The larva of Polythore spaeteri Burmeister & Börzsöny, with comparison to other polythorid larvae and molecular species assignment (Zygoptera: Polythoridae)

2006·2.9 MB·English

by  Etscher, V

Odonatologica35(2):127-142 JuneI,2006
Thelarva of Polythorespaeteri
Burmeister& Börzsöny, with comparison
to otherpolythorid larvaeandmolecular
species assignment
(Zygoptera: Polythoridae)
V.Etscher,M.A.MillerandE.-G. Burmeister
ZoologischeStaatssammlungMünchen,Münchhausenstrasse 21,D-81247München,Germany
[email protected]
ReceivedJanuary20,2005 / RevisedandAcceptedOctober27,2005
Thelarva fromtheareaofPanguana(Huanucoprov.,Peru)isdescribed.Thiscon-
stitutesthefirstdescriptionofaPolythore.P-distancemeasuringofa790bplongfrag-
mentofthemitochondrial COIgenewasusedasatoolfortheassignmentofthelarva.
Thelowdegreeofsequencedivergencesbetweenlarval andimaginalCOIsequences
leavesnodoubtaboutconspecificity.Theuseofscanningelectron microscopygives
animpressionofsomemorphologicalcharactersnotmentioned sofar concerning
polythoridlarvae.ComparisonoftheP.spaeterilarvawith thefewcurrentlyavailable
descriptionsofpolythoridlarvaeshows thatcharacterisation ofthelarvaeatgeneric
level isnotpossibleuntilmorelarval specimensofthe familyareexamined.
INTRODUCTION
ThegenusPolythore Calvert, 1917ismainly distributedinwestern SouthAmer-
ica,whereitis mostcommoninPeru(BICK&BICK, 1986).BURMEISTER &
BORZSONY (2003) described anewPeruvianspecies, P. spaeteri, whichis cur-
rently theonlyrecordofPolythoridae fromtheregion ofPanguana (anAmazo-
nianareacloseto theAndes,elevation250m, coveredwithprimary rainforest).
Thisspecies is welldistinguishable fromothers inthegenusby wingcolourpat-
tern andby charactersofthemale genitalia.
During a systematic searchforthe immaturestagesofP. spaeteri, larvaewere
foundclinging to thelowersurfacesoflarger stones inasmallforeststreamwith
relatively strongcurrent. Thelarvaewereequipped withlateroventral, serialgill-
like appendages onthe abdomen, whichis aspecial featureof thelarvaeof only
128 V.Etscher,M.A.Miller& E.-G.Burmeister
two familieswithintheOdonata:the Polythoridae andthe orientalfamily Eu-
phaeidae(LIEFTINCK, 1962; WICHARD, 1979;BECHLY, 1998).
So far,larval descriptions of only six polythorid species andsubspecies exist:
Cora chirripa Calvert, 1907 (CALVERT, 1911), C. cyane Selys 1853 (DE
MARMELS, 1982), C. marina Selys, 1868 (NOVELO-GUTIERREZ
& GONZALES-SOR1ANO,1985),Chalcopteryx rutilans Rambur, 1842(DOS
SANTOS & COSTA, 1987), Chalcothore montgomeryi Racems, 1968 (DE
MARMELS,1988)andEuthorefastigiatameridanaSelys, 1879(DE MARMELS,
1995).
BecausenootherPolythoridae were foundinthestudy area,thelikelihoodof
species identity withthe imagines of P.spaeteri appeared tobe high. Attempts
atkeeping collectedlarvae alive untiltheirlastmoultwere unsuccessful. Thus,
anothermethod, makinguseofmoleculartechniques intaxonomy,was applied
to testforspecies identity. Afragment ofthegenecoding forthemitochondrial
cytochrome oxidase subunitI(=COI)was compared betweenlarvaeandimag-
ines. Calopteryxsplendens (Harris)was usedas referencespecies. Moreadequate
referencespecies (possibly fromthesame family or even the same genus) could
have advanced the comparability and the recognition of presumptive species
boundaries, but atthe timeofthis study C. splendens offered theonly material
readily available and showing good PCR results. ThefamiliesCalopterygidae
andPolythoridae are generally placed intothesame super-family Calopterygoi-
dea(e.g. FRASER, 1957; REHN, 2003).
