロショウジョウバエのOr83bと呼ばれるタンパク質の機能から、昆虫に特異的な嗅覚シグナル伝達機構があることがわかった。

Or83bのアミノ酸配列は嗅覚受容体と似ているものの、Or83bは嗅覚受容体としては機能しなかった。このタンパク質は昆虫種を超えて、アミノ酸配列がよく保存されている（Krieger et al., 2003、Jones et al., 2005）。キイロショウジョウバエでは、Or83bが嗅覚受容ニューロンの大多数で発現しており、Or83b欠損変異体は匂いに応答をしなかった（Vosshall et al.,1999、Larsson et al., 2003）。形質転換体キイロショウジョウバエを用いた研究から、Or83bの機能は嗅覚受容体の膜への輸送や保持であると推定された（Benton et al., 2006）。培養細胞を用いたin vitroの実験から、Or83bは嗅覚受容体とヘテロ複合体を形成していることが明らかにされた（Neuhaus et al., 2005、Lundin et al., 2007）。Or83bファミリータンパク質はカイコガでも見つけられており、アフリカツメガエル卵母細胞で性フェロモン受容体と共発現させると受容体の性フェロモンに対する応答感度が上昇した（Nakagawa et al., 2005）。その後、昆虫の嗅覚受容体では、Gタンパク質を介してシグナル伝達されるのではなく、Or83bファミリータンパク質と共にチャネルを形成し、リガンド作動性のカチオンチャネルとして機能することが明らかにされた（Sato et al., 2008、Wicher et al., 2008）。このように、昆虫において匂いのシグナルは、匂い結合型のイオンチャネルを通して、ORNにシグナルを伝達することが示された。

その他の嗅覚受容体（イオンチャネル型グルタミン酸受容体）

キイロショウジョウバエでは、一般臭に対する嗅覚受容体やフェロモン受容体のほかにイオンチャネル型グルタミン酸受容体（ionotropic receptor；IR）が嗅覚受容体として機能していることが報告されている。（Benton et al., 2009）IRは、NMDA、AMPA、Kainate受容体と類似性があるが、グルタミン酸を受容する部位が欠失しており、嗅覚感覚子（Coeloconica sensillum）の感覚ニューロンの樹状突起で発現しているという特徴がある。異所発現させた遺伝子組換えキイロショウジョウバエによる機能解析からこれらIRがアンモニア、フェニルアセトアルデヒドを含む一般臭を受容することが示されている。しかし、現在のところ、カイコガを含む他の昆虫種では未だ単離、同定されていない。

フェロモン受容機構

フェロモンについて

一般臭とは対照的に、個体から発せられ同種の他個体に特異的な行動もしくは生理的変化を引き起こす情報化学物質をフェロモンと呼ぶ（Karlson and Luscher, 1959）。フェロモンは昆虫に誘引する効果によって、性フェロモン、警報フェロモン、集合フェロモン、道しるべフェロモンなどに分類されている。この中で、昆虫の繁殖に欠かせないものが配偶行動を引き起こす性フェロモンである。性フェロモンは、1959年に初めてカイコガ（Bombyx mori）でボンビコール（(E,Z)-10,12-hexadecadien-1-ol）の化学構造が決定されて以来（Butenandt et al., 1959）、現在までに、農業害虫を含む1500種以上の昆虫種で化学成分の化学構造が決定され、データベースに登録されている（The Pherobase; http://www.pherobase.com/、The Pherolist; http://www-pherolist.slu.se/pherolist.php, Byers 2002）。同定された性フェロモン成分の化学構造は多様であるが、蛾類の性フェロモン成分は化学構造が種の間で類似している（Byers, 2005）。この類似性は、蛾類の性フェロモンが蛾類に共通な2つの生合成経路で合成されている、ことに由来すると考えられている（Ando et al., 2004）。すなわち、蛾がde novo合成した脂肪酸を出発物質として生合成する経路（TypeⅠ）と、植物由来のリノール酸やリノレン酸を出発物質として生合成する経路（TypeⅡ）、の2つが知られている。前者からはボンビコールのようなアルコールやアルデヒド、アセテートなどが、後者からは直鎖炭化水素が、それぞれ性フェロモン成分として合成される（Ando et al., 2004）。本項では、このように種に特異的に生合成された性フェロモンの受容機構について紹介する。

性フェロモンの可溶化と輸送

フェロモンは、触角（antenna）上にあるフェロモンの受容に特化した嗅覚感覚子（Trichodea sensillum）によって受容される。感覚子に吸着したフェロモン分子は嗅孔を通り、感覚子リンパに入る。昆虫の性フェロモン成分は親油性が高いため、一般臭と同様、可溶性タンパク質（Pheromone binding protein；PBP）によって可溶化され輸送される（Vogt, 2003）。PBPは、これまでに多種の蛾類から単離され、性フェロモン成分と結合することが確かめられている（Pelosi et al., 2006）。PBPは性フェロモン識別の第一段階であり、性フェロモンを特異的に受容すると考えられてきた（Plettner et al., 2000、Bette et al., 2002、Maida et al., 2003）。これに対して、PBPが他種の性フェロモン成分やそれ以外の匂い物質とも結合する例も報告されている（Campanacci et al., 2001、Grater et al., 2006）。また、PBP遺伝子の嗅覚組織における発現解析をおこなって、性差は見られるものの、メスとオスいずれの触角でも発現していることが報告されている（Abraham et al., 2005、Forstner et al., 2006、Watanabe et al., 2007、Xiu et al., 2007）。これらの研究結果から、PBPは性フェロモンの特異的な受容には関わっていない、とも考えられている。
しかし、最近、キイロショウジョウバエで雄の放出するフェロモン成分であるcis-vaccenyl acetate（cVA）の受容にOBP（LUSH）が関与することが報告されている（Xu et al., 2007、Laughlin et al., 2008）。lush変異体ではcVAを受容する嗅覚感覚子の応答がなくなる一方で、部位特異変異によるLUSHの立体構造の変化は嗅覚感覚子でフェロモン受容と同等以上の応答を引き起こす。これらの報告により、受容体は性フェロモンではなく、LUSHの性フェロモン結合による構造変化を認識していることが示唆されている。しかし、現在までに、PBPと性フェロモン受容体の関係については明らかにされておらず、今後の研究成果が望まれている。

