Atypical vitellins in ponerine ants (Formicidae: Hymenoptera)

Journal of Insect Physiology 45 (1999) 287–293

Atypical vitellins in ponerine ants (Formicidae: Hymenoptera)

Diana Wheelera,*, Jurgen Liebigb, Bert Holldobler b

a Department of Entomology, University of Arizona, Tucson, AZ 85721, USAb Biozentrum, Zoologie II, Universita¨t Wurzburg, Am Hubland, Wu¨rzburg D-97074, Germany

Received 14 July 1997; accepted 8 July 1998

Abstract

Higher hymenopteran vitellogenin/vitellins have been characterized as containing one large apoprotein. We show that in the antsubfamily Ponerinae, species in the tribes Odontomachini, Platythyrini, and Amblyoponini, also have a vitellin with this simplestructure, containing a single apoprotein of 180–190 kDa. Species in tribes Ponerini and Ectatommini, however, have vitellinscontaining multiple subunits. The size and number of the subunits varies, with differences even among species within the samegenus. This is the first report of diversity in vitellogenin structure in the higher Hymenoptera. Vitellin and vitellogenin inHarpeg-nathos saltator(Ponerini) contain two large subunits of about 165 kDa and two small subunits of about 45 and 43 kDa. N-terminalsequence analysis suggests that provitellogenin is cleaved at two different sites, producing two large and two small subunits differingslightly in size. Diversity of vitellin types in Ponerini and Ectatommini is similar to that found in the more primitive tenthredinoidsawflies (Hymenoptera, Symphyta), and may indicate polyphyly in the Ponerinae. Insect vitellogenins and yolk proteins showconsiderably more diversity than originally believed, and the possibility of the functional significance of these differences shouldbe considered. 1999 Elsevier Science Ltd. All rights reserved.

Keywords:Vitellin; Vitellogenin; Harpegnathos saltator; Ponerinae; Phylogeny

1. Introduction

Egg proteins of ants are of interest not only becauseof the unusual nature of aculeate hymenopteran egg pro-teins (Harnish and White, 1982a; Kageyama et al., 1994)but also because they are a currency of power in antsocieties. The primary organizing principle in insectsocieties is control of female reproduction, especiallyegg development (Wilson, 1971). Vitellogenins/vitellinsin the order Hymenoptera were first characterized ascontaining only one large apoprotein (Harnish andWhite, 1982a, b). This categorization was later supportedby additional descriptions of vitellogenins/vitellins in themajor suborder, Apocrita. Honeybee vitellogenin con-tained only one type of large subunit (| 180 kDa)(Wheeler and Kawooya, 1990) as did the ichneumonidwasp,Pimpla nipponica(Nose et al., 1997), and the antsCamponotus festinatus(Martınez and Wheeler, 1991),Solenopsis invicta(Vargo and Laurel, 1994) andMono-

* Corresponding author. Tel.:1 1-520-621-3273; Fax:1 1-520-621-1150; E-mail:[email protected]

0022–1910/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved.PII: S0022-1910 (98)00124-3

morium pharaonis(Jensen and Borgesen, 1995). Thefirst exception to this pattern was found in a sawfly,which belongs to the other major hymenopteransuborder, Symphata. Hatakeyama et al. (1990) showedthat vitellogenin in the sawflyAthalia rosaecontainedboth small and large vitellogenin subunits and suggestedthat the two suborders of Hymenoptera, Symphyta andApocrita, expressed vitellogenins of different types, withthe symphytans resembling those of most other insects.

During a study of physiological correlates of domi-nance behavior in the antHarpegnathos saltator(Ponerinae: Ponerini), we discovered that vitellin in thisspecies contained subunits of more than one molecularweight. The ant subfamily Ponerinae is often charac-terized as primitive, but members of this taxon arediverse, displaying an array of basal as well as highlyderived characters (Baroni-Urbani et al., 1992). Anunusually diverse collection of live ponerine ants col-lected all over the world were at the University of Wu¨rz-burg at the time of this study. We were able to examinevitellins of ponerine ants in five tribes to begin to assessthe taxonomic extent of this apparently atypicalvitellogenin/vitellin structure.

