Subunit Composition and O2 Binding of the Crustacean Hemocyanins: Interspecific Relationships

13 Pages·1994·5.5 MB·English

by  J E Reese

Reference: Biol. Bull 187: 385-397. (December. 1994)
O
Subunit Composition and Binding of the Crustacean
2
Hemocyanins: Interspecific Relationships
JOHN E. REESE AND CHARLOTTE P. MANGUM*
Department ofBiology. College ofWilliam & Mary. Williamsburg. Virginia 23187-8795
Abstract. The monomeric subunit composition and Usingelectrophoretic heterogeneityand immunological
oxygen bindingpropertiesofhemocyaninswereexamined relatedness in combination, Markl (1986) described dis-
in 9 taxonomic groups of 43 crustacean species and 1 tinct patterns ofmonomeric subunit composition in dif-
hybrid. In native polyacrylamide electrophoresis the ferent arthropod families, both crustacean and arachnid.
bandingpattern washighly speciesspecific, even inclosely He formulated three discrete immunological categories
related congeners. In less closely related taxa, there was ofchains, designated alphas, betas, and gammas. When
little apparent relationship between phylogenetic affinity separated by native polyacrylamide gel electrophoresis
and banding pattern. (PAGE), membersofeachcategoryexhibit migration rates
Within a taxonomic group, pH dependence was the that are characteristic oftheir category.
mosthighlyconservedandO:affinitywasthe mostdiverse Subunit composition can influence intrinsic O:-
O
ofthe : binding properties investigated. In congeneric binding properties. Artificial components such as iso-
but not sibling species, O: affinity was more highly cor- lated monomers (Jeffrey and Treacy, 1980) or homo-
related with an environmental variable such as temper- hexamers of them (Johnson et ai, 1987) differ in O2
aturethan with phylogenetic affinity. Only in veryclosely binding. More important in the present context, the
related groups found in similar environments were dif- respiratory properties ofnatural Hcswith different sub-
O
ferent : binding properties correlated with differences unit compositions can differwithin a polymorphic spe-
in particular electrophoretic bands. cies or between sibling species. In addition, the differ-
ences can be attributed to particular chainsand, in one
case, their effects on oligomerization (Mangum and
Introduction
Rainer, 1988; Mangum et a/.. 1991; Mangum, 1993a,
Crustacean hemocyanins(Hcs)existinvivoashexamers b, 1994). On theotherhand, notall different Hemorphs,
(1X6 monomers)and multiplesofhexamers. most often either within or between species, have appreciably dif-
apair(2 X 6-mers). Although thepolypeptidecomposition ferent functional properties (Mangum, 1993a, b).
of both artificial and natural 1 X 6-mers can be homo- Markl's(1986)investigationsweredesignedtoelucidate
geneous, thenativeblood inaspeciescontains morethan broad phylogenetic relationships ofHcsrepresentingtaxo-
one, and up to a dozen or more different, monomeric nomic categories ranging from families to subphyla. Be-
chains(reviewed by Markl, 1986, and Markl and Decker, cause his sample wascomposed of, at most, a few species
1992; see also Callicott and Mangum, 1993: Mangum per family, the patterns he described may or may not be
and Greaves, unpub. data). This condition, which is characteristic ofthe families. Few interspecific compari-
knownasmonomericsubunit heterogeneity, isa necessary sons of PAGE banding patterns, much less respiratory
prerequisite to differences in polypeptide chain compo- properties, have been made undercommon experimental
sition between individuals ofa species, which is known conditions. In addition, the level ofevolutionary diver-
as monomeric subunit polymorphism. gence at which subunit composition and respiratory
properties begin to differ is not known.
Here we present PAGE and Orbinding data for nine
Received 2 December 1991; accepted 3 October 1994. taxonomic sets of crustaceans, primarily decapods, of
* Authortowhom correspondence should headdressed. various degrees ofrelatedness. The taxonomic sets differ
385
386 J. E. REESE AND C. P. MANGUM
intheircomposition. Theseparation rangesfrom anatural In most cases, the best separation was obtained on
hybrid and its sibling parental species, to sibling species, 12.5%gels. Higherand lowergelconcentrationswerealso
to nonsibling and also sympatric congeners, to different employed when we encountered dense bands that sug-
genera, to closely related families. gested more than one chain with similar migration rates.