性フェロモン受容体

性フェロモンを末梢で識別している分子機構としては、嗅覚受容ニューロンで発現している受容体が第一候補と考えられる。動物の性フェロモン受容体は、カイコガのボンビコール受容体遺伝子が単離、同定されたのが最初の例である（Sakurai et al., 2004）。カイコガのオス触角で特異的に発現している遺伝子群をディファレンシャルスクリーニング法によって分離した。それらの配列解析によって嗅覚受容体候補遺伝子を単離し、アフリカツメガエル卵母細胞発現系を用いた電気生理学実験によってボンビコールに応答することを確認した。カイコガ性フェロモンのもう1つの成分であるボンビカールの受容体も同様に同定された（Nakagawa et al., 2005）。また、オオタバコガではいくつかの性フェロモン受容体候補遺伝子が単離されており、それらはカイコガの性フェロモン受容体と同様にオス触角で特異的に発現している（Krieger et al., 2004）。最近、キイロショウジョウバエのempty neuronで受容体を発現させた形質転換体を用いて、オオタバコガのHR13が性フェロモン成分であるZ11-16:Aldに応答することが報告された（Kurtovic et al., 2007）。また、培養細胞を用いたカルシウムイメージング法を用いても、HR13の機能解析が行われている（Grosse-Wilde et al., 2007）。これまでに同定されてきた昆虫の嗅覚受容体はいずれも、1つの受容体がいくつもの異なる匂いに応答するので、リガンド特異性が低いことが共通の特徴である（Hallem et al., 2004、Carey et al., 2008）。それらに対して、同定された3つの蛾類の性フェロモン受容体は嗅覚受容体でありながら、いずれもリガンド特異性が高いことが明らかになった。
最近では、コナガ（Plutella xylostella）、アワヨトウ（Mythimna separata）、ウリノメイガ（Diaphania indica）、アワノメイガ（Ostrinia）性フェロモン受容体が性フェロモンの主成分に対する受容体が同定されてきている（Mitsuno et al., 2008、Miura et al., 2009）。これら受容体は、昆虫の嗅覚受容体の中でカイコガの性フェロモン受容体と同じクラスターに分類されることが示されている。そのため、チョウ目の性フェロモンは配列の類似した受容体により認識されていると考えられている。
性フェロモンの受容に関しては、SNMP（sensory neuron membrane protein）も関与していることが報告されている（Benton et al., 2007）。SNMPはカイコガを含む多くの昆虫種のフェロモンを受容する嗅覚感覚子に存在し、フェロモンの受容に関わることが示唆されてきた（Rogers et al., 1997、2001）。Bentonらは、キイロショウジョウバエを用いてsnmp変異体のフェロモンに対する応答を測定し、一般臭の嗅覚受容体に対する応答は変わらなく、性フェロモンの応答が減少することを示した。オオタバコガの性フェロモン受容体HR13を組み換えたショウジョウバエについても同様の結果が得られたことから、SNMPが脂肪酸由来の性フェロモンの受容機構において、PBPから性フェロモン受容体への輸送の間で機能していることが示唆されている。

参考文献

Abraham D., Lofstedt C. & Picimbon J.F. (2005) Molecular characterization and evolution of pheromone binding proteingenes in Agrotis moths. Insect Biochem. Mol. Biol. 35, 1100-1111.

Anderson A.R., Wanner K.W., Trowell S.C., Warr C.G., Jaquin-Joly E., Zagatti P., Robertson H. & Newcomb R.D. (2009)Molecular basis of female-specific odorant responses in Bombyx mori. Insect Biochem. Mol. Biol. 39, 189-197.

Ando T., Inomata S. & Yamamoto M. (2004) Lepidopteran Sex Pheromones. Topics Curr. Chem. 239, 51-96.

Ben-Arie N., Lancet D., Taylor C., Khen M., Walker N., Ledbetter D.H., Carrozzo R., Patel K., Sheer D. & Lehrach H. (1994) Olfactory receptor gene cluster on human chromosome 17: possible duplication of an ancestral receptor repertorie. Hum. Mol. Genet. 3, 229-235.

Benton R., Sachse S., Michnick S.W. & Vosshall L.B. (2006) Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLOS Biol. 4, e20.

Benton R., Vannice K.S., Gomez-Diaz C. & Vosshall L.B. (2009) Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149-162.

Benton R., Vannice K.S. & Vosshall L.B. (2007) An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature 450, 289-293.

Bette S., Breer H. & Krieger J. (2002) Probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence. Insect Biochem. Mol. Biol. 32, 241-246.

Boekhoff I., Seifert E., Goggerle S., Lindemann M., Kruger B.W. & Breer H. (1993) Pheromone-induced second-messenger signaling in insect antrnnae. Insect Biochem. Mol. Biol. 23, 757-762.

Bohbot J., Pitts R.J., Kwon H.W., Rutzler M., Robertson H.M. & Zwiebel L.J. (2007) Molecular characterization of the Aedes aegypti odorant receptor gene family. Insect Mol. Biol. 16, 525-537.

Buck L.B. & Axel R. (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175-187.

Butenandt V.A., Beckmann R., Stamm D. & Hecker E. (1959) Uber den sexsuallockstoff des seidenspinners Bombyx mori. Reindarstellung und konstitution. Z. Naturforschg. 14b, 283-284.

Byers J.A. (2002) Internet programs for draing moth pheromone analogs and searching literature database. J. Chem. Ecol. 28, 807-817.

Byers J.A. (2005) Chemical constraints on the evolution of olfactory communication channels of moths. J. Theor. Biol. 235, 199-206.

Campanacci V., Krieger J., Bette S., Sturgis J.N., Lartigue A., Cambillau C., Breer H. & Tegoni M. (2001) Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay. J. Biol. Chem. 276, 20078-20084.

Carey A.F., Wang G., Su C.Y., Zwiebel L.J. & Carlson J.R. (2010) Odorant reception in the malaria mosquito Anopheles gambiae. Nature 464, 66-71.

