identification of vitellogenin in the ant, camponotus festinatus: changes in hemolymph proteins and...

Archives of insect Biochemistry and Physiology 17:143-155 (1991)

Identification of Vitellogenin in the Ant, Camponotus festinatus: Changes in Hemolymph Proteins and Fat Body Deve I o p m e n t i n Workers Teresa Martinez and Diana Wheeler Department of Entomology, University of Arizona, Tucson, Arizona

Vitellogenin has been identified in the ant Camponotus festinatus, both in queens and workers. In the workers, it is already present before adult eclosion in low concentrations (<I bg/bl hemolymph). Vitellogenin andvitellin are immunologically identical and are composed of a single type of apoprotein with an apparent M, 185,000. The molecular weight of the native molecules was estimated as -460,000 by pore limiting gradient electrophoresis. Vitellogenin was detected as a major protein in the hemolymph of young workers, both under queenright and queenless conditions. Thus, in spite of their sterility in the presence of the queen, C. festinatus workers are able to syn- thetize vitellogenin which i s identical both in size and immunologically to the queen vitellogenin. About 6-7 weeks after adult eclosion, however, vitellogenin was usually undetectable in the hemolymph of queenright workers, particularly the minor workers, while it constituted about 30% of total protein in queenless workers. Protein concentration in the hemolymph of queenless insects increased up to 20-fold as compared to I-day-old insects. Queenless workers also developed large amounts of perivisceral fat body, while queenright workers, particularly the minor workers, showed a dramatic fat body regression about 6 weeks after emergence.

Key words: reproduction, vitellin, Hymenoptera, caste

INTRODUCTION

In social Hymenoptera, egg development in workers is usually inhibited when reproductive females (queens) are present in the colony. Although this

Acknowledgments: Thiswork was supported by NlAlD award 27403. We thank H. H. Hagedorn and J. A. Veenstra for critically reading the manuscript. This paper i s publication no. 7304 of the Arizona Agricultural Experiment Station.

Received December 17,1990; accepted April 1,1991.

Address reprint requests to T. Martinez, Department of Entomology, University of Arizona, Tucson, AZ 85721.

0 1991 Wiley-Liss, Inc.

144 Martinez and Wheeler

is a genera1 phenomenon in social insects, little is known about the physiological mechanisms involved in the inhibition of oogenesis in workers. In the bumble bee Bornbus ferresfris, research supports a model in which a pheromone produced in the queen mandibular glands would inhibit the corpora allata of workers, and thus oocyte development [l-41. A similar situation has been pro- posed recently in the honey bee Apis mellifirn, in which it has been further suggested that the influence of the queen pheromone on the corpora allata of workers may be mediated by median neurosecretory cells [ 5 ] . Apart from some research done in social wasps [6,7], the information available on the regulation of worker reproduction in other social Hymenoptera is very scarce.

Vitellogenin synthesis by the fat body and its uptake into developing oocytes lead to egg maturation and oviposition in vitellogenic females; thus, any of these processes might be inhibited in the workers. Research on the identification of vitellogenins in social Hymenoptera has been limited to A . melliferu [8-101. In this species, it has been recently reported that the queens inhibit vitellogenin synthesis in workers, since synthesis of that protein in vitro was found to be twice as high in queenless workers as compared to queenright individuals [11]. There is no information on vitellogenin in ants.

As a first approach to understand the physiological mechanisms involved in the inhibition of worker reproduction, we have identified vitellogenin in Curnponotus festinntus queens and workers (including soldiers and minor workers), and have examined changes in hernolymph proteins and fat body development in workers under queenright and queenless conditions.

MATERIALS AND METHODS Insects

Camponofus fistinufus colonies were maintained in the laboratory at 30°C and 16L:8D photoperiod. Ants were fed cockroaches (Nnuphoeta cinerea) and a solution of honey, and water was provided in test tubes to maintain humidity. Groups of workers (which in this species include two distinct castes: soldiers and minor workers) were removed from the colony as late pupae and kept in Petri dishes together with minor workers added as nurses. After adult emergence, some workers were marked and returned to the colony as queenright controls. Other workers of the same age were kept outside the colony as queenless individuals, in small groups of about 6 soldiers and 30 minor workers.