Three or more amounts of He were first examined to
Materials and Methods determine the optimal level for each species; the level
chosen wassimilar within ataxonomic set, but it differed
The species investigated are listed in Table I. Most rep- between sets. The Hcs from each set were subsequently
resentativeswerecollectedandbledbythepresentauthors. analyzed on the same gel.
Pagiirus impressus, P. lacustris, and Uca virenswere pur- MostgelswerestainedwithCoomassie Bluealone. After
chased from a commercial supplier and then bled by us. the banding pattern for a species was ascertained, a gel
The Pacific coast species of Cancer, the two species of was prepared for the detection ofCu in the bands. The
ChaceonandMenippe, andtheMenippehybridwerebled presence ofCu wasexamined by notingthequenching of
by colleagues and the fresh material was shipped to us fluorescence generated by bathocuproine sulfonate
within 24 h. Callinectes arcuatus, C. toxotes, Portimits (Bruyninckx et ai. 1978). In the present investigation,
spinnemanus, Panopeus lacustrus. Cataleptodius flori- EDTA was omitted from the dissociating buffer (N.B.
danus, Uca inversa, and the two speciesofGnathophausia Terwilliger, pers. comm.). Due to the lowersensitivity of
were bled by colleagues and the material was shipped to the bathocuproine sulfonate procedure, the He concen-
us frozen. tration was raised by a factor of6. The positions ofthe
Cu-positive bands were marked and the gels were then
Bloodsamples stained with Coomassie Blue to verify their correspon-
Blood was taken from the infrabranchial sinuses into dence to those previously noted with Coomassie Blue
a hypodermic syringe and allowed to clot in a tissue alone.
grinder. Blood from all available individuals ofa species The number ofbands in a species was ascertained by
(see Table III for the actual numbers) was pooled. The visual inspection ofthe gels on a light table. In most spe-
pool wasthen homogenized and centrifuged, and theclot cies, the relative amounts ofmaterial in each band were
was discarded. A small aliquot ofserum was frozen for estimated from densitometric scans. A few species were
PAGE, and O2 binding ofthe remainder wasdetermined investigated in alaboratoryinwhich nodensitometerwas
immediately. available. Gels were initially scanned with a Gelman in-
MostoftheO:-binding measurementswere performed tegrating densitometer. Although the width of the scan
within 1-2 daysofbleeding, on material that had notbeen produced by this instrument is not adjustable, it gave the
frozen. Because freezingcan lowercooperativity (Morris, best resolution of the individual peaks. It expresses the
1988), we have chosen not to report cooperativity values areaofeach peak asan integram (a numberofspikesthat
formostoftheHcsthat had been frozen. Wereportvalues variesdirectly with peak area), which permitscalculation
forCallinectesarcuatusand C. toxotes, however, because ofthe percentage ofthe total material. Thesecalculations
an investigation of Callinectes sapidits He revealed no (in Table II) include only the Cu-positive material. To
effect of similarly brief periods of freezing (Mangum el aid in theexamination ofgelphotographs,additionalscans
were made with a Shimadzu densitometer. The widthsof
al, 1991).
these scans were precisely compressed so that the peaks
were aligned with their corresponding bands on the gel.
Electrophoresis
Photographsofthegelsandtheircorrespondingscanscan
The frozen material was thawed, and its He concen- be found in Reese (1989).
tration was estimated by absorbance ofdissociated sub-
units at i. .;-iive site (338 nm). It was then diluted to O
about 5 mg . with a buffer (0.05 mol 1~' Tris : binding
+ 0.05 moll \. PH 8.9) that dissociates native Hcs were stripped of organic modulators by 12 h di-
oligomers into th nstituent monomers. The He alysis with Spectrapor high-speed membranes, against a
monomerswerethen v.-puruled by native PAGE;thegels, physiological saline. Where possible, the saline wasbased
reagents, and other conditions were those detailed by on the ionic composition ofa blood representative ofa
Hames and Rickwood (1985). This is the most sensitive taxonomic set. If no information on ionic composition
procedureavailable forseparatingthe monomersofthese was available, seawater was used. The dialyzed Hcs were
proteins;forcomparison withalternative methods, please then diluted to the optimal absorbance range with
see Markl el al. (1979), Brenowitz et al. (1981), and 0.05 mol 1 ' Tris-buftered saline, and the pH wasadjusted
Stocker el al (1988). with HC1 and NaOH.
