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中国腹刺斑螟生物学和线粒体COI基因序列初步记述(鳞翅目:螟蛾科

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环境昆虫学报Journal ofEnvironmental Entomology,December 2016,38(6):1178—1184 ISSN 1674-0858 doi:10.3969/i.issn.1674—0858.2016.06.14 Preliminarily notes on biology and mitochondrial COI gene of Sacculocornutia sinicolella Caradja (Lepidoptera:Pyralidae: Phycitinae) XIAO Yun.Li ,FANG Cheng ,REN Ying—Dang ,YANG Lin—Lin ,ZHONG Yu—Lin (1.Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive,Utilization Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal University,Huanggang 438000,Hubei Province,China;2.Institute of Plant Protection,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China) Abstract:Sacculocornutia sinicolella Caradja was firstly reported feeding on the leaves of Castanea mollissima Blume in Luotian County,Hubei Province,China.On the basis of both field survey and laboratory rearing,the larva and pupa morphologies of Sacculocornutia sinicolella were described and photographed,the ecological habits of the species were primarily given and photographedand molecular ,analyses based on the 629 bp portion of the mitochondrial DNA from the cytochrome oxidase I(mt COI) region was conducted to supplement morphological characteristics in identifying this species. Key words:Sacculocornutia sinicolella;larva;pupa;biology;COI gene;Castanea mollissima Blume;China 中国腹刺斑螟生物学和线粒体COI基因 序列初步记述 (鳞翅目:螟蛾科:斑螟亚科) 肖云丽 ,方 程 ,任应党 ,杨琳琳 ,钟玉林 (1.经济林木种质改良与资源综合利用湖北省重点实验室,大别山特色资源开发湖北省协同创新中心,黄冈师范学院生命科学学院 湖北黄冈438000;2.河南农业科学院植物保护研究所,郑州450002) 摘要:首次发现中国腹刺斑螟幼虫取食板栗叶片,描述了幼虫和蛹的形态特征,拍摄了幼虫和蛹的生活特征图 和外形等结构特征图,初步描记了其在湖北省罗田县栗园的生活习性;对幼虫线粒体COI基因片段序列进行了 提取、扩增、测序和分析,其5’端629 bp的DNA序列可以作为中国腹刺斑螟鉴定的分子标记。 关键词:中国腹刺斑螟;幼虫;蛹;生物学;COI gene;板栗;中国 中图分类号:Q968;¥433 文献标识码:A 文章编号:1674—0858(2016)06—1178—07 The genus Sacculocornutia Roesler,1 97 1 is on the roots,stems,leaves,flowers,fruits and s0 on, belonged to the subfamily Phycitinae Ragonot in the family Pyralidae.The larvae of Phycitinae species feed vast majority becoming the pest insects of agricultural, forest,grass,stored grain,dried fruit,herbs,oil and 基金项目:湖北省教育厅科学研究项目(D20152904);湖北省自然科学基金项目(2015CFA158);湖北省重点实验室项目(2013000703) 湖北省协同创新中心创新团队项目(2015TD06) 作者简介:肖云丽,女,1978年生,湖南新化人,博士,主要研究方向为昆虫系统学与害虫防治,E—mail:xiaoyunli0817@126.con 通讯作者Authors for correspondence,Email:swzyl@hgnu.edu.CI1 收稿日期Received:2016—08—26;接受日期Accepted:2016—10—12 6期 肖云丽等:中国腹刺斑螟生物学和线粒体COI基因序列初步记述(鳞翅目:螟蛾科:斑螟亚科) 1179 food processing products(Li&Ren,2009).The species of the genus Sacculocornutia were distirbuted in Tianjin,Hebei,Shanghai,Henan,Zhejiang,Anhui, Hubei,Hunan,Guizhou,Shaanxi,Gansu Provinces, China and Japan.It consisted of four species,the type species S.monotonella(Caradja,1 927),S.zhengi (Du&Wang,2002),S.sinicolella(Caradja,1926) and S.flavipalpella(Yamanaka,1990).The adults 0f all species have been detail described from China (Caradja,1926,1927;Roesler,1971;Yamanaka, 1990;Du et a1.,2002;Li&Ren,2009,2012),but the other information such as the biology and molecular characters of the genus is unknown to data.Based on the larval rearing in the laboratory and the investigations in the chestnut orchard in Luotian County,Hubei Province,China,the larvae,pupae of S.sinicolella were described on detail,the ecological habits were briefly narrated,and the COI molecular characters were analyzed. 