ZLOTYetal. (1993)madefirstattempts to useproteinelectrophoresis toiden-
tify Hetaerinalarvae. Thereforethey are thefounders inusing moleculartech-
niques inodonatology. Werefined theirapproach tothe levelof matching DNA
sequences betweenlarvaeandadults.As faras weknow, thismethodofspecies
identification isapplied forthe firsttimewithinOdonata.
Thefollowing description ofthepresumptive larvaofP. spaeteriisthefirstof
its kind in the genusPolythore. It containsdrawingsofthe habitand scanning
TableI
Morphologicalvoucher specimens and extracted DNA deposited in the frozen DNA and tissue
collection ofZSM
SSppeecciimmeenn DDNNAATTAAXX--IIDD CCoolllleeccttiinnggddaattaa
PP..ssppaaeetteerriiiimmaaggoo DDNNAATTAAXX0022771100
PP..ssppaaeetteerriiiimmaaggoo DDNNAATTAAXX0022771111
PPssppaaeetteerriiiimmaaggoo DDNNAATTAAXX0022771133 PPeerruu,,HHuuaannuuccoopprroovv..,, nnrrRRiioo YYuuyyaappiicchhiiss((LLlluullllaappiicchhiiss)),,
PP.ssppaaeetteerriillaarrvvaa DDNNAATTAAXX0022771144 PPaanngguuaannaa,,99°°3377''SS7744°°5566'EE;;0066--1IVV-- 1177--1IVV--22000033
PP.ssppaaeetteerriillaarrvvaa DDNNAATTAAXX0022771166
PP.ssppaaeetteerriillaarrvvaa DDNNAATTAAXX0022771188
CCaalloopptteerryyxxsspplleennddeennssDDNNAATTAAXX0022771122 GGeerrmmaannyy,,BBaavvaarriiaa,,MMuunniicchh,,OObbeerrmmeennzziinngg,,ZZSSMMggrroouunnddss,,
iimmaaggoo((rreeffeerreennccee)) 22--VVII--22000033
Polytore spaeterilarva: molecularspeciesassignment 129
electronicmicroscope (SEM) photographs of somespecial features. A diagnosis
againstthe hithertodescribedlarvaeofotherpolythorid species ispresented.
MATERIAL&METHODS
SPECIMENS.— 19larvaewerecollectedinPeru,Huanuco prov.,neartheRioYuyapichis(Llull-
apichis),atthebiologicalstationPanguana,9°37'S and74°56'E,between06-IVand 17-IV-2003,H.,
J.&E.-G.Burmeister leg.Ninelarvae werepreservedinapprox. 100%ethanolformolecular analy-
sis, tenin70%ethanol. AllarestoredintheZSM(= ZoologischeStaatssammlungMiinchen).Nine
larvae(inapprox. 100%ethanol)wereusedforthemolecular identification,five ofthese (39,2c?),
preservedingoodcondition,wereselectedforthemorphologicaldescription.Imagineswerecollected
byE.-G.Burmeisteratthesamelocality, between 28-IXand06-X-2000,and byH.,J. andE.-G.Bur-
meisterbetween06-IV and 17-IV-2003,Theimaginesofthereference-species,Calopteryxsplendens,
werecollected byE.-G.BurmeisterinthesurroundingsoftheZSM,on02-VI-2003.
DESCRIPTION.—Somepartsweredissectedand examinedinaLEO 1430VPscanningelectron
microscope(SEM)at 15kV aftercritical-pointdryingintheCPD 030 apparatus(BAL-TEC),with
C02asexchangemedium andsubsequentcoatingwithgoldinasputter-coaterapparatus(Polaron,
SEMCOATINGSYSTEMS)atthefollowingparameters: 2.4kV, 20mA and sputtering-time120
sec.,resultinginacoatingthickness ofapprox. 25nm.