INTERSPECIFIC RELATIONSHIPS OF HEMOCYANINS 387
Table I
Theclassification, geographicdistribution andhabitatsofthespecies
Species Habitat Geographic Range
OrderDecapoda
InfraorderAnomura
Family Diogenidae
Clibinanus vittatus(Bosc) subtidal. to22 m Virginiato Brazil
Family Paguridae
Pagtirus impressus(Benedict) subtidal, I to 33 m North Carolinato Florida
PagiiruslongicarpusSay intertidal to 200 m MassachusettstoFlorida
Paguntspollicaris(Say) subtidal to 200 m New Brunswick to Florida
Infraorder Brachyura
FamilyCancridae
CancerantennariusStimpson to20m BritishColumbiato BajaCalifornia
CanceranthonyiiRathbun to20 m Monterey BaytoGulfofBajaCalifornia
CancerboreahsStimpson intertidal to 800 m NovaScotiatoFlorida
CancerirroralnsSay subtidal, to 575 m Labradorto Florida
Cancermagister(Dana) shallow subtidal Aleutiansto Monterey Bay
Cancerproductiis(Randall) shallowsubtidal Kodiak to BajaCalifornia
FamilyGeryonidae
Chaceonjennen(Manning& Holthius) 300 m + GulfofMexico
Chaceonquinquedens(Smith) 300to 700 m + GulfofMexico
FamilyGrapsidae
Sesarmacinereum(Bosc) semiterrestrial Maryland to Mexico
Sesanna reticulatum(Say) semiterrestrial Massachusettsto Florida
Family Majidae
Libiniadubm H. Milne-Edwards subtidal, to46 m MassachusettstoCuba
Libinia emarginata(Leach) subtidal, to49 m NoviaScotiatoGulfofMexico
FamilyOcypodidae
Ocypodequadrala(Fabricius) semiterrestrial Rhode Islandto Brazil
Ucacrenitlalacoloradensis(Lockington) semiterrestrial GulfofBajaCalifornia
L'ca inversa Hoffman semiterrestrial Indian Ocean and Arabian Sea from Natal to Pakistan
L'ca musica mttsica Rathbun semiterrestrial BajaCaliforniatoSan Bias
UcaprincepsmoniliferaCrane semiterrestrial GulfofBajaCalifornia
Ucapugilalor(Bosc) semiterrestrial MassachusettstoFlorida
Ucapugnax(Smith) semiterrestrial MassachusettstoFlorida
Ucaminax(LeConte) semiterrestnal Massachusettsto Florida
Uca virens(Salmon & Atsaides) semiterrestrial Floridato Mexico
Family Portunidae
Ovalipesace//alus(Herbst) subtidal, to95 m Northumberland StraitstoGeorgia
Portuniiigibbesii(Stimpson) subtidal, to 393 m Ma. toGulfofMexico; French Guiana
Portunusspmimanus(Latreille) subtidal, to91 m NewJersey to Brazil
CallinectesarcuatusOrdway subtidal, to 28 m Southern Californiato Peru
Cal/ineclesbellicosusStimpson subtidal. to 18 m San Diegoto Matzatlan
CallinectesornalusOrdway subtidal, to 75 m Virginiato Brazil
Callinectessapidus Rathbun subtidal, to90 m NovaScotiatoArgentina
CallinectessimilisWilliams subtidal. to 392 m Delaware BaytoColombia
CallinectestoxoiesOrdway subtidal. to 27 m BajaCaliforniatoPeru
Family Xanthidae
Menippeadina Williams& Felder shallowsubtidal Northern and western GulfofMexico
Menippcmercenaria(Say) subtidal. to 54 m North Carolinato Mexico;Jamaica
Hybrid Northwestern Florida
Panopeusherbstii H. Milne-Edwards intertidal, to 22 m Massachusettsto Brazil
Panopeitsobesus(Smith) intertidal North Carolinato Florida
PanopeuslacustrisDesbonne intertidal toshallow Bermuda, southern Florida to Brazil
subtidal
Eurypanopeusdepressus(Smith) intertidal to48 m Massachusettstotropics
Cataleplodiusflondanus intertidal Bermuda, southern Florida to Brazil
(Gibbes)
Order Mysidacea
Gnathophausiagigas(Willemoes-suhmj mesopelagic North PacificOcean
Gnathophausia ingens(Dorhn) mesopelagic North PacificOcean
388 J. E. REESE AND C. P. MANGUM
O: binding ofall but a few Hcs was determined tono- 1. Anomwan hermitcrabs: DiogenidaeandPaguridae.