1 Material and Methods 1.1 Collecting and rearing The larvae of Sacculocornutia sinicolella were collected from the leaves of Castanea mollissima Blume in Kuanghe,Baimiaohe,Luotouao,Hepu,Shengli Towns,Luotian County,Hubei Province,China from May to August in 2014.Partial larvae were reared to get pupae and adults putting into the plastic mess tins covered the pricking holes lids in the laboratory at the room temperature.The larvae and pupae are preserved in 100%alcohol for morphology and molecular studies,the adults were made in dry specimen for identiifcation study followed in the method of Li& Zheng(1996). 1.2 Morphology and ecology The larval and pupal habitats were observed by both field survey and laboratory rearing.The ecological,morphological bodies of the mature larvae, pupae and adult were photographed by Canon EOS 70D camera.The larval characteristic parts including the head.prethorax and mesothorax,third abdominal segment and caudal abdominal segments were photographed under Nikon SMZ 745T stereoscopic microscope by Nikon CooLPIX P7 1 00 camera.The adult specimens were dissected and mounted on the glass slides following the method of Li&Zheng (1996),the male genitalia structures were photographed same as the larval part.The nomenclature of larvae setae follows Hinton(1946). 1.3 Molecular analysis Total DNA was extracted from abdominal epidermis of five larval specimens(Sample ID: LT14001,LT14002,LT14003,LT14027,LT14049) with the E.Z.N.A.@Insect DNA Kit.The region of COI(sets of 629 bp)was ampliifed with the primer I_ ̄pF1/LepR1(Hebert et a1.,2004),PCRs were conducted in a 50 L reaction mixture containing 25 L 2×Taq PCR Master Mix,1.5 L forward and 1.5 tzL reverse primers(10 mmol/L),10 L DNA template,1 2 L sterile water.The PCR program included in order an initial denaturation step of 3 min at 9422,35 cycles of 9422 for 30 s,annealing at 5522 for 30 s and 72℃for 30 s.and a final extension at 7222 for 10 min.All ampliifcations were confirmed by gel electrophoresis electrophoresis on a 1.5%W/V agarose gel in TAE buffer(40 mmol/L Tris—acetate, 2 mmol/L Na2 EDTA・2H2O)and PCR products were direct—sequenced in forward direction at Biotech (Shanghai)Limited by Share Ltd,utilizing the same primers used for PCR ampliifcation. The sequences were manually trimmed to remove primers and ambiguous regions rfom the 5 and 3 ends in chromos 2.0 softwares,then a BLAST search was conducted in GenBank to determine the closest sequence match.Sequence data were analyzed using MEGA 6.0 softwares. The all specimens of larvae,pupae and adults of the species are deposited in the Insect specimen room of College of Life Science,Huanggang Normal University. 2 Results 2.1 Adult identification 1 Based oni’tH苦morphology of adult and the male genitalia sturctures(Fig.1),this insect was identiifed as Sacculocornutia sinicolella Caradja(Li&Ren, 2009,2012). 