Sample Preparation Hemolymph from larvae, pupae, and adults was collected in 1 pl capillary

tubes through a dorsal thoracic incision, and immediately diluted with cold Tris-buffered saline (20 mM Tris, 0.15 M NaCI, 5 mM EDTA, pH 7.5) containing the following protease inhibitors: 1 mM PMSF,* 5 mM benzamidine, 0.7 pM pepstatin A, 8 pM chymostatin, 10 pM leupeptin, 8 pM antipain, and 0.8 pM aprotinin. For quantitative analysis, 0.5 pl hernolymph from individual

"Abbreviations used: DFP = diisopropylfluorophosphate; kD = kilodalton; PMSF = phenyl- methylsulfonyl fluoride; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS = Tris-buffered saline.

Vitellogenin in Ants 145

soldiers or 0.25 p1 from minor workers were mixed with 20 pl TBS. Whole samples or aliquots, as indicated, were used for PAGE.

Perivisceral fat body was dissected from the abdomen of adult workers, rinsed in cold 0.9% NaC1, and homogenized in TBS. Mature eggs (<24 h old) were collected from C. fistinatus queens and homogenized in the above saline. Sam- ples of hemolymph, and fat body and egg homogenate were centrifuged at 12,0009 for 20 min at 4"C, and the supernatants used for polyacrylamide gel electrophoresis and immunochemical analysis.

Polyacrylamide Gel Electrophoresis Samples were either subjected to electrophoresis or stored at - 20°C until

used. Hemolymph, fat body, and egg extracts were mixed with an equal vol- ume of sample buffer (62 mM Tris-HCl, pH 6.8, containing 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.005% bromphenol blue). SDS and mercaptoethanol were not included in the buffer when samples were subjected to native electrophoresis. SDS-PAGE was performed according to Laemmli [12], using a 6-15% gradient separating gel with a 4% stacking gel. Electrophoresis was performed at 20 mA constant current. Gels were stained with 0.1% Coomassie brilliant blue R 250 in 50% methanol, 10% acetic acid, and destained in 10% methanol, 50% acetic acid.

Large differences in protein concentration in the hemolymph between queenright and queenless workers were detected by SDS-PAGE. Gels were scanned at 633 nm with a laser densitometer (LKB 2222-020, Ultroscan XL) to quantify those differences. Total Coomassie staining in hemolymph proteins and the percentage of Coomassie staining in vitellogenin were determined.

Two-dimensional electrophoresis was performed to determine the subunit composition of vitellogenin and vitellin. Samples of hemolymph, fat body, and egg extracts were first subjected to electrophoresis under nondenaturing conditions using a 5% acrylamide separating gel with a 3% stacking layer. The gels were subsequently stained, destained partially, and rinsed with distilled water for about 30 min to remove the methanol and acetic acid. The areas of the gel containing the proteins of interest were excised, incubated in SDS- sample buffer for about 20 min, and layered on top of a 6-15% gradient SDS-gel.

Molecular Weight Determinations The molecular weight of native vitellogenin and vitellin was estimated by

nondenaturing pore limiting gradient electrophoresis (4-20% acrylamide). To confirm that migration was completed, electrophoresis was performed for 2,000, 2,800, and 3,500 volt-hours at 4°C. A standard curve was made by plotting the log M,. of protein markers versus their corresponding Rf values. The molecular weight standards (Pharmacia) used were thyroglobulin (M, = 669,000), ferritin (M, = 440,000), catalase (M, = 232,000), lactate dehydrogenase (M, = 140,000), and bovine serum albumin (M, = 67,000). The molecular weight of vitellogenin and vitellin subunits was determined by SDS-PAGE (6-15% gradient) using Bio-Rad HMW electrophoresis calibration kit.

Immunology Vitellin antigen was prepared as follows: Newly laid eggs collected from a

C. fistinatus queen were homogenized in phosphate-buffered saline (0.1 M


sodium phosphate, 0.15 M NaC1, pH 7.0) containing 0.02% sodium azide, 5 mM glutathione, and 1 mM benzamidine, 1 mM DFP, and 1 mM PMSF, as protease inhibitors. The homogenate was centrifuged at 12,OOOg for 20 min at 4"C, and the supernatant subjected to SDS-PAGE. Polyacrylamide gel slices containing the vitellin band were used for rabbit immunization according to the method by Boulard and Lecroisey [13]. A New Zealand white rabbit was injected