MOLECULARANALYSES. - Total genomicDNAofthreeP.spaeteriimaginesandninelar-
vaewas extractedfrom thoracicmuscles ofwholeindividuals bymeansofthe DNeasyTissueKit
(QIAGEN)accordingtothemanufacturer's protocols.Thethreelarvae showingthehighesteffects
onDNAextractionswereusedforthefurtherprocedure.TheyaredepositedintheDNA-TAXDNA
and vouchercollection atZSM,alongwith theimaginesofP.spaeteriand C. splendens(collection
numbers.Tab.I).
Table II
Positions ofsequence divergences(inboldface)among andbetween P.spaeterilarvaeandimagines,
respectively. — [Positionsofsequence divergences(inboldface)amongandbetween P.spaeterilar-
vaeandimagines,respectively]
DDNNAATTAAXX--IIDD PPoossiittiioonnssooffddiivveerrggeennttnnuucclleeoottiiddeess,,rreellaattiinngg ttootthheeccoommpplleetteeCCOOII--ggeennee
BBaassee 11 11 11 11 11 11 11 11 1i
ppoossiittiioonn 77 77 88 99 99 99 99 99 99 11 I! 11 33 33 33 33 33 33
9999 00 88 88 88 9999996666666 66 66 86 88 88 8
88 99 00 55 66 77 66 77 88 44 55 66 55 66 77 00 11 22
CCooddoonn
ppoossiittiioonn 11 223311 22 33 11 22 33 11 22 33 11 22 33 11 22 33
DDNNAATTAAXX0022771100PP..ssppaaeeteterir-i-
iimmaaggiinneessGG GGTTCCTTAA AA GG CC GG GG AA AA TT TTCCTTTT
DDNNAATTAAXX0022771111 GG GGTTCCTTAA AA GG TT GG GG GG AATTTT Cc TT CC
DDNNAATTAAXX0022771133 GG GGTTCc TT AA AA GG TT GG GG AA AATTTTCcTTCc
DDNNAATTAAXX0022771144PP..ssppaaeeteterir-i-
llaarrvvaaee GG GGTTCc TT AA AA GG TTGGGG AAAATTCCCcTTCc
DDNNAATTAAXX0022771166 GG GGcCTTTT AA AAGG TTGGGG AAAATTCCCcTTCc
DDNNAATTAAXX0022771188 GG GGTTCc TT AA AAGG TTGGGGAAAATTCCCcTTCc
ccooddeeddaammiinnooaacciidd GGllyy LLeeuu SSeerr GGllyy HHee LLeeuu
130 V.Etscher,M.A.Miller&E.-G.Burmeister
Fortheamplificationofthe790 bplongfragmentofthe COIgene, thefollowinguniversal insect
primerswereselectedfrom SIMONetal.(1994);
Cl-J-2195 (forward):
5' TTGATTTTTTGGTCATCCAGAAGT3'
andTL2-N-3014 (reverse):
5' TCCAATGCACTAATCTGCCATATTA 3'
ThePCRwasperformedonaPTC220 DYAD thermocycler(MJRESEARCH) with atotalre-
action volumeof25piusingtheExpand-PCRSystem (ROCHEDIAGNOSTICS)atthe following
parameters:25 pmol ofeachprimer; 25 pmol DNTPs; 12.5 pmol MgCI,and 0.88 units of taq-
-polymerase.ThePCR ranatthefollowingparameters;94°C for4min;45cyclesat94°Cfor 1.5min;
48°C forImin;72°C for 1.5min and afinalelongationstepat72°Cfor3min.