环境昆虫学报Journal of Environmental Entonmlog) 38卷 C Fig.1 Aduh I|lOl'])hology of Sac( 11locorntltia sinicolella A,female;B,male;C,male genitalia(Axy12013016) I)isli’ibuliim:Chi[1a(Anhlli,Gansu,Guizhou, Ht bet,l lenan,I hillei,Huuan,Shanxi,Shanghai, Head capsule with anterior bristles group A I,A2 and A3 arranged in a right—angled triangle,A 1 posterior t()antenna, A2 posterior to A 1, A3 dorsoposterior to first ocellei;posterior bristle PI Fiattjili,ZIleiiang);Japan. 2.2 Immature morphologies Larvae(Fig.2).Mature larvae length 14.0— beside apex angle of fron,P2 posterior to P1 and beside 1/2 of epicranial suture;lateral bristle LI 1 6.0 lUln.Head(Fig.2 D—E)green to yellowish gt’eetl,sI)mP luming reddish])rown before pupatton: (1ark lll‘own strips ai’i’anged in a somewhat circle shape on IIn Vt、i’tex al1(I hileral si(1es of head capsule.the ventroposterior to A3;ocellei bristles group O l,02 and 03 arranged in an obtuse—angled triangle,O 1 ventral to middle between second and third ocellei,02 liast t)f1 lit’istles 1)lack.anti fimning one large and one sltlal lel’tllack roun(1ed spots tieside verlex of fron and al st ot’I 1 llristle.Ocellei six.arranged in form of an a r(’.Iliack.Adfi‘()|1 about 4/7 length ot’capsule. carlTittg fliti1 lilac’k pinacula at 4/7 and 1/2.Fron aliotil 2/5 letlgtl1(’f capsule.1aterally bearing four Ilia(‘k spots at I)asc kill(1 1/2,and two black pinacula at I/4.1nisally will1 1wo bla(・k caves at center.Labrum ventroposterior to first ocellei,03 ventroposterior of 02;SO l,S02 and S03 arranged in an acute・angle triangle.Adfron bristle Afl and Af2 at 1/2 and apex of adfron respectively.Labrum with inner lateral bristles M l,M2 and M3 forming an acute—angled triangle, outer lateral bristles E 1.E2 and E3 arranged in a somewhat arc.Mandible with outer side carrying two bristles under first tooth of cutting edge.Labium bearing bristle LB 1 at center of postmentum. Submaxilary with MX2 anteriorouter to MX 1. 【 t1l1‘・dv‘-II to a1)1)iiI 1/3 of depth in middle of anterior nlat gin,fortni tg atI arc.MaMil)le with four apical teed1 Oil sl1一()l gly sclel’otize(I and menalised cutting edge,alni)ng heiIi,second f()()th largest,third tooth largeI’,finirth tooth sma]lest;inner suYface carrying Body green to yellowish green,some turning reddish brown before pupation,with ten yellowish white longitudinal bands along both sides of dorsal (ine hit’gt .rOHIidly trialigular tooth in center.Labium wiih stlitInerel length about 6.3 times of width of base, Mlial fnilpi aliout 1/3 length(if’spinneret,apically line,subdorsal line,lateral line,spiracle line and subventral line;each segment caudally yellow on dorsal and lateral sides,base of bristles and spiracles bristle t‘Iosed 10 etld tlf spinncl‘e1. all blank(Fig.2 A—C);prethorax covered with dark 6剐 『Li ijIJ :I…1 q腹刺斑蚁 物学和线粒体COl基闪序列仞步记述(鳞翅只:螟蛾科:斑螟业科) I】8 Fig.2 1 ̄lrval morphoh)gy of Sacculocornutia sinit’olella A—C,gem ral stl u( ̄ttll’P;D—E,head;F,aria1 sldehl;G,crochet;H,prethorax an(1 mesothrax; segmenl:J,eighth and ninth a1)dominal segmenls brown spots i ll dorsal anti lateral sides,a large blank spot,surrounding SD 1 and SD2 bristles(Fig.2-H); eighth abdominal segment laterally with a blank loop subrectangulal’or su],rhom1)ic spot carl’ying SD2 bristle existed dol。