Clyne P.J., Warr C.G., Freeman M.R., Lessing D., Kim J. & Carlson J.R. (1999) A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22, 327-338.

de Bruyne M., Clyne P.J. & Carlson J.R. (1999) Odor coding in a model olfactory organ: the Drosophila maxillary palp. J. Neurosci. 19, 4520-4532.

de Bruyne M., Foster K. & Carlson J.R. (2001) Odor coding in the Drosophila antenna. Neuron 30, 537-552.

Engsontia P., Sanderson A.P., Cobb M., Walden K.K., Robertson H.M. & Brown S. (2008) The red flour beetle’s large nose: an expanded odorant receptor gene family in Tribolium castaneum. Insect Biochem. Mol. Biol. 38, 387-397.

Firestein S. (2001) How the olfactory system makes sense of scents. Nature 413, 211-218.

Forstner M., Gohl T., Breer H. & Krieger J. (2006) Candidate pheromone binding proteins of the silkmoth Bombyx mori. Invert. Neurosci. 6, 177-187.

Fox A.N., Pitts R.J., Robertson H.M., Carlson J.R. & Zwiebel L.J. (2001) Candidate olfactory receptors from the malaria vector mosquito Anopheles gambiae and evidence of down-regulation in response to blood feeding. Proc. Natl. Acad. Sci. USA 98, 14693-14697.

Fox A.N., Pitts R.J. & Zwiebel L.J. (2002) A cluster of candidate odorant receptors from the malaria vector mosquito, Anopheles gambiae. Chem. Senses 27, 453-459.

Gao Q. & Chess A. (1999) Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics 60, 31-33.

Grater F., Xu W., Leal W. & Grubmuller H. (2006) Pheromone discrimination by the pheromone binding protein of Bombyx mori. Structure 14, 1577-1586.

Grosse-Wilde E., Gohl T., Bouche E., Breer H. & Krieger J. (2007) Candidate pheromone receptors provide the basis for the response of distinct antennal neurons to pheromonal compounds. Eur. J. Neurosci. 25, 2364-2373.

Hallem E.A., Ho M.G. & Carlson J.R. (2004) The molecular basis of odor coding in the Drosophila antenna. Cell 117, 965-979.

Hallem E.A. & Carlson J.R. (2006) Coding of odors by receptor repertoire. Cell 125, 143-160.

Hill C.A., Fox A.N., Pitts R.J., Kent L.B., Tan P.L., Chrystal M.A., Cravchik A., Collins F.H., Robertson H.M. & Zwiebel L.J. (2002) G protein-coupled receptors in Anopheles gambiae. Science 298, 176-178.

Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T.: Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori, Genetics, Vol. 165 (2003) pp. 1329–1340.

International Silkworm Consortium. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem. Mol. Biol. 38, 1036-1045 (2008).

Jones W.D., Nguyen T.T., Kloss B., Lee K.J. & Vosshall L.B. (2005) Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Curr. Biol. 15, R119-R121.

Jordan M.D., Anderson A., Begum D., Carraher C., Authier A., Marshall S.D., Kiely A.,　Gatehouse L.N., Greenwood D.R., Christie D.L., Kralicek A.V., Trowell S.C. & Newcomb RD. (2009) Odorant receptors from the light brown apple moth (Epiphyas postvittana) recognize important volatile compounds produced by plants. Chem. Senses. 34, 383-394.

Karlson P. & Luscher M. (1959) Pheromones’: a new term for a class of biologically active substances. Nature 183, 55-56.

Keil T. (1997) Comparative morphogenesis of sensilla: a review. Int. J. Insect Morphol. Embryol. 26, 151-160.

Krieger J., von Nickisch-Rosenegk E., Mameli M., Pelosi P. & Breer H. (1996) Binding proteins from the antennae of Bombyx mori. Insect Biochem. Mol. Biol. 26, 297-307.

Krieger J. & Breer H. (1999) Olfactory reception in invertebrates. Science 286, 720-723.

Krieger J., Grosse-Wilde E., Gohl T., Dewer Y.M.E., Raming K. & Breer H. (2004) Genes encoding candidate pheromone receptors in a moth (Heliothis virescens). Proc. Natl. Acad. Sci. USA 101, 11845-11850.

Krieger J., Klink O., Mohl C. & Breer H. (2003) A candidate olfactory receptor subtype highly conserved across different insect orders. J. Comp. Physiol. A 189, 519-526.

Krieger J., Raming K., Dewer Y.M.E., Bette S., Conzelmann S. & Breer H. (2002) A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens. Eur. J. Neurosci. 16, 619-628.

Kurtovic A., Widmer A. & Dickson B.J. (2007) A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542-546.

Kwon H., Rutzler M. & Zwiebel L.J. (2006) Olfactory responses in a gustatory organ of the malaria vector mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. USA 103, 13526-13531.

Larsson M.C., Domingos A.I., Jones W.D., Chiappe M.E., Amrein H. & Vosshall L.B. (2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43, 703-714.

Laue M.L., Maida R. & Redkozubov A. (1997) G-protein activation, identification and immunolocalization in pheromone sensitive sensilla trichodea of moth. Cell Tissue Res. 288, 149-158.

Laughlin J.D., Ha T.S., Jones D.N. & Smith D.P. (2008) Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell 133, 1255-1265.

Leal W.S., Nikonova L. & Peng G. (1999) Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori. FEBS Lett. 464, 85-90.

Leal W.S., Chen A.M., Ishida Y., Chiang V.P., Erickson M.L., Morgan T.I. & Tsuruda J.M. (2005) Kinetics and molecular properties of pheromone binding and release. Proc. Natl. Acad. Sci. USA 102, 5386-5391.

Lu T., Qiu Y.T., Wang G., Kwon J.Y., Rutzler M., Kwon H.W., Pitts R.J., van Loon J.J.A., Takken W., Carlson J.R. & Zwiebel L.J. (2007) Odor coding in the maxillary palp of the malaria vector mosquito Anopheles gambiae. Curr. Biol. 17, 1533-1544.