metrically at atmospheric pressure (Burnett, 1979), with The He ofthe diogenid Clibinarius vittatits consistently
precision mixed, humidifiedgasespassedthroughtonom- exhibited two electrophoretic bands in apparently equal
eters in a rapidly shaken water bath ( <0.5C); absor- quantities. Visual inspection ofthe gels clearly revealed
bance wasdetermined with a Milton Roy Spectronic 501 alightarea inthe middleand acorrespondingconstriction
spectrophotometer. The remaining Hcs, designated as at the edges ofthe single dark band in Fig. 1A (lane 1);
such in Figure 2 and Table III, were examined with the however, the separation doesnot showin thisphotograph.
cellrespiration procedure(Mangum and Lykkeboe, 1979). Regardless, thepattern differed unambiguously from that
In both cases P50 and n50 were estimated from Hill plots, of the three pagurid Hcs, each of which had additional
using linear regions ofthe data in the region of50% ox- bands(Fig. 1, lanes2-4). Mostbandsin thepaguridswere
ygenation. Bohr plots (log P50 vs. pH) were described by more cathodic than those in C. vittatus. Each pagurid
regression lines, and 95% confidence intervals were con- species clearly contained at least one band that did not
structed around the lines and their slopes. Many ofthe co-migrate with a band found in the others. Although
data sets differ significantly throughout the pH range ex- several ofthe bands in C. vittatus. P. pollicarus, and P.
amined; a smaller number are statistically indistinguish- longicarpmdid co-migrate, the proportions ofthe co-mi-
able throughout the range examined. These relationships grants differ considerably (Table II).
are clear from the Bohr plots. Many, however, differ sig- 2. Brachyurans:Cancridae. Thesubunitcompositions
nificantly in only a portion of the pH range, which is of the Cancer Hcs did not resemble one another very
indicated in the Resultssection. Cooperativity valueswere closely (Fig. IB, lanes 1-6). Thus, it is unlikely that a
compared by using a Student's t test. morph ofC. productus will prove to resemble that ofany
otherspecies. C. borealisand irroratus Hcseach separated
//; vivo respiratory variables intothreedifferentbands, whereasanthonyiandproductus
Blood was taken anaerobically into a hypodermic sy- Hcs separated into four, and antennarius and magister
ringe from thepericardia! and infrabranchial sinuses, and Hcsseparated into fivebands(seealso Larsen elal.. 1981).
pH and PO were measured with a Radiometer BMS1 Each He exhibited electrophoretically unique bands.
BloodGasSy2stem. Becausethesampleswereoften small, As in Cancer pagiirus (Markl, 1986), the bands in
the suction line was disconnected from the pH capillary the present sample ofcancrids fell into three more or
electrode, and a syringe was used instead to position the less distinct groups: an anodic group, an intermediate
group, and a cathodic group. They may correspond to
material.
Markl's ( 1986) immunological categories, in the order
Results beta, alpha, gamma. Within each group, some of the
bands migrated at identical rates, and the migration
Hesubunit composition rates ofothers differed only slightly. In all cases, how-
We have attempted to accurately represent the species ever, the proportions ofthe co-migrants are consider-
investigated by choosing high-frequency phenotypes for ably different (Table II).
those known to be polymorphic (Mangum, 1990, 1993a; 3. Grapsidae. Sesarmacinereum Heseparated intotwo
Callicottand Mangum, 1993; Mangum and Greaves, un- bands, whereas 5. reticulatiim He separated into three
pub. data; C. P. Mangum and K. T. White, unpub. data). (Fig. 1A, lanes 13-14). Only band 2 in each species co-
Asdiscussedbelow, in nocaseknownthusfarcanamorph migrated.