Theresults ofthePCR werevisualised under UVlightusingethidium-bromide stainedagarose
gels.ThefollowingpurificationofthePCRproductswasperformedusingtheMinElutePCR-purifi-
cationkit(QIAGEN),accordingtothemanufacturer'sprotocols.TheamplifieddsDNAfragmentof
the COIgeneofeachspecimenwasusedastemplateforthesequencingreactionwith therespective
PCRprimers,usingthe ReadyReaction DyeDeoxy Terminator Cycle SequencingKit (“BigDye”,
APPLIED BIOSYSTEMS)atthefollowingparameters:94°Cfor2min;25cyclesat94°Cfor20 sec;
52°C for 10sec; 70°C for4min. The fragmentsweresequencedin both directions. Thefilteredand
resuspendedproductswereelectrophoresedonanABI377automatedsequencer. Thesequences con-
tained inthe ABIfileswerecheckedmanually;sequences ofthe P.spaeteriimagineswerealignedto
thoseofthelarvae,andthesequencesofbothwerealignedtothatofthereference species,C. splend-
ens.Thepairwisedistancesbetween larvalandadultsequences werecalculated withtheformulap=
nd/nt(=p-distancemeasuring,whereas ndisthe number ofnucleotide divergencesbetweentwo se-
quences,and n,isthetotalnumber ofcomparednucleotides;seeHEBERTetal.,2003b)
MOLECULAR ANALYSES
Inthe790bp long COI fragmentcompared betweenP. spaeteriimagines and
larvae, sequencedivergence wasalmostnegligible.Varianceamongthelarvaland
imaginal sequences, respectively, was small as well(seeTab. II).
Positionsdivergent between P spaeteriandthereferencespecies, C. splendens,
are not given indetailhere, butthe p-distance of 22.4%showsthe expected di-
vergence(see Tab.III).
TableIII
p-distancevalues accordingtothe formulap=nd/ntafterHEBERTetal.(2003b);
see“Material &methods”
p-distanceamong p-distanceamong p-distance between larvaeand p-distanceto
larvae imagines imagines reference
0.25 % 0.38% 0.76% 22.4%
The sequences are deposited in EMBL/GenBankunderaccession numbers;
AM_180640 for DNATAX02710, AMJ80641 for DNATAX02711,AM_
180642for DNATAX02713,AMJ80643forDNATAX02714,AM_180644for
Polytore spaeterilarva:molecular speciesassignment 131
DNATAX02716,AM_180645forDNATAX02718,andAM_180646for DNA-
TAX02712.
On the basisof these results (seealso Discussion) the larvadescribed in this
paperwillbereferredto as P.spaeteri-larvafrom nowon.
DESCRIPTION OF THE P.SPAETERILARVA
Figures 1-8
Measurements (inmm):Bodylength:~15.0,presumptiveultimateinstar(~11.0,presumptive
penultimateinstar);lengthoftheparaprocts: 3.5(2.8);lengthoftheepiproct:3.0(2.3);length(with-
Fig. 1.Polythorespaeteri,habit ofthepresumptivepenultimateinstar indorsal (a),ventral(b) and
lateral (c)views. — (drawingsbyR. Kiihbandner)
132 V.Etscher,M.A.Miller& E.-G.Burmeister
Figs 2-3. Polythorespaeteri,larval structural features: (2)(a)head,dorsal view (arrows indicate 7
ofthe 13symmetricallyarrangedbare spots); (b) fringyincised integumentalborderclose tocom-
poundeyes, ventralview; - (3)(a)labium,dorsal view (dottedlines indicateaslightembossment
with aspike-like tooth oneachside); (b)right labial palp,dorsal view,with amovable claw onthe
right; (c) marginofthelabial prementum(whitecirclesindicatespike-like teeth,presumptiverelics
ofparts ofthe former second maxillae;arrowspointto thecorrugatedpresumptivefusion line of
the former second maxillae;onboth sides ofthe fusion line,small,rathersymmetricallyarranged
setaearevisible).
Polytorespaeterilarva: molecular speciesassignment 133
outpalps)/width (atwidestposition) ofthelabialprementum:3.3/2.5(2.3/2.0);length/widthof
thehead: 3.1/4.2(2.6/3.5);nodifferencesbetween femaleand male dimensions detectable.