Lundin C., Kall L., Kreher S.A., Kapp K., Sonnhammer E.L., Carlson J.R., Heijine G. & Nilsson I. (2007) Membrane topology of the Drosophila OR83b odorant receptor. FEBS Letters 581, 5601-5604.

Maida R., Steinbrecht A., Ziegelberger G. & Pelosi P. (1993) The pheromone binding protein of Bombyx mori: purification, characterization and immunocytochemical localization. Insect Biochem. Mol. Biol. 23, 243-253.

Maida R., Ziegelberger G. & Kaissling K.E. (2003) Ligand binding to six recombinant pheromone-binding proteins of Antheraea polyphemus and Antheraea pernyi. J. Comp. Physiol. B 173, 565-573.

Mitsuno H., Sakurai T., Murai M., Yasuda T., Kugimiya S., Ozawa R., Toyohara H., Takabayashi J., Miyoshi H. & Nishioka T. (2008) Identification of receptors of main sex-pheromone components of three Lepidopteran species. Eur. J. Neurosci. 28, 893-902.

Miura N., Nakagawa T., Touhara K. & Ishikawa Y. (2010) Broadly and narrowly tuned odorant receptors are involved in female sex pheromone reception in Ostrinia moths. Insect Biochem. Mol. Biol. 40, 64-73.

Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P. and Bamberg, E. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940-13945 (2003).

Nakagawa T., Sakurai T., Nishioka T. & Touhara K. (2005) Insect sex-pheromone signals mediated by specific combinations of olfactory receptors. Science 307, 1638-1642.

Nef S., Allaman I., Fiumelli H., De Castro E. & Nef P. (1996) Olfaction in bird: differential embryonic expression of nine putative odorant receptor genes in the avian olfactory system. Mech. Dev. 55, 65-77.

Neuhaus E.M., Gisselmann G., Zhang W., Dooley R., Stortkuhl K. & Hatt H. (2005) Odorant receptor heterodimerization in the olfactory system of Drosophila melanogaster. Nat. Neurosci. 8, 15-17.

Ngai J., Dowling M.M., Buck L., Axel R. & Chess A. (1993) The family of genes encoding odorant receptors in the channel catfish. Cell 72, 657-666.

Pelosi P., Zhou J.J., Ban L.P. & Calvello M. (2006) Soluble proteins in insect chemical communication. Cell. Mol. Life Sci. 63, 1658-1676.

Plettner E., Lazar J., Prestwich E.G. & Prestwich G.D. (2000) Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar. Biochemistry 39, 8953-8962.

Robertson H.M. & Wanner K.W. (2006) The chemoreceptor superfamily in the honey bee, Apis mellifera: expansion of the odorant, but not gustatory, receptor family. Genome Res. 16, 1395-1403.

Rogers M.E., Sun M., Lerner M.R. & Vogt R.G. (1997) Snmp-a, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins. J. Biol. Chem. 272, 14792-9.

Rogers M.E., Steinbrecht R.A. & Vogt R.G. (2001) Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus. Cell. Tissue Res. 303, 433-446.

Sakurai T., Nakagawa T., Mitsuno H., Mori H., Endo Y., Tanoue S., Yasukochi Y., Touhara K. & Nishioka T. (2004) Identification and functional characterization of a sex pheromone receptor in the silkmoth Bombyx mori. Proc. Natl. Acad. Sci. USA 101, 16653-16658.

Sato K., Pellegrino M., Nakagawa T., Nakagawa T., Vosshall L.B. & Touhara K. (2008) Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452, 1002-1006.

Sandler B., Nikonova L., Leal W.S. & Clardy J. (2000) Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein-bombykol complex. Chem. Biol. 7, 143-151.

Scaloni A., Monti M., Angeli S. & Pelosi P. (1999) Structural analysis and disulfide-bridge pairing of two odorant binding proteins from Bombyx mori. Biochem. Biophys. Res. Commun. 266, 386-391.

Sengupta P., Chou J.C. & Bargmann C.I. (1996) odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84, 899-909.

Shanbhag S.R., Muller B. & Steinbrecht R.A. (1999) Atlas of olfactory organs of Drosophila melanogaster 1. Types, external organization, innervation and distribution of olfactory sensilla. Int. J. Insect Morphol. Embryol. 28, 377-397.

Shanbhag S.R., Muller B. & Steinbrecht R.A. (2000) Atlas of olfactory organs of Drosophila melanogaster 2. Internal organization and cellular architecture of olfactory sensilla. Arthropod Struct. Dev. 29, 211-229.

Stange G. (1992) High resolution measurement of atmospheric carbon dioxide concentration changes by the labial palp organ of the moth Heliothis armigera (Lepidoptera: Noctuidae). J. Comp. Physiol. A 171, 317-324.

Steinbrecht R.A., Laue M. & Ziegelberger G. (1995) Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx. Cell Tissue Res. 282, 203-217.

Stortkuhl K.F. & Kettler R. (2001) Functional analysis of an olfactory receptor in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 98, 9381-9385.

Tamura, T., Kuwabara, N., Uchino, K., Kobayashi, I., & Kanda, T. An improved DNA injection method for silkworm eggs drastically increases the efficiency of producing transgenic silkworms. J. Insect Biotechnol. Sericol, 76, 155-159 (2007).

Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P.: Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector, Nat. Biotechnol. 18, 81-84 (2000).

Tanaka K., Uda Y., Ono., Y., Nakagawa T., Suwa M., Yamaoka R. & Touhara K. (2009) Highly selective tuning of a silkworm olfactory receptor to a key mulberry leaf volatile. Curr. Biol. 19, 881-890.

Touhara K. & Vosshall L.B. (2009) Sensing odorants and pheromones with chemosensory receptors. Annu. Rev. Physiol. 71, 307-332.

Troemel E.R., Chou J.H., Dwyer N.D., Colbert H.A. & Bargmann C.I. (1995) Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207-218.

van der Goes van Naters W.M. & Carlson J.R. (2007) Receptors and neurons for fly odors in Drosophila. Curr. Biol. 17, 606-612.

Vogt R.G. & Riddiford L.M. (1981) Pheromone binding and inactivation by moth antennae. Nature 293, 161-163.

Vogt R.G., Prestwich G.D. & Lerner M.R. (1991) Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. J. Neurobiol. 22, 74-84.