288 D. Wheeler et al. / Journal of Insect Physiology 45 (1999) 287–293

2. Materials and methods

2.1. Insects

To obtain vitellin from ponerine ants, eggs less than24 h old were collected from colonies of the following:Tribe Ponerini: Harpegnathos saltator(coll. near Ban-galore, India, by J. Liebig;Diacammasp., coll. Nakijin,Japan by K. Tsuji).Diacammasp. (Malaysia, coll. unk.),Diacammasp. (coll. near Jog Falls, Karnataka, India byJ. Liebig), Pachycondyla obscuricornis(coll. Bahia,Brazil, by B. Trunzer);Tribe Ectatommini: Ectatommatuberculatum (coll. Paragominas, Brazil, by P.S.Oliveira), E. ruidum (coll. Barro Colorado Island, Pan-ama, by S. Pratt);Tribe Platythyrini: Platythyreapunctata (coll. south Florida, USA, by K. Schilder);Tribe Odontomachini: Odontomachus chelifer(coll.Sta. Genebra Reserve, Campinas, SP Brasil, by P.S.Oliveira), Odontomachus clarus(coll. Florida, USA);Tribe Amblyoponini: Amblyopone sp.nr reclinata(coll. Jog Falls, Karnataka, India, by J. Liebig).

To confirm that the structure of vitellogenin/vitellinsin the major ant subfamilies conformed to the typicalhymenopteran pattern, we examined newly laid eggsfrom three genera of Dolichoderinae (Conomyrmainsana, C.nr. bicolor, Liometopum apiculatum, Tapin-oma sessile), two genera of Myrmicinae (Crematogasteropuntiae, Pheidole spadonia, P. dentata, Leptothoraxrugulatus, Monomorium minimum and Solenopsisxyloni), one genus of Formicinae (Camponotusrep-resenting four subgenera—C. festinatus, C. ocreatus, C.sayi, S. rasilis, C. mina, C. pennsylvanicus, C. texanus)and one genus of Pseudomyrmicinae (Pseudomyrmexapache). These colonies were collected as foundingqueens or as colonies near Tucson AZ, Lafayette LA,and Austin TX (USA) by D. Wheeler.

Eggs obtained from a nest ofPolistes dominulus(supplied by P.F. Ro¨seler, J. Gadau, Wu¨rzburg,Germany) provided material for a comparison withanother member of the aculeate Hymenoptera.

2.2. Gel electrophoresis

Eggs were prepared by disrupting them in buffer con-taining protease inhibitors, as described in Wheeler andBuck (1995). The mixture was then added to sampleloading buffer for either denaturing or non-denaturinggel electrophoresis. Electrophoresis was run accordingto the general method of Laemmli (1970). Pore-limitednative gels were run for 2500 vh at 4°C. Molecularweights of protein in SDS–PAGE gels were estimatedwith reference to standards in the high molecular weightcalibration kit from Bio-Rad. Values were estimatedfrom at least three sample lanes and two gels. Two-dimensional electrophoresis, with non-denaturing con-ditions in the first dimension and SDS-denaturing con-

ditions in the second, was carried out as described inMartınez and Wheeler (1991) and Wheeler and Buck(1995) to determine the relationship between the nativeprotein and its subunits.

2.3. Preparation of antibody

Freshly laid eggs (n 5 20) of Harpegnathos saltatorwere gently ground in inhibitory buffer and electrophor-esed on a 6% native polyacrylamide gel. The gel wasstained for about 30 s in Coomassie and rinsed in dH2Oovernight at 4°C. The prominently stained band was cutout with a razor blade, macerated, and mixed with adju-vant. The antigen was emulsified in an equal volume ofFreund’s complete adjuvant and injected into the breastmuscle of a chicken (Meisterhybriden/Rhodla¨nder). Fourand eight weeks later the chicken was boosted with simi-larly prepared batches of antigen emulsified in Freund’sincomplete adjuvant. Ten days after the last booster,antibody was purified according to the methods of Akitaand Nakai (1992).