Main colour ofthelarvamid-brown, brighterventralsidebecause ofthe
thinnerintegument ofthe sternitesandthelucent-whitelateroventral, abdomi-
nal gillappendages.
Head atitswidestpart slightly broaderthanthorax, length-width ratioof
head ~%on average;external, posterior angle ofcephalic loberounded(Fig.
la); on the upperside ofthe head with 13 symmetrically arranged barespots
visible(Fig. 2);clypeus also freeofsetae,as wellas proximalhalfoflabrum; re-
maining surfaceofheadcoveredwithscale-like setae;fringy incised integumen-
tal border close to compound eyes, inventral view;each fringe representsthe
stemof ashort seta.
Antenna7-segmented, length ofthesegments decreasingfrompedicel to 7th
segment, except forthe second segmentafter thescape, which is insignificantly
longer thanthefirst;joints3-7significantly thinnerthanthefirst two,no hyper-
trophied pedicel; also hairiness of the antennae decreases from segment 1-7;
first two segments after the scape internally setae withslightly longer than on
the remaining antenna surface; interiorsideof pedicel bearing a groupof no-
ticeably longer setae,presumably withsensory function, nearthe distalmargin
ofthe segment.
Articulationoflabialprementumreachesprothoracical coxae,length-widthra-
tio oflabium(with closedpalps, atwidestposition)approx. 3/2(Fig. lb); distal
marginofprementumwithsmall,roundedteethof varying sizeandwithtrun-
catedsetaebetweenthem;thesmallteethbearing 1-3transversebands, depending
on thesizeofthetooth; mediancleftatdistalmarginofprementumvery slight
(only around0.36%oftotalpremental length, angle: 30°); alternating corruga-
tionproximad fromthe cleft, visiblefromdorsalview; spike-like teeth, situated
onaslightembossmentbelowthedistalmarginoftheprementumonbothsides
of the cleft (Fig. 3); no conspicuous setae on dorsal side of prementum,only
aboutten very smallandtiny, symmetrically arranged setaevisiblefrom dorsal
view; internaledges ofpalps very finelydenticulated; externalclawmovable;dis-
talmargins ofpalpswith3pointed teeth,eachcurvedinward; innertoothmore
strongly curved, givingtheimpression ofbeing roundedand shorter;midtooth
longer thanthe othertwo.
Maxillaebiramousandcoveredwithlong, thinsetae,presumably withsensory
function;palpus absent.
Mandiblesuniramous, butwithonelarge, movabletooth, standing outfromthe
remaining teeth, facing ventralsideofhead(Figs 4a-b);external sideofmandi-
bleswithpresumably sensoryhairs; dorsalbaseof theisolatedtoothwitharasp-
likearea,coveredwithsmall,jagged teeth(Fig.4c).
Thorax broaderthanabdomen, butnarrower thanheadatitswidestpart;
surfaces of visible partsofthoraxwidely coveredwith scale-likesetae,butwith
134 V.Etscher,M.A.Miller&E.-G.Burmeister
Figs4-5. Polythorespaeteri,larval structuralfeatures: (4)mandibles; (a)left mandible,internal view
(whiteellipse marksthe rasp-likearea);(b)rightmandible,internal view(movablesite oftheinner
tooth shown bydotted line);(c) close-upoftherasp-likearea,showingsmalljaggedteeth; — (5)
surface oftibiae:(a)alternatingjaggedandrounded flattened setaeonthecarina;(b)feather-shaped
setae, ventral,ondistalpartoftibia.
Polylorespaeterilarva; molecularspeciesassignment 135
crescent-shaped bareareas oneachlateralsideofthepronotum. Mesothoraci-
calepisternum separated by transverse suture; two smallbumps onthemostlat-
eral points, wheretheanteriorandtheposterior halves oftheepisternum touch
each other;spiracle at anteriormargin ofmesothoraxvisiblemoreclearlythan
atepisternal plates ofmetathorax.
Hindwingpads reachbeyond abdominalsegment 10(reach posterior margin
ofsegment3onaverage); forewingpads reachbeyond segment10(reach poste-
rior marginofsegment4onaverage).