soallfel‘ior to spiracle (Fig.2-H); mesothol‘ax beat’ing a blank broad opell—loop shaped shaped spot,encircling SD I(Fig.2一J);anal shield covered with small blank spots,arc shaped in caudal 38巷 I)1 iII t'J()…of D2:SI)I ll…spirHt・I :L1 a『1(I 12 ,boll1 in d l}ina{’ulUI11;SV ~一.~_n . ~,一. 二.~一a川二 ~_一也.…三  __jr~. 三-一v三川 一~~一 .m一~a三Ⅲ .一三…_ 一_~圳Ⅲ三. _一¨三 _~.…三 =h~川乏 ~●一垮 isth-si ngle. pa{I vt n【l Oall[(}l’ior to s1) ht’isth sill lP.Ninlh seg;ll{ nl:1)1 Velltl‘t}antel’iot’to 1)2:l 1)fistles three,alI exis|e{{in a pinaeulure:S\/ Pupa(Fig.3).Ptl㈨length 7.5—8.0nIlll,wing l【‘hing anleriot’111 ̄11’gi ll llf fifth seglhen[.Bo{h~¨ _ _-三, . 州 ~~三^H ¨ 叫…/l . . 一.一 一一一一Ⅲ川¨ 一一~_ ●   . ~一U d一川薹一、.一 . ……… 一 . ,-^,  ”, .mstl',grt el1.gradual1) lul’ning to IlrOWII.I}la{‘k in etitl(1al t)ortion(Fig.3一A). l'enth at)donfinal segmental tel’grinl with small spiny hart(ts al I)asaI I/4 all{1 lateral 111 ̄11’g]‘r1 ;( ̄tlLl(tal lllal’gil1 latn’ially with 2 tooth—like Sl1jnt s.111( dially with 4 long l}J‘istle f'ail‘s t‘urled al apex (Fig.3一I;).All|IS.male al1《t ti ̄male genital pol’es all ril>shaped,existed in middle ol tenth,nitnh and t ighth ahdolninal segmental ='-;IeI‘Illllll ot ptlI)|J. I'eSp{ ti x I、(Fig.3 C—I)). 6期 肖云Ⅲ等:巾周腹刺斑蜞生物学和线粒体COl基 序列初步记述(鳞翅目:螟蛾科:斑螟亚科) 1183 Fig.4 Living ha1)its of Sacculocornutia sinicolella A,la,’va inhat,it in nests consist I)f two leaves;B,larva inhabit on upper side of leaf;C・D,larvae inhabit in empty nests 【lf D hocrocis chlorophanta Butler;E-F,larvae inhabit in empty nests of Rhodoneura vittula Guen6e;G—H,mature larva spinning all(1 forming a sparse thread cocoon r pupating. 2.3 Ecological habits larvae directly pupate(Fig.2-A)or inside a sparse thread cocoon(Fig.4 G—H).In the chestnut orchard of Luotian county.Hubei Province.1arvae firstly occu ̄ed at the end of April and pupated in the midmonth of May,adult appeared at the end of May, which were sustained to the end of August. 2.4 COI toolecular characters The 629 bp potion of the mt COI gene of the species was amplified by PCR and sequenced from the C.mollissimtl Blume (Fagaceae) is firstly reported as the host of S.sinicolella Caradja.The larvae of the species often spin threads to join two leaves and form a nests(Fig.4-A),or spin threads to knot a net on upper side of leaf and form a nests, feeding mesophyll and excreting faeces in the nest (Fig.4-B).some like to inhabit the empty nests of DichocrocL ̄chloropbanta Butler(Fig.4 C—D)or Rhodonelf(1 vittula Guen ̄e(Fig.4 E-F). Mature larval specimens(Samples ID:LT14001,LT14002, l】84 环境昆虫学报Journal of Environmental Entomology 38卷 LT14003.IJT14027,LT14049),and the mt COI sequences were deposited in the GenBank database with Accession No.K煳ll47,K煳ll48,K煳1149, KX091 150,KX091 151 respectively.Among them,only one nucleotide is different between group 1(LTI4001, LT14003,I 14027) and group 2(L1’14002, LT14049),that is,third nucleotide of the Codon coding the Amino acid glycine,A of group 1 was transferred to G of group 2 at No.376 nucleotide,with the nucleotide identity reaching 99.84%.The proportions of A:C:G:T was 31.5%一31.6%. 