Vogt R.G. (2003) Biochemical diversity of odor detection: OBPs, ODEs and SNMPs. In Insect Pheromone Biochemistry and Molecular Biology Blomquist G.J., Vogt R.G. (eds) pp391-446. Elsevier Academic Press.

Vosshall L.B., Amrein H., Morozov P.S., Rzhetsky A. & Axel R. (1999) A spatial map of olfactory receptor expression in the Drosophila antennae. Cell 96, 725-736.

Wang G., Carey A.F., Carlson J.R. & Zwiebel L.J. (2010) Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. USA 107, 4418-23.

Wanner K.W., Anderson A.R., Trowell S.C., Theilmann D.A., Robertson H.M. & Newcomb R.D. (2007) Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori. Insect Mol. Biol. 16, 107-119.

Watanabe H., Tabunoki H., Miura N., Sato R. & Ando T. (2007) Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type 2 sex pheromone components. Invert. Neurosci. 7, 109-118.

Wetzel C.H., Behrendt H.J., Gisselmann G., Stortkuhl K.F., Hovemann B. & Hatt H. (2001) Functional expression and characterization of a Drosophila odorant receptor in a heterologous cell system. Proc. Natl. Acad. Sci. USA 98, 9377-9380.

Wicher D., Schafer R., Bauernfeind R., Stensmyr M.C., Heller R., Heinemann S.H. & Hansson B.S. (2008) Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452, 1007-1011.

Wistrand M., Kall L. & Sonnhammer E.L.L. (2006) A general model of G protein-coupled receptor sequences and its application to detect remote homologs. Protein Sci. 15, 509-521.

Yamagata, T., Sakurai, T., Uchino, K., Sezutsu, H., Tamura, T. and Kanzaki, R. GFP labeling of neurosecretory cells with the GAL4/UAS system in the silkmoth brain enables selective intracellular staining of neurons. Zool. Sci. 25, 509-516 (2008).

Yao C.A., Ignell R. & Carlson J.R. (2005) Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. J. Neurosci. 25, 8359-8367.Xiu W.M. & Dong S.L. (2007) Molecular characterization of two pheromone binding protein and quantitative analysis of their expression in the beet armyworm, Spodoptera exigua Hubner. J. Chem. Ecol. 33, 947-961.

Xu P., Atkinson R., Jones D.N. & Smith D.P. (2005) Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons. Neuron 45, 193-200.

Sensory Systems of Insects

Olfaction in insects

Insects perceive odorant mainly with sensory organs called sensilla on their antennae. Olfactory sensilla are generally characterised by bearing tiny pores on their cuticular surface and occur in a bewildering variety of shapes, of which the most common one is probably the hair. There are several olfactory receptor cells in the sensillum. Their dendrites extend into a sensillar lumen and their axons project to the first order olfactory information processing center of the brain, the antennal lobe (see also the chapter on the insect antenna). An odorant can be adsorbed on the cuticular surface of the sensillum, diffuse towards a pore and through it into the inside of the sensillum. Since the sensillum is filled with sensillum lymph, it is thought that volatile and insoluble odorants such as pheromones bind to specific odorant binding proteins (OBP), are thereby solubilized and transferred to the olfactory receptors on the dendrites of the olfactory receptor cells. Recently it has been reported for a special class of OBPs, the pheromone-binding proteins (PBP) that they are necessary for the activation of pheromone-sensitive odorant receptors. When the solubilized odorant binds to odorant receptors in the dendritic membrane of the olfactory receptor cell, the cell is depolarized and generates action potentials, which transmit the olfactory signal to the antennal lobe. In this chapter, we introduce the current knowledge concerning the molecular mechanisms of olfaction.

Insects olfactory receptor neurons (ORNs) can roughly be classified into two types: those are responsive to general odors such as floral odors or food-related odors and those that are responsive to pheromone, i.e. species-specific odorants. General odorant receptor proteins (ORs) show low specificity and respond to various odorants hence they are called generalists. They have partially overlapping odorant response spectra. On the contrary pheromone receptor proteins show high specificity and respond to only one ligand (a specific pheromone) hence they are called specialists. Genera ORs are well investigated in fruit fly, cockroach and honeybee and categorized into a number of types based on their response spectra^1-3).

The domestic silkmoth (Bombyx mori) is the species in which the chemical structure of a pheromone was first identified¹⁶⁾ and it has become a model organism of pheromone reception for which much information has been accumulated. Pheromone (specific) receptor neurons are housed in a type of hair sensillum, the long sensillum trichodeum, of which a large number covers the side branches of the antennae of the male silkmoth. Silkmoth pheromone is composed of bombykol, the major component [(E, Z)-10, 12-hexadecadien-1-ol], and bombykal, a minor (or accessory) component [(E, Z)-10, 12-hexadecadien-1-al]^16,17). Bombykol alone is sufficient to elicit mating behavior in the male silkmoth. On the other hand bombykal raises the threshold for the induction of male mating behavior by bombykol and can thus effectively inhibit mating behavior¹⁷⁾. Bombykol and bombykal receptor neurons are housed in pairs in the same type of trichoid sensillum. These receptor cells respond with high specificity to their pheromone ligands^{17, 18, 22, and 23)}

Insect odorant receptor genes

Buck and Axel first isolated olfactory receptor genes in rat in 1991 and showed that the genes belong to the G-protein coupled receptor (GPCR) family¹⁹⁾. In the following, olfactory receptor genes belonging to the GPCR family were also isolated from fish and the nematode Caenorhabditis elegans

In insects, it has been shown that the levels of second messenger inositol triphosphate (IP3) increase transiently upon olfactory stimulation in olfactory receptor neurons and their dendrites have also been shown to have ion channels opened by IP3. [I don’T understand the part on the reduction of responses in Drosophila, is it for an experiment with IP3 antagonist?] These lines of evidence led to the hyopthesis that insect odorant receptors also belong to the GPCR family. Then Vosshall et al. (1999) first identified a candidate insect odorant receptor gene family, DOr (Drosophila odorant receptor), by searching for GPCR genes in the genomic DNA sequence²⁰⁾.