2.4. Western blots

SDS–PAGE gels were blotted onto nitrocellulosepaper. After blotting was complete, the nitrocellulosewas rinsed in distilled water, air dried and stored at220°C. The membrane was incubated with anti-H. salt-ator vitellin IgY antibody (1:5000) and rabbit anti-chicken IgG (1:50 000). Alkaline phosphatase conjugatewas used as the detection agent.

2.5. N-terminal sequences

Subunits of vitellin fromH. saltatorandC. festinatuswere isolated by SDS–PAGE 9–15% gradient gels forN-terminal sequence analysis. Proteins were transferredto PVDF membranes and the N-terminals sequenced bythe University of Arizona Laboratory for ProteinSequencing and Analysis using an Applied Biosystem477A Protein/Peptide Sequencer (Edman chemistry)interfaced with a 120A HPLC (C-18 PTH column,reverse-phase chromatography) analyzer to determinephenylothiohydantoin (PTH) amino acids. Sequenceswere aligned with the aid of the ClustalW 1.6 tool formultiple sequence alignments.

3. Results

Eggs of species sampled in two tribes, the Poneriniand the Ectatommini, released more than one majorpolypeptide in SDS–PAGE (Fig. 1).Harpegnathos salt-ator eggs produced lanes with protein bands indicatingone to two large subunits of about 165 kDa and a doubletof smaller polypeptides at 45 and 43 kDa. Typically the

289D. Wheeler et al. / Journal of Insect Physiology 45 (1999) 287–293

Fig. 1. Vitellins of Ponerini and Ectatommini. Ponerini: D1–D3 rep-resentDiacammaspecies from three different localities (Japan, India,Malaya). Pa5 Pachycondyla obscuricornis; Ha 5 Harpegnathos salt-ator; Ectatommini: Ecru5 Ectatomma ruidum; Ectu 5 E. tubercula-tum. Leftmost lane5 molecular weight standards. 6–15% SDS–PAGE.

smaller bands were clearly distinct but the larger bandstended to coalesce.Pachycondyla obscuricorniseggscontained two large polypeptides (184 and 148 kDa) andone small (46 kDa). Vitellins of all three species ofDiacammaexamined were similar to each other but dis-tinctly different from the other genera in the tribe Poner-ini. Diacammaeggs produced numerous bands, with themost abundant polypeptides at about 88, 64, and 49 kDa.Vitellin in the tribe Ectatommini, in two species ofEcta-tomma, also revealed more than 2–3 major polypeptidesin SDS–PAGE:E. ruidum(163 and 32 kDa) andE. tub-erculatum(160, 118, 40 kDa)

In contrast, eggs from three other ponerine tribes, theOdontomachini (O. chelifer 191, O. clarus 189 kDa),Platythyrini (Platythyrea punctata 186 kDa), andAmblyoponini (Amblyoponenr reclinata, 188 kDa) con-tained a single major polypeptide with a molecular massof 180–190 kDa (Fig. 2).

On pore-limited non-denaturing gels, most speciesexamined showed a single major band of protein with

Fig. 2. Vitellins of Amblyoponini, Odontomachini, and Platythyrini.Am 5 Amblyopone; Od 5 Odontomachus clarus; Pl 5 Platythyreapunctata. S 5 molecular weight standards. 6–15% SDS–PAGE.

Fig. 3. Non-denaturing 4–20% pore-limiting polyacrylamide gel ofvitellins from six species of ponerine ants representing four tribes.Only Amblyoponewas not included due to scarcity of material. Allshow an estimated molecular weight in the range of 400–500 kDa. Ec5 E. tuberculatum; Ha 5 Harpegnathos saltator; Od 5 Odontom-achus clarus; Pa 5 Pachycondyla obscuricornis; Pl 5 Platythyreapunctata. S 5 molecular weight standards of 669, 440, 232, and140 kDa.

relative mobilities of 400 000–500 000 (Fig. 3). This sizeis consistent with the model that the native moleculecontains two each of a large and a small apoprotein. Todetermine if all subunits were derived from a singlenative molecule, vitellin from Ponerini and Ectatomminivitellins were examined using 2D electrophoresis (Fig.4). In most cases, vitellin subunits were derived fromdiscrete, if somewhat fuzzy, single native bands. Thesingle exception was theDiacammaprotein, which didnot form a discrete band under short-term non-denatur-ing conditions, and could not be analyzed by thismethod.

Polyclonal antibodies raised against the major proteinin H. saltatoreggs recognized the major subunits foundin eggs and the hemolymph of young adult females on

Fig. 4. Second dimension SDS gel showing that the single bands ofvitellin produced in non-denaturing gel electrophoresis (Fig. 3) pro-duced multiple apoproteins under denaturing conditions. Pa5 Pachy-condyla obscuricornis; Ha 5 Harpegnathos saltator; Ectatommini:Ecru 5 Ectatomma ruidum; Ectu 5 E. tuberculatum. S 5 molecularweight standards.


Fig. 5. Lanes A and B: Hemolymph (H) and egg (E) samples ofHar-pegnathos saltator, 6–15% SDS–PAGE. Lanes C and D: Western blotof gel shown in lanes A and B, probed with antibodies toH. saltatorvitellin. Vitellin subunits are indicated by arrowheads. The distinctnessof the two large subunits is especially clear in this preparation.

Western blots (Fig. 5). Minor proteins in the egg showedcross reactivity with the antibody, though these may rep-resent small of amounts of degraded yolk. The antibodywas highly specific against the array of hemolymph pro-teins. The pattern of subunits observed in the hemo-lymph confirms that both small and large apoproteins arefound in vitellogenin as well as vitellin.

N-terminal analysis ofH. saltatorvitellin showed thatthe couplet of small vitellin subunits (Fig. 1) began withidentical N-terminal sequences. Comparison with otherinsects indicated some degree of conservation, evenamong distantly related groups (Fig. 6). Vitellogeningenes contain both conserved and unconserved regions(Hagedorn et al., 1998). The N-terminals of the largesubunit of H. saltator vitellin produced two differentsequences (SWKAEMHNMGLVLISQ andPSILGLNLSDLSKMLDPLEM). Alignment tools foundno significant homologies when these were compared tofull cDNAs available for Pimpla nipponica(Hymenoptera),Aedes aegypti(Diptera), Bombyx mori(Lepidoptera) andAnthonomus grandi(Coleoptera) sug-gesting these sequences lie in an unconserved region.

Few hymenopteran vitellogenin/vitellins have beendescribed biochemically. Given that the results reportedabove showed unanticipated variability in the ponerineants, eggs of representatives from four other ant subfam-ilies were examined to determine if Formicidae were

Fig. 6. N-terminal sequences for the antsHarpegnathos saltator(Ponerinae) (underlined) andCamponotus festinatus(Formicinae) are comparedwith the same region of the honeybee (Apis mellifera), an ichneumonid wasp (Pimpla nipponica), a sawfly (Athalia rosae), a mosquito (Aedesaegypti), and the boll weevil (Anthonomus grandi). Bold font indicates identity among at least four of these taxa at a given position.

more variable throughout the family than previouslyassumed. The eggs of all the Myrmicinae, Formicinae,Dolichoderinae and Pseudomyrmicinae we have exam-ined on SDS gels to date (see Section 2) show a single,large vitellin subunit in the range of about 180–190 kDa.In addition, eggs fromPolistes dominulus, representinganother major Hymenoptera family (Vespidae) alsorevealed a vitellin of this type, structure and weight. Sev-eral of these ant species and thePolistes sample areshown in Fig. 7.

4. Discussion

4.1. Diversity in insect and ant vitellins

Previously, vitellogenin and vitellins of Hymenopterawere characterized as having only one large subunit orapoprotein (Harnish and White, 1982a, b). Morerecently, Kageyama et al. (1994) and Takadera et al.(1996) showed that the two subunits in Symphyta werederived from a common transcript, in which the smallsubunit was at the 59 end. Thus the subunit compositionof a symphatan vitellogenin appeared to be similar tothat of most other insects (Hagedorn and Kunkel, 1979).The basal position of Tenthridinoidea and Xyloideawithin the paraphyletic suborder Symphyta and the orderHymenoptera has been supported by recent phylogeneticanalyses (Whitfield, 1998).

Fig. 7. Vitellins of the vespid waspPolistes dominulus(Po) and ofthe ants representing subfamilies Myrmicinae (Ps5 Pheidole spa-donia, Pr 5 Pheidole rhea, Cr 5 Crematogaster opuntiae), Dolichod-erinae (Co5 Conomyrmanr. bicolor), Formicinae (Cs5 Camponotussayi). S 5 protein standards. 6–15% SDS–PAGE.


Further examination of vitellogenin/vitellins of 21sawfly species (12 genera, 2 families) revealed diversityin subunit composition. These Tenthridinoidea taxa con-sistently showed subunits of two sizes, although specificsizes varied somewhat even within a genus. In severalspecies, intermediate-sized subunits were also found(Takadera et al., 1996). The difference between the sin-gle large and the smaller multiple subunits of hymenop-teran vitellogenin/vitellin could be simply a differencein post-translational processing, similar to that reportedfor mosquitoes (Dhadialla and Raikhel, 1990). Heilmannet al. (1993) showed that the amino acid sequence ofthe N-terminal of honeybee vitellogenin matched the N-terminal of the small subunit of boll weevil vitellogenin,and the sequence of the N-terminal of an internal frag-ment was homologous to a region within the large beetlesubunit. This suggested that the single large honeybeeapoprotein represented an uncleaved product of a singlevitellogenin transcript. In Symphyta (Hymenoptera),Kageyama et al. (1994) demonstrated that both subunitsin the sawflyAthalia rosaeare indeed derived from onemRNA transcript. The fact that the two small subunitsin H. saltatorhave the same N-terminal sequences whilethe large subunits reveal two different sequences sug-gests that the small subunits occupy the 59 end of provit-ellogenin and there are two different cleavage sites.

Results reported here for the subfamily Ponerinaedemonstrate diversity within the apocritan Hymenoptera.The nature of the diversity is similar to that seen in saw-flies in that the size of the large and small subunits variesbetween taxa, even within genera, and intermediate-sized as well as small subunits are present in somegroups. InPachycondyla, which like Harpegnathosisa member of the Ponerini, both an uncleaved product(184 kDa) and cleaved products (148 and 46 kDa)appear to be present. Vitellogenin/vitellin of the threeDiacammaspecies (also in the Ponerini), with the mostabundant peptides of 50–90 kDa in size, may be pro-duced by multiple cleavage sites in a provitellogenin.Alternatively, this pattern of multiple bands is remi-niscent of vitellogenin banding described without thebenefit of protease inhibitors (Winkler and Schmidt,1988). In our experience, a protease inhibitor cocktail(see Section 2) can prevent the action of most proteindegradation under our experimental conditions. Thepossibility remains, however, thatDiacammaeggs con-tain a protease that is not curtailed by our cocktail andthat degrades vitellogenin in the same specific way inall three species we examined (Fig. 1).Diacammais asomewhat unusual genus in lacking a morphologicallyspecialized queen caste. Mated workers serve as thefemale reproductives (Moffett, 1986; Peeters and Tsuji,1993). It remains to be determined if their unusual pat-tern of peptides in the egg reflects some functional dif-ference in their reproductive biology or whether it is anunrelated fluke of taxonomy.

Clearly, the initial system of categorizing ofvitellogenin/vitellin (Harnish and White, 1982a, b) hasproven inadequate to accommodate the diversity offorms now known in insects. Other insects, in additionto Hymenoptera, have vitellogenin/vitellins that do notconform to the original categories and that vary withinorders. For example, in Hemiptera,Oncopeltus fasciatusappears to have two large subunits (200 and 170 kDa)(Martınez and Garcera´, 1987), even when samples areprocessed with protease inhibitors (Martı´nez, pers.comm.). In Homoptera the sweet potato whitefly(Bemisia tabaci) has a vitellogenin/vitellin with singlelarge subunit of 190 kDa (Tu et al., 1997), and the cochi-neal scale insect (Dactylopius confusus) produces a yolkprotein made of long ribbons of 45 kDa subunits (Ziegleret al., 1996). Vitellin of Derobrachus geminatus(Coleoptera) is composed of three apoproteins of about160, 140 and 50 kDa (Osir and Law, 1989). Two largesubunits of 199 and 162 kDa have been reported for vit-ellogenin of the Colorado potato beetle,Leptinotarsadecemlineata (Koopmanschap et al., 1992).Vitellogenin/vitellins of at least some Coleoptera do con-form to the more typical pattern of one large and onesmall apoprotein (e.g. Curculionidae, Heilmann et al.,1993; Cerambycidae, Azuma et al., 1993). The egg pro-tein of the fall webworm,Hyphantria cunea, deviatesfrom the typical lepidopteran structure in being made upof three yolk proteins of low molecular weight (35.5,34, 18 kDa) (Lee et al., 1995). Furthermore, even thosevitellogenin/vitellins with two apoproteins, one of highmolecular weight and one of low, can be produced bydifferent mechanisms. For example, in mosquitoes(Dhadialla and Raikhel, 1990), the boll weevil(Heilmann et al., 1993), and sawfly (Kageyama et al.,1994), the small apoprotein is derived from the 59 endof the provitellogenin message, but in the gypsy moththe small subunit is generated from the 39 end (Hiremathand Lehtoma, 1997). Molecular data and phylogenetictools will ultimately provide the bases for a new systemof classification.

In addition, the existence of supplementary yolk pro-teins such as microvitellogenin in Lepidoptera (Kawooyaet al., 1986) and the use of hexamerin storage proteinsas yolk (Chinzei et al., 1992) underscores the diversityof protein types that can fulfil a yolk function. The possi-bility of functional significance of fundamental differ-ences in macrostructure among insect yolk proteins andvitellogenins deserves exploration.

4.2. Implications for ant phylogeny

Agreement does not exist for the phylogeneticrelationships among ants in general and among Poneri-nae in particular (Grimaldi et al., 1997). The subfamilyPonerinae is large and diverse (1300 species, Bolton,1995). Holldobler and Wilson (1990) placed them most


closely to the Myrmicinae, with both groups many nodesdiverged from the Dolichoderinae and Formicinae. Theanalysis of Baroni-Urbani et al. (1992) divides the antsinto two major clades, with the Ponerinae in one and theother major ants subfamilies in the other. Members ofthe Ponerinae show the highest number of polymorphiccharacters in the Baroni-Urbani data matrix, indicatingdiversity and perhaps polyphyly. The discovery of anunusual ponerine or near ponerine from Madagascarcasts further doubt on the monophyly of the Ponerinae(Ward, 1994). Reanalysis of the data in Baroni-Urbaniet al. (1992) after the addition of fossil material placesthe Ponerinae as a monophyletic group that springs froman intermediate position, rather than basally in the clado-gram (Grimaldi et al., 1997). A recent phylogeneticanalysis of ants based on molecular data indicates poly-phyly in the Ponerinae, with Amblyopone and Poneriniarising distinctly from more basally than Ectatommini(Sullender, pers. comm.).

The evidence presented here for ponerine vitellins alsosupports a major division in the subfamily. Mapping cur-rent information regarding vitellogenin/vitellin structureonto a hymenopteran phylogeny (e.g. Whitfield, 1998)raises the question of whether some tribes of ponerineants have retained a primitive pattern because they arerelatively basal in the Hymenoptera or because they haveresumed the expression of what appears to be a lessderived form of vitellogenin/vitellin. The division ofponerine tribes expressing single vs multiple subunitsdoes not map simply onto any cladogram currently pro-posed.

Acknowledgements

D.W. owes special thanks to D.A.A.D. and Universityof Paris-Nord for providing funds that enabled thesestudies of ponerine ants, and to C. Peeters, J. Heinze,M. Obermayer, B. Ehmer, J. Gadau, P. Ro¨seler, D. Palm,D. Karbach, S. Sternbeiss, K. Schilder, B. Trunzer forproviding their time, support and ants so generously. Wealso thank S. Cover for identifying theAmblyopone. TheE.K.W Gift to the U of A funded N-terminal sequences.

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Atypical vitellins in ponerine ants (Formicidae: Hymenoptera)

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