ofone species be confused with a morph in another. We 4. Afajidae. The HcsofLibiniadubiaand emarginata
note that one species (Cancerproditctus) is polymorphic each exhibited four bands (Fig. IB, lanes 9-10). In both
(Wache ct ai, 1988), but the polymorphism has not been speciesthe mostcathodicband predominated. Moreover,
characteriz In all likelihood, other members of our the position ofthis predominant band wasquitedifferent
sample wili o prove to be polymorphic. in each species, although some of the minor bands co-
Figures 1A B summarize the banding patterns migrated.
found in each nd allowdirect comparison ofthe 5. Ocypodidae. Ocypode quadrata He (Fig. 1A, lane
electrophoretic beh k>r of the monomers composing 19) contained four distinct bands (see also Markl. 1986;
most ofthe Hcs in < -.ample. A few species were inves- Johnson, 1987), none of which co-migrated with any
tigated after these gels were prepared; they are shown in bands found in thegenus Uca(Figs. 1A, lanes 15-18, and
Fig. 1C. All bands were assigned numbers in order from Fig. 1C, lanes 5-7). The present material from U. pugi-
the top (anode) Eo the bottom (cathode) ofthe gel, with lator, U. pugnax, and U. virens all contained four Cu-
no implication that the same number in different species positive bands that migrated similarly but not exactly.
reflects co-migration. The relative amounts of the Cu- The relative amountsofmaterial arequitedistinct in each
containing bands in each species are given in Table II. species(Table II). Despite repeatedattemptsandwithdif-
INTERSPECIFIC RELATIONSHIPS OF HEMOCYANINS 389
A
444254 462 34234 444
1
PPCEPPPMMMSSUUUl)^
v p | j f d I o h a a m c r v m p p q CvPpiP jP fC dE lP oP hP aMaMmmMcSrSvUmUpUpUq
, 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 , 234S678910H 12 13 14 15 16 17 18 19
G Gb . Q33454 sGG 445565676 565
+
C C mC g 'NLdLfCsCtCaCoCgPsPcI!C "ands CbiC CaaC pCmC g ' LedL CsCstC aC oC gP sP ofCqC
a p
'
23456 789 10 11 12 13 14 15 16 17 18 19 20 23456 78 ,0 11 12 13 14 15 16 17 18 19 20
1
-
U U C C U U
mm mp b b mm p
fn
* fl
1 2 3
Figure I. Native PAGE banding patternsofdissociated hemocyaninsexamined in thepresent investi-
gation. Left: Photographs ofgels summarizing the nine taxonomic clusters. Lane nos. at bottom. Right:
Diagrammatic representationsofphotographs. Lane nos. at bottom. NumberofCu-positive (Cu +)bands
attop. Cu-negative bandsareblackened.
Panel A. from left to right beginning with lane 1: [1] Clibinariux villains. [2] Paguruspollicaris, [3] P.
longicarpux, [4]P. impressus. [5]Caialeptodiusflaridanus. [6]Eurypanopeusdepressus. [7]Panopeitslacustris,
[8]P ohemix. [9]P.herbslii, [10]Mcnippcadina. [11]Menippeadlna-mercenariahybrid,[12]A/, mercenaria.
V
[13]Sesarmacinereum, [14]S. reticulalum. [15] L'cavirens. [16] t'. minax. [17] I',pugnax. [18] pugilalor.
[19P]anOeclypBo,dleefqtutaodrrailgah.t: [1] Cancer borealis, [2] C irroratm. [3] C. anthonyi. [4] C. antennarius. [5] C
p[1ro1d]ucCailnlgi.ne[c6t]esC.sampaigdinssi.er,[12[]7]CGn.aVt//hH/o//p.Vh.au[1s3i]aCg.igtaosx,ot[e8s],G[.14i]ngCensa.re[n9]a/Luisb,in[i1a5]emCarogrinnaatttias,. [[1106]]LPordluubniau.s
gibbesu. [17] P. spinncmanus, [18] Ovalipesocellatus. [19] Chaceonfenneri. [20] C. quinquedens.
Panel C. left to right: Callinecies hellicosus(high cone.). C bellicosus(low cone.), C toxotes (high). C.