14.6%.14.6—14.8%,39.1%,with no insertions, deletions.nonsense,or stop codons in the COI sequences from all the samples.The blast searches from NCBI showed 92%一93% similarity with Phycitinae species such as Salebriopsis albicilla,Sciota subcaesiella,Ceutholopha petalocosma,etc.There were no other more similar sequences of COl for Sacculocornutia sinicolella Caradja,the present study reports for the first COI sequences of the species. 3 Conclusion The larvae are characterized by the head with one large and one smaller black rounded spots beside vertex of fron and at base of P1 bristle,respectively, A1,A2 and A3 arranged in a fight-angled tirangle; the body carrying ten yellowish white longitudinal bands in dorsal and lateral sides,a large blank subrectangular or subrhombic spot in prethorax and a blank broad open—loop shaped spot in mesothorax; crochets of third to sixth abdominal segments triordinal,arranged in a circle.The pupae are characterized by tenth abdominal segmental tergum with small spiny bands at basal 1/4 and lateral margins;caudal margin laterally with 2 tooth—like spines,medially with 4 long bristle fairs curled at apex.The 629 bp portion of the mt COI gene of the species is firstly reposed. References Caradja A.Nachtr ̄ige zur Kenntnis ostasiatischer Pyraliden[J]. Deutsche entomologische Zeitschriif Iris,1926,40(4):168—170. Caradja A.Die Kleinfaher der Stt ̄tzner'schen Ausbeute nebst Zutr ̄ige aus meiner Sammlung (Zweite biogeographische Skizze: “Zentralasien”)[J].Memoriile Sectiunii Stiintiifce.Academia Romana(& 3),1927,4(8):361—428. Du YL,Li ttH,Wang SX.The genus Sncculoeornutia Roesler from China(Lepidoptera:Pyralidae:Phycitinae)[J].Animal Science, 2002,135—139.[杜艳丽,李后魂,王淑霞.中国腹刺斑螟属 Sacculocornutia研究(鳞翅目:螟蛾科:斑螟亚科)[J].动物 科学,2002,135—139] Hebert PDN,Penton EH,Bums JM,et a1.Ten species in one:DNA barcoding reveals cryptic species in the neotmpical skipper butterlfy Astraptes fulgerator[J].Proceedings of the National Academy of Sciences ofthe United States ofAmerica,2004,101(41):14812— 148l7. Hinton HE.On the homology and nomenclature of the setae of Lepidoptera larvae,with some notes on the phylogeny of the Lepidoptera【J].n。mⅡef 0m of the Entomological Society of London.1946.97:1—37. Li HH,Ren YD,et a1.Insect Fauna of Henan(Lepidoptera: Pyraloidea)[M].Beijing:Science Press,2009:1—440.[李后 魂,任应党等.河南昆虫志(鳞翅目:螟蛾总科)[M].北京: 科学出版社,2009:1—440] Li HH,et a1.Microlepidoptera of Qinling Mountains(Insecta: Lepidoptera)[M].Beijing:Science Press,2012:1—1271.[李 后魂等.中国秦岭小蛾类(昆虫纲:鳞翅目)[M].北京:科 学出版社,2012:1—1271] Li HH,Zheng ZM.Research methods of small moth insect specimens [J].Journal of Shaanxi Normal University(Natural Science Edition),1996,24(3):63—7O.[李后魂,郑.小蛾类昆 虫标本研究方法[J].陕西师范大学学报(自然科学版), 1996,24(3):63—7O] Roesler RU.Phycitinen-Studien 9.(Lepidoptera,Pyralidae)[J]. Entomologische Zeitschrift,1971,81:177—192. Yamanaka H.Descriptions of three new species of Phycitinae (Lepidoptera,Pyralidae)from Japan[J].Tinea,1990,12(26): 23】一238. 

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