In 2000 the Drosophila genomic DNA was completely sequenced. It has been shown that the DOr family is composed of 60 genes which encode 62 receptor proteins. Of these 60 genes, 42 are expressed in adult olfactory receptor neurons. DOr putative amino acid sequences possess seven hydrophobic regions that are inferred to be transmembrane regions and they are thought to belong to GPCR family as the odorant receptors of vertebrates and C. elegans do. As they have little sequence homology with known GPCR amino acid sequences, they form a separate GPCR family. Drosophila olfactory receptor neurons express two types of receptors, one of which is Or83 that is expressed almost every olfactory receptor neuron²¹⁾.

Pheromone receptor genes were first identified by Sakurai et al., 2004²²⁾ in the silkmoth. Silkmoth pheromone receptor neurons are only present on male antennae and females do not respond to the pheromone they release.. Sakurai et al. performed differential screening on a male cDNA library to isolate genes that were specifically and abundantly expressed on male antennae, and they obtained pheromone receptor gene candidates. One of the cDNA clone obtained showed a conspicuous homology with known insect odorant receptor genes in its amino acid sequence. This clone was named BmOR1 (Bombyx mori olfactory receptor 1) after the nomenclature for the silkmoth²²⁾. Subsequent experiments in which BmOR1 was expressed in Xenopus oocytes showed that BmOR1 specifically binds bombykol and activates a signal transduction system via BmGaq. Based on the sequences of BmOR1 and known insect odorant receptors, 29 odorant receptor-like sequences were discovered. Of these 29 sequences four genes were identified that were expressed male-specifically or male-dominantly. However, there was no receptor that responds to bombykol in the Xenopus oocyte expression system except BmOR1²²⁾.

Female antennae responded to bombykol electrically when BmOR1 was expressed in female antennae by infection with a recombinant baculovirus. The study by Sakurai et al. cited above revealed the identity of the silkmoth pheromone receptor almost fifty years after Butenandt et al. resolved the chemical structure of silkmoth pheromone²²⁾.

Transduction Mechanisms in the reception of General Odors in Insects

General Odors

Odorants are volatile chemical substances that have molecular weights less than about 300 and are recognized as odor by olfactory systems of living organisms (Touhara and Vosshall, 2009). Among them odorants that are, for example associated with the existence of food, fire, or a predator are called general odors (about pheromone, see chapter pheromone). Most odorants are lipophlic and insoluble in water, thus different mechanisms of odorant solubilization, reception, and recognition have evolved in insects and mammals. . In this section we provide an introduction to the molecular transduction mechanisms of general odor reception in insects. (sorry but most of the information is for Drosophila).

Solubilizaiton of odorants and their transport mechanisms

Insects perceive odorants through olfactory sensilla mostly located on the antennae. There is also a smaller number of olfactory sensilla on the maxillary palp and labial palp that belong to the mouth parts ([cite Altner!] Stange, 1992; de Bruyne et al., 1999; Kwon et al., 2006). An olfactory sensillum has a characteristic structure generally with numerous pores on its cuticular surface (see also Antenna). The lumen of the sensillum is filled with sensillum lymph surrounding the dendrites of olfactory receptor neurons (ORNs). ORN soma is surrounded by support cells (thecogen, thecogen, and trichogen cell) that secrete odorant binding proteins (OBP) or pheromone binding protein (PBP) (Steinbrecht et al., 1995; Shanbhag et al., 1999; Shanbhag et al., 2000). The ORN is a bipolar cell that extends its single dendrite into the sensillum and its single axon projects to the brain . The ORN is excited and can transmit an electrical signal to the brain when an odorant binds to the receptor protein in its dendritic membrane. Olfactory sensilla are categorized into several types according to their morphology and size (cite Altner and Prillinger, maybe 1988; Shanbhag et al., 1999; see also Antenna) and the odor response of each cell type can be recorded by single sensilum recording under favorable conditions in some types of sensilla (de Bruyne et al., 2001). In Drosophila the olfactory response spectra of most types of sensilla have been recorded and each type has a distinct olfactory spectrum (de Bruyne et al., 1999, 2001; Hallem et al., 2004; Yao et al., 2005; van der Goes van Naters and Calrson, 2007). Each sensillum houses two to four ORNs with different odorant receptor and the olfactory response spectrum of each sensillum represents these ORN spectra. (de Bruyne et al., 1999, 2001; Hallem et al., 2004). It has been shown that these response spectra of ORNs are derived from the ligand affinity of their olfactory receptor proteins.

Odorants adsorbed on the sensillum are solubilized by a high concentration of OBP in the sensillum lymph and transported (or do they diffuse rather?) to the receptor proteins in the dendritic membrane. Insect OBPs were first discovered in the silkmoth Antheraea polyphemus (Vogt and Riddiford, 1981). Since then insect OBPs have been isolated in more than 40 species and genomic analysis implies that the silkmoth has 44 types of OBP, Drosophil 51, Anopheles gamgiae 57 (Maoda et al., 1993; Krieger et al., 1996; Pelosi et al., 2006). OBPs isolated so far are categorized into four types (PBP, General odorant binding protein; GOBP1. GOBP2, Antennal binding protein X; ABPX) (Vogt et al., 1991, 1999; Pelosi et al., 2006). OBPs are soluble proteins of about 15 kDa molecular weight and have six cysteine residues and three disulfide bonds (Scaloni et al., 1999; Leal et al., 1999). Since X-ray crystallography of silkmoth PBP was first performed, this method was also applied to six other OBPs including Drosophila OBP (LUSH) (Sandler et al., 2000; Pelosi et al., 2006). Based on these data, a model has been proposed according to which the OBP-ligand complex dissociates at acidic pH, such as found the dendritic membrane, making the odorant available for binding to an OR.

General odorant receptor

genes were first identified in rat (Buck and Axel., 1991), and it has been shown that they are G-protein coupled receptors (Firestein, 2001). After that the odorant receptor genes of human (Ben-Arie et al., 1994), fish (Ngai et al., 1993) and bird (Nef et al., 1996) were identified. In invertebrates, nematode odorant receptor genes that contain seven transmembrane domains were identified (Troemel et al., 1995; Sengupta et al., 1996) by genomic analysis. In insects the genomic analysis have revealed a species-specific number of candidate odorant receptor genes: 62 in the fruit fly (Drosophila melanogaster), 170 in the honeybee (Apis mellifera), 79 in the Malaria mosquito (Anopheles gambiae), 131 in the Yellow fever mosquito (Aedes aegypti), 341 in a flour beetle (Tribolium castaneum) and 66 in the silkmoth (Bombyx mori) (Clyne et al., 1999; Vosshall et al., 1999; Gao and Chess, 1999; Fox et al., 2001. 2002; Hill et al., 2002; Krieger et al., 2002, 2004; Robertson and Wanner, 2006 Wanner et al., 2007; Bohbot et al., 2007; Engsontia et al., 2008; Tanaka et al., 2009). It has been shown that insect odorant receptors are of the seven transmembrane domain types through hydrophobicity analysis. However, insect odorant receptors have little similarity with mammalian odorant receptors and do not have similarity sequences such as the DRY amino acid motif. Besides, the insect odorant receptors are different from vertebrate odorant receptors in terms of topology, which is reversed compared to vertebrates, with the C terminus intracellular and the N terminus extracellular. Insect odorant receptors lack many features that are characteristic of G-protein coupled receptors (Wistrand et al., 2006; Benton et al., 2006; Lundin et al., 2007).

Functional analyses of insect odorant receptors are performed by electrophysiological experiments using the Xenopus oocyte expression system, Drosophila transgenesis, or calcium imaging in the Barathra ovarian cell derived Sf9 cell expression system (Wetzel et al., 2001; Stortkuhl and Kettle, 2001; Hallmen et al., 2004; Lu et al., 2007; Anderson et al., 2009; Tanaka et al., 2009; Jordan et al., 2009). Among the insect odorant receptors, Drosophila Or43a was first analyzed in detail with respect to its functional properties. Using the Xenopus oocyte expression system it has been shown that Or43a responds to benzaldehyde and cyclohexanol (Wetzel et al., 2001). Using Or43a ectopically expressed in Drosophila antennae, the response characteristics of Or43a were investigated in vivo and it was shown that the antenna responds to benzaldehyde and cyclohexanol as well (Stortkuhl and Kettler, 2001). These two studies revealed that the odorant receptor receives and discriminates odorants.

The Carlson lab carried out a largescale functional analysis of the odorant receptor using empty neurons of Drosophila transformants (de Bruyne et al., 1999, 2001; Hallem et al., 2006). Using these methods, response measurements for 24 Drosophila odorant receptors to 110 odorants were performed to functionally characterize this set of odorant receptors (Hallem et al., 2004, 2006). Recently, using this method, the response characteristics of 50 odorant receptors of Anopheles gambiae were revealed (Carey et al., 2010). It was shown that the response characteristics of these receptors are identical with those of the ORNs and therefore, the response properties of ORNs are determined by the odorant receptors.

The identification of odorant receptors to general odors in the silkmoth is also rapidly progressing. Anderson et al. functionally identified three odorant receptors (BmOR19, BmOR45, and BmOR47) specifically expressed in females (Anderson et al., 2009). It has been shown that BmOR19 responds to linalool and BmOr45 and BmOr47 respond to benzoic acid. Linalol and benzoic acid are odorants derived from plants, therefore, it has been suggested that these receptors are related to the indentification of potential oviposition sites or male pheromone. Tanaka et al. functionally analyzed 23 odorant receptors expressed in silkmoth larvae (Tanaka et al., 2009). Of these receptors, BmOR59 was shown to specifically respond to cis-jasmone that is contained in mulberry leaves eaten by silkmoth larvae. This implies that BmOR59 activation by cis-jasmone is involved in attracting silkmoth larvae to leaves of the food plant.

Recently, peculiar molecular mechanisms have been discovered in insect odorant receptors. As mentioned above, G-proteins are present in insect antennae (Laue et al., 1997), and IP3, a second messenger activated via a trimeric G-protein, transiently increases in response to odorant (Boekhoff et al., 1993). Also, IP3-activated ion channels are known in insects. From these lines of evidence it has been hypothezised that insect olfactory transduction occurs though a trimeric G-protein-coupled receptor (Krieger and Breer, 1999). This resembles the scheme also found in vertebrates. However, the function of Or83b, a Drosophila odorant receptor family protein, indicates that olfactory signal transduction mechanisms in insects are different.

Although the amino acid sequence of Or83b is similar to other odorant receptors, Or83b does not function as an odorant receptor. The amino acid sequences of this protein and its homologs are well conserved across insect species (Krieger et al., 2003; Jones et al., 2005). In Drosophila, Or83b is expressed in almost all olfactory receptor neurons and Or83b deletion mutants do not respond to odorants (Vosshall et al., 1999; Larsson et al., 2003). From a study using Drosophila Or83b transformants it has been inferred that the functions of Or83b are membrane trafficking of odorant receptors and retention of the receptor proteins in the membrane (Benton et al., 2006). In vitro experiments using cultured cells demonstrated that Or83b forms heteromers with other odorant receptor proteins (Neuhaus et al., 2005; Lundin et al., 2007). An Or83b-family protein is also found in the silkmoth. When it is coexpressed with sex pheromone receptor protein the response sensitivity of the receptor mechanism is increased (Nakagawa et al., 2005). After that it was shown that the insect odorant receptor forms a ligand-gated cation channel in conjuction with the Or83b-family protein and does not function as a G-protein-coupled receptor (Sato et al., 2008: Wicher et al., 2008). Thus, rather than being a classical G-protein-mediated mechanism, odorant binding appears to gate channel activity directly in insects, providing a chemo-electrical transduction with only two components.

Atypical odorant receptors (ionotropic glutamate receptor family?)

In Drosophila it has been reported that a member of the ionotropic glutamate receptor family (ionotropic receptor; IR) functions as an odorant receptor other than general odorant receptors and pheromone receptors (Benton et al., 2009). The IR shares sequence homology with known NMDA, AMPA, and Kainate receptors but are devoid of a glutamate receptor site and expressed in the dendrites of sensory neurons in sensilla coeloconica. The functional analysis of recombinant Drosophila ectopically expressing this IR shows that this receptor is responsive to specific general odors including ammonia and phenylacetaldehyde. However, this type of IR has so far not been isolated and identified from other insect species.