Mvii/i'v(low). tVa im-cr\u. L'. musica musica. L'.princepsmonilifera.
ferent gelconcentrations, we were unable todemonstrate material from U. pugilator (Mangum, 1993a; Callicott
morethan oneCu-positivebandin t'. minaxHe,although and Mangum, 1993) to ascertain relative positions ofthe
several additional bands thatwere not Cu-positiveclearly bands. U. musica musica He separated into eight bands.
appeared on the gels. U. princeps monilifera He separated into seven bands,
Hcs from C'. musica musica. U. princeps monilifera, and U. inversa He separated into four bands (Fig. 1C,
and U. inversa wereelectrophoresed on thesamegel with lanes 5-7). The major bands were diagnostic of each
390 J. E. REESE AND C. P. MANGUM
Table II
Ri'latircamountsf^)ofelectrophoretichands
Species(abb;#1*
INTERSPECIFIC RELATIONSHIPS OF HEMOCYANINS 391
7-8)thatwechosenottocharacterizethehandingpatterns differ significantly throughout the pH range investigated.
further. The gels are included in Fig. 1B to call attention In addition, the few significant differencesare small from
to the distinctive behavior. The He hands migrated at a a physiological point ofview. All Bohr factors are statis-
much slower rate than any of the other Hcs, all from tically indistinguishable except those for C. magisterand
decapods. Investigation ofbetterpreserved material would irroratus Hcs (Table III, lines 5-10). The cooperativity
be ofinterest. values, however, differsignificantly inall comparisonsex-
ceptthatofC. productuswitheithermagisterorirroratus.
O
O: binding 5. Portunidae. In the following comparisons, the 2
affinity data are statistically indistinguishable throughout
Bohr plots of the data for each species are shown in the pH range examined: ( 1) Callinectes similis and sapi-
Figure 2. Values forthe Bohrfactor, obtained astheslopes dus. (2) C. arcnatusand toxotes, and (3) C. ornatus, Ova-
ofthe regression lines in Figure 2, are given in Table III, lipes ocellatus, and Portimus spinnemanus. O2 affinity of
along with estimates ofcooperativity. O. ocellatusHediffersfrom P.gibbesiiHeonlyabovepH
1. Diogenidaeand Pagnridae. O: affinity ofthe four 7.4: in the remaining comparisons O2 affinity differs sig-
anomuran Hcs (Fig. 2: Table III. lines 1-4) differs sig- nificantly, either throughout the pH range or at all but
nificantly throughout most ofthe pH range examined, the two pH extremes.
although the difference between P. impressus and P. Bohrfactors (Table III, lines 22-30) forthetwo most
pollicarus Hcs disappears at low pH. The Bohr factor pH-sensitive Hcs (C. arciiatus and toxotes) differ sig-
for Clibinarius vittatus He differs considerably from nificantly from those forthe two least pH-sensitive Hcs
those for P. longicarpus and P. pollicarus Hcs. which (P. spinnemanus and C. ornatus): the Bohr factors for
alsodiffersignificantly from oneanother(Table III. lines C. arciiatus and toxotes also differ significantly from
C
1-4). Although the estimate ofcooperativity for C. vit- the value for sapidus. The remaining values are not
latus He is distinctive as well, the values for the three significantly different. Most ofthe cooperativity values
pagurids do not differ significantly from one another are statistically homogeneous, even though Callinectes
(Table III, lines 2-4). arciiatusand toxotesHcs had been frozen andthe others
2. Geryomdae. Majidae. Grapsidae. and Mysidacea. had not. The values for C. ornatus and similis Hcs,
The Hcs ofthe two members ofeach congeneric pair of which do not differ from one another, are both signif-
crabs had O: affinities that differ significantly from one icantly larger than the rest.
another throughout the pH range examined (Fig. 2). The 6. Xanihidue. The O2 affinity values for Cataleptodius
two mysid Hcsalsodiffer, but only at high pH. We report floridanus and Eurypanopeus depressus Hcs are statisti-
these values, despite the poor condition ofthe material, cally indistinguishable throughout the pH range exam-
primarily because they closely resemble the results for ined, asare those forthetwo species ofMenippe. C. flori-
fresh (aswell aspreviously frozen) material from G. ingens danus He differed significantly from that for M. mercen-
(Sanders and Childress, 1990). Only in the grapsids do aria except at high pH. and the Menippe hybrid He dif-
values forcooperativity and pH dependence differ signif- fered from the two parent species except at low pH. The
icantly(Table III, lines 11-12. 13-14. 39-40.and41-42). difference between Panopeus herhstii and obesus He O2