Legs decreasing inlength fromthefirst to thethirdpair; tibiaea littlelonger
thancorresponding femora; femora laterallycompressed, withtwo ventraland
one dorsalcarina; tibiaewithtwo ventralandtwo dorsalcarinae, whichcontrast
with the remaining surfaces of thefemora by embossment and darker colour.
Femoraandtibiaecoveredwithscale-like,jaggedsetae,carinaeoftibiaecovered
withalternating roundedandjagged setae(Fig. 5a); ventral transitionbetween
tibiaeand tarsiwithsuccessive formationof strong,feather-shaped setae (Fig.
5b). Tarsithree-jointed; pretarsi bearing simpleclawswithexpanded bases, and
anempodium appearingbetweentheclawsandoriginatinginsidethepraetarsus
(Figs 6a-b); tarsiwithlong, thinsetae withpresumably sensoric function, espe-
cially onthepraetarsus(Fig. 6b).
Abdominal tergites 2-9withmedian, slight caudadthorns, whereasthorn
onlyhintedon segment 1, lackingon 10. Posteriormarginofeachsegment ap-
pearing ciliatedwithsmallsetae,posterior marginof 10withmedianincisional-
lowing verticalmovement ofthe mediancaudalappendix (=epiproct), whichis
originates underneath; depthofincisionequalshalfthe lengthof 10thsegment,
width corresponds to width of base of atergal thorn. Caudal appendages (=
procts) saccoid andpetiolated; length ofepiproct onaverage about26%ofto-
talbody length, length oflateralcaudal appendages (= paraprocts) on average
about35%; epiproctwithbilateralsymmetry, withtheshape ofaninvertedtrian-
gle; caudaledge ofepiproct ending inthree equidistant pointed protuberances,
mid protuberance is longest; one moreprotuberance, directedcaudad, oneach
lateralsideofepiproct, nearthe dorsalside, atmid-length ofepiproct(Fig. 8c).
Shape ofparaprocts approximately cylindric, withoneprotuberance atthecau-
dalend, threefurtherprotuberances distributedindistalhalfofparaproct (Fig.
8c). Thus, the resulting protuberance “formula”is 4(lateral)-5(median)-4(later-
al). Surface ofepiproct widely coveredwithvery flatscale-likesetae thatare di-
rected caudad(Fig. 8b), only ventraledge withelongate, capillary setae;surface
ofparaprocts differs fromthatof epiproct by presentingawider areawithlong
capillary setae whichincreasein length caudadandare condensedto abrush at
thecaudalend(fig.8a). Both, theepiproct andthe paraprocts, showsmallerand
largerbareareas withoutsetae; largerbarearea ofepiproct onlateralsides re-
stricted to proximal 2/3 of the length and to proximal 2/3ofparaproct height;
smallerbarearearestrictedto periphery ofpetiolated baseofepiproct (Fig. 8c);
136 V.Etscher,M.A.Miller&E.-G.Burmeisler
Table IV
Comparisonof differingcharacters between previously described polythorid larvae and the pre-
sumptive larva ofP.spaeteri; where comparisonis not possible(e.g.due to lack ofinformation
orimpreciseillustrations),cellscontain interrupted horizontal black lines; 1 p.u. i.; presumptive
ultimateinstar
SSppeecciieess CCoorraa CCoorraa CCoorraa EEuutthhoorree CChhaallccootthhoorree CChhaallccoopptteerryyxx PPoollyytthhoorree
CChhaarraacctteerr cchhiirrrriippaa ccyyaannee mmaarriinnaa ffaassttiiggiiaatlaa mmoonnttggoommeerryyii rruuttiillaannss ssppaaeetteerrii
mmeerriiddaannaa
TToottaallbbooddyy 1188..55 1133..55 1199..55 --2200 --1111..55 --1133..55 ((pp..uu..ii..llaarrvvaa::
lleennggththooffffiinnaall uuppttoo~-1155..00))
iinnssttaarreexxuuvviiaaee
iinnmmmm
PPoosstteerriioorraannggllee wweellll
ooffhheeaaddssiiddee aanngguullaarr rroouunnddeedd rroouunnddeedd pprroonnoouunncceedd aanngguullaarr rroouunnddeedd
lloobbeess aanngglleess
LLeennggtthhooffsseecc..
aanntteennnnaalljjooiinntt// 11..22 22..33 ~-11
lleennggtthhooffppeeddiicceell
SSeettaaaarrrraannggeemmeenntt lloonnggeerr ccoonnssttaanntt,. ccoonnssttaanntt,. tthhrreeeelloonnggeerr lloonnggeerrsseettaaee lloonnggeerrsseettaaee
oonn tthheeffiirrssttttwwoo sseettaaeeoonn lloonnggeerr sshhoorrtt aannddtthhrreeee oonntthheeiinnnneerr oonntthheeiinnnneerr
aanntteennnnaalljjooiinnttss tthheeiinnnneerr sseettaaee-- sseettaaee-- sshhoorrtteerrsseettaaee ssiiddeeoofftthhee ssiiddeeooffbbootthh
bbeeyyoouunnddtthhee ssiiddeeooff lleennggtthhoonn lleennggtthhoonn oonntthheeffiirrsstt ffiirrssttjjooiinntt jjooiinnttss,.
ssccaappee bbootthhjjooiinnttss., bbootthhjjooiinnttss bbootthhjjooiinnttss jjooiinntt,,ssiinnggllee aanndd sshhoorrtteerr sshhoorrtteerroonn
sshhoorrtteerroonn sseettaabbaassaall sseettaaeeoonn tthheesseeccoonndd
tthheesseeccoonndd aannddmmiiddwwaayy tthheeiinnnneerr jjooiinntt,,iissoollaatteedd
jjooiinntt oonntthhee ssiiddeeoofftthhee oonntthheeffiirrsstt
sseeccoonnddjjooiinntt sseeccoonnddjjooiinntt jjooiinntt
AAbbddoommiinnaall ffoorreewwiinngg:: ffoorreewwiningg:: ffoorreewwiinngg:: ffoorreewwiinngg:: ((pp..uu..ii..llaarrvvaa.,
sseeggmmeenntt IIVVttooVVII;; IIVV;;hhiinndd-- IIVV;;hhiinndd-- hhiinnddwwiinngg:: hhiinnddwwiinngg:: IIIIII;; ffoorreewwiinngg::ttoo
rreeaacchheeddbbyy hhiinnddwwiinngg:: wwiinngg::VV wwiinngg::VV VVII VV hhiinnddwwiinngg:: bbeeyyoonnddXX;;
wwiinnggppaaddss VVttooVVIIII IIVV hhiinnddwwiinngg::ttoo
bbeeyyoonnddXX))
PPrreesseenncceeooff
eemmppooddiiuumm yyeess yyeess yyeess yyeess yyeess nnoo yyeess
PPrreesseenncceeooff
ltoonngguuee--sshhaappeedd nnoo yyeess nnoo nnoo nnoo nnoo nnoo
eexxtteennssiioonnoonn
ggiillll--bbaassee
““FFoorrmmuullaa””ooff 44--55--44 66--66--66 55--66--55 44--55--44 44--55--44 33--33--33 44--55--44
pprroottuubbeerraanncceess
PPrroottuubbeerraannccee sshhoorrtt,, vveerryy sshhoorrtteerr vveerryylloonngg
ssiizzee ssiimmiillaarrttoo tthhaannCChh.. ((aapppprrooxx,,hhaallff sshhoorrtt,,ssiimmiillaarr
vveerryysshhoorrtt PP. ssppaaeetteerrii,, mmoonnttggoommee-- tthheelleennggtthh ttooiCC..cchhiirrrriippaa
bbuutt mmoorree rryyii,,lloonnggeerr oofftthhee
ppooiinntteedd tthhaannCC. aappppeennddaaggee)),.
cchhiirrrriippaa aallmmoossttbbaarree