Transduction Mechanisms of Pheromone receptors

Pheromones

In contrast to general odors, chemical substances that are released by an individual and that elicit specific behaviors or physiological changes in conspecific individuals are called pheromones (Karlson and Luscher, 1959). Pheromones are classified as sex pheromones, alarm pheromones, aggregation pheromones, or trail pheromones by their effect on conspecifics. Sex pheromones elicit mating behavior and are thus crucial for insect reproduction. Since the chemical structure of silkmoth sex pheromone, bombykol ( (E, Z)-10, 12-hexadecadien-1-ol), was first identified, the chemical structure of sex pheromones of more than 1500 species, including numerous economically important insects, has been identified and registered in databases (Pherobase; http://www.pherobase.com/; Pherolist; http://www.pherolist.slu.se/pherolist.php; Byers, 2002). Although the chemical structure of identified sex pheromone components includes a wide range of chemicals, sex pheromones of moths share some homologies among species (Byers, 2005). It is thought that these homologies are due to two common biosynthetic pathways that are shared by moths (Ando et al., 2004). In one pathway, moths synthesize the sex pheromone component from de novo fatty acids, (Type I), in the other the component is synthesized from linoleic acid or linolenic acid derived from plants (Type II). Through the former, alcohols such as bombykol as well as aldehydes and acetate are produced, the latter permits the synthesis of straight-chain carbohydrates (Ando et al., 2004). Generally, information on pheromones, pheromone-sensitive sensilla, and molecular mechanisms has in particular been obtained for sex pheromone systems in moths. Therefore, the present material is mostly representative for moths.

Solubilizaiton of pheromones and their transport mechanisms

Insects perceive pheromone through olfactory sensilla trichodea that are specialized for pheromone binding and localized on the antennae. Pheromone is adsorbed to the cuticle and can diffuse laterally to finally enter a sensillum trichodeum through one of the pores on its surface and be solubilized into the sensillar lymph. Insect sex pheromone components are highly lipophilic and as a result, in analogy to general odorants, they are solublized by pheromone binding proteins (PBP) and transported [transport or diffusion??? see also above under “general odors”!] (Vogt, 2003). PBPs have been isolated from many moth species and it has been confirmed that PBPs can bind sex pheromone components (Pelosi et al., 2006). It has been thought that binding to PBPs is the first step to sex pheromone recognition and that PBPs bind sex pheromones specifically (Plettner et al., 2000; Bette et al., 2002; Maida et al., 2003). However, it has been reported that PBPs can also bind sex pheromones of other species and even general odorants (Vampanacci et al., 2001; Grater et al., 2006). Gene expression analysis of PBPs in sensillar tissues revealed that PBP are expressed in the antennae of both sexes although with different expression levels (Abrahma et al., 2005; Forstner et al., 2006; Watanabe et al., 2007; Xiu et al., 2007). This implies that PBP does not bind sex pheromone exclusively. On the other hand, an OBP (LUSH) appears to be involved in the reception of Drosophila male pheromone, cis-vaccenyl acetate (cVA) (Xu et al., 2007; Laughlin et al., 2008). In lush mutant, trichoid sensilla normally sensitive to cVA does not respond to cVA stimulation whereas a conformational change of LUSH induced by site-directed mutagenesis causes the sensilla to respond to cVA. These reports suggest that the pheromone receptor recognizes a structural change in the LUSH-sex pheromone complex and not the sex pheromone itself. However, the relationship between PBPs and sex pheromone receptors is still poorly understood.

Sex pheromone receptor

Odorant receptors expressed in olfactory receptor neurons are the first candidate of the peripheral molecular mechanisms that recognize sex pheromone. The bombykol receptor of the silkmoth has been the first sex pheromone receptor isolated and identified in any animal species (Sakurai et al., 2004). Genes specifically expressed in male silkmoth antennae were isolated using differential screening methods. From these genes, candidate olfactory receptor genes were isolated by sequence analysis and were confirmed to be olfactory receptors, by electrophysiological experiments using the Xenopus oocyte expression system. The receptor for another sex pheromone component in silkmoth, bombykal, was identified in the same way (Nakagawa et al., 2005). In a hawkmoth, several candidates of sex pheromone receptors were isolated and they are specifically expressed on male antennae (Krieger et al., 2004). Recently, using aDrosophila mutant with “empty” olfactory receptor neurons devoid of their native receptors, the gene product of the pheromone receptor candidate gene HR13 of hawkmoth responds to a sex pheromone component, Z11-16:Ald (Kurtovie et al., 2007). A functional analysis of HR13 has also been done using Calcium imaging of cultured cells (Grosse-Wilde et al., 2004). Generally, insect odorant receptors identified so far respond to several odorants and thus have low ligand-specificity (Hallem et al., 2004; Carey et al., 2008). In contrast, the three moth sex pheromone receptors identified have high ligand specifity.

Recently, sex pheromone receptors of the diamondback moth (Plutella xylostella), Mythimna separate, Diaphania indica, and Ostrinia responding to major sex pheromone components have been identified (Mitsuno et al., 2008; Miura et al., 2009). It has been shown that these receptors can be classified into the same cluster to which silkmoth sex pheromone receptor belongs. Thus it is thought that sex pheromones of Lepidoptera are recognized by receptors that have the similar sequences.

Another type of protein that appears to be involved in sex pheromone reception is SNMP (sensory neuron membrane protein) (Benton et al., 2007). It is suggested that SNMP occurs in olfactory trichoid sensilla of many insect species including silkmoth (Rogers et al., 1996, 2001).Benton et al. measured the pheromone response of Drosophila snmp transgenic line and show that its response to general odorant does not change but the response to sex pheromone decreases. The same result is obtained from recombinant Drosophila that expresses the hawkmoth sex pheromone receptor HR13. The function of SNMP is thought to be a transfer of the fatty-caid-derived ligand from the PBP to the odorant receptor protein.

Reference