3. Ocypodidae. In Ocypode quadrala, O2.affinity dif- affinities becomes insignificant at pH 8.
fered significantly from that in the Uca species, either Many of the Bohr factors (Table III. lines 31-38) do
above pH 7.2 (pugilator and princeps monilifera) or notdiffersignificantly, theexceptions being(1)P. lacustris
throughout the pH range examined (all other species). In He, which is different from any other, (2) P. herbstii He,
the genus L'ca. He O: affinities were generally distinctive which differs from E. depressus and the three Menippe
but often become indistinguishable at high pH. Of par- Hcs, and (3) the Menippe hybrid He. which differs from
ticular interest (see Discussion) is a difference between M. adina. The cooperativity values forM. adina and the
Ucapugnax and virens below pH 7.4. Menippe hybrid Hcs do not differ significantly from one
M
Bohr factors are fairly diverse (Table III. lines 15-21 ). another. Both differ from mercenaria He, although
Ifthe values are arranged in series, each differs from all the differences are small. The cooperativity value for P.
others except the next higher or lower one. The cooper- ohesusHe issignificantlygreaterthan that forE. depressus
ativity figures fall into two significantly different groups: He,again byasmall margin: neitherdiffersfromP. herbs-
(1) the three lowest (U. minax. pugnax, and virens) and til He.
(2) the four highest (other) values.
4. C'ancridae. He O: affinities were more alike in this Blood PO: amipiI
than in any other genus. In 23 ofthe 30 possible inter-
specific comparisons between the six cancrid Hcs, the O2 The information required to assess the physiological
affinitydataare indistinguishable. In nocasedothe values importance ofdifferent O: binding properties is available
392 J. E. REESE AND C. P. MANGUM
ocypodidae
diogemdae,pagundae 2 geryonidae
v
*
|
".*****"
72 74 79 78 6.0
pH pH
5 mysidacea cancndae
"
|
PH PH
xanthidae
7 portunidae .3 porlunidae
22-
-
I
. t
PH
grapsidae
10 xanthidae
'
1
" M
' > *- "".
PH
INTERSPECIFIC RELATIONSHIPS OF HEMOCYANINS 393
in the literature for either a single member ofthe taxo- As a first example, Callinectes bellicosm is regarded as
nomic setsexamined hereormorethan one member, but difficult to distinguish from C. toxotes (Brusca, 1990).
not under common environmental conditions. Their essentially non-overlapping but contiguous geo-
In Table IV. data obtained underconditions very sim- graphic ranges (Table I) raise the possibility that they
ilar to those ofthe O; binding measurements are shown are siblings; several bands in these two species co-mi-
forseveral ofthe mud and fiddlercrabs in oursample. In grated. C. similis is exceptionally difficult to distinguish
all cases the predicted oxygenation at the gill appears to from C. ornatus (Ruppert and Fox, 1988), although in
be essentially complete even though experimental tem- thiscasethegeographic rangesdooverlap. In both cases,
peratureswere fairly high. Although the predicted deoxy- the differences in subunit composition are unambigu-
genation atthetissuesisappreciable in mostofthespecies, ous, qualitative, and fargreater than the differences be-
the venous reserve is high in both Uca minax and U. tween the He morphs found within Callinectes sapidus
pugnax. perhaps because these very active runners were (deFuretal.. 1990; Mangum, 1994).
held in small aquaria that constrained locomotion. As a second example, Williams 1983) has shown that
(
Panopeus herbstii (sensu lato Rathbun) is in fact a com-
Discussion plex ofno less than six cryptic species, which can be dis-
tinguished by careful morphological examination as well
Sitbimit composition as ecological preference (Reames and Williams, 1983;
The clearest findings of the survey ofelectrophoretic Williams, 1983). Sullivan et al. (1983) reported that at
separation ofcrustacean He subunits are (1) The PAGE least fourofthe six differ in He subunit composition (the
bandingpatternsare highlyspeciesspecific, and(2)above other two were not examined). Our data for P. herbstii
the level of sibling species, the PAGE banding patterns sensu stricto H. Milne Edwards, obesus, and lacustris
are not especially characteristic ofa taxon. strongly support this conclusion. Sullivan et al. (1983)
The specificity is best illustrated by following inter- briefly mentioned the existence ofintraspecific polymor-
specific examples in which morphological differences phism in P. herbstii. Although we have notyetcompleted
are small, suggesting that divergence of the He O: our own investigation ofthis species, data for hundreds
transport system may occur quite early in speciation. ofindividuals from the same locality and others as well
Figure2. O;bindingofhemocyaninsexaminedhere.20C,tonometricmethod,unlessspecifiedotherwise
Panel 1: Diogenidaeand Paguridae. CHbinanus villains( ), Pagurusimpressus(E3),P.po/licari.s(*), P.
longicarpus (). 0.05 mol 1"' Tris-buffered saline containing 460mmol1 ' NaCl, llmmoll 1 KG,
13 mmol I"' CaG2, 20mmol T1 MgCl2, 29mmol T' NaSO4 and 3 mmol I'1 NaHCO,.
Panel 2:Geryonidae. Chaceonfenneri(), C. quinquedens(A).0.05 mol1~' Tris-bufferedsalinecontaining
455 mmol I'1 NaCl, 11 mmol1"' KG, 13 mmolr1 CaG2, 18mmol I'1 MgG2. 22 mmolI'1 Na2SO4 and
3mmoir'NaHCO
3.
Panel 3:Ocypodidae. Ucacrenulatacoloradensis(). U. minax(O), U.princepsmoni/ifera(A), U.pugilator
(0), U.pugnax(t), U. \irens().Ocypodequadrata(A).ThedataforV crenulaiacolnradensisandprinceps
monilifera were collected with the cell respiration method. 0.05 moll"1 Tris-buffered saline containing
383 mmol I'1 NaCl, 11 mmol r1 KCI, 11 mmol r1 CaCl2, 45 mmol r1 MgCU, 40 mmol T1 Na2SO4 and
3 mmolr1 NaHCO3.
Panel 4: Majidae. Libinia duhia (CD), L emarginata (*). 0.05 mol 1~' Tris-buffered saline containing
350mmol r1 NaCl, 8 mmol r1 KG, 10mmol I'1 CaG2.42mmolI'1 MgSO4and 3mmolI'1 NaHCO3.
Panel 5: Mycidacea. Gnathophaiisiagigas(H), G ingens(*). Buffered salineasin panel 2.
Panel6:Cancndae.Cancerantennarius(D),C anthonyi().C. borealis(E),C irroratits(*),C.productus
(X).0.05 mol I"1 Tris-bufferedsalinecontaining441 mmol r' NaCl, 15 mmol I'1 KG. 11 mmolT'CaG,,
40mmolI'1 MgG2, 10 mmol r' Na2SO4 and 3 mmol I'1 NaHCO3.
Panel 7: Portunidae. Callinectesarcuatus(D), C. bellicosm(),C. ornatus(V), C. sapidus(),C. similis
(A), C. toxotes(T). Datacollectedat25Cwiththecell respiration method. Buffered salineas in panel 2.
Panel 8: Portunidae. Ovalipes ocellalus (D), Portunus spmnemanus (*), P gibbesii (). Buffered saline
asin panel 2.
Panel9:Xanthidae.Menippeadina(+),M.mercenaria(B).M.adina-mercenariahybrid().0.05 mol I*1
Tris-bufferedsalinecontaining350mmolI'1 NaCl, 7mmolI"1 KG. 11 mmol r1 CaG2, 33 mmolT1 MgG2,
20mmol r1 Na2SO4and 3 mmolT' NaHCO3.
Panel 10: Xanthidae. Cataleptodiiisfloridanm (). Eurypanopeiis depressiis(D), Panopeus herbstii (*).
P. lacustris('".),P. obesus (D). Buffered salineasin panel 9.
Panel 11:Grapsidae.Sesannacinereum(D),S. reticulalum(*).0.05 mol1~' Tns-bufferedsalinecontaining
383 moir' NaCl, 11 mmoir1 KG, 16 mmolr1 CaCI2, 45 mmolr1 MgG2, 40 mmol r1 Na2SO4 and
3mmolr1 NaHCOj.
394 J. E. REESE AND C. P. MANGUM
Table III
Bohrfactorsandcooperalivityvalues
Numberof BohrFactor
individuals Alogp/^pH Cooperativity
Species (n) (95%CI) n50(x SE)
OrderDecapoda
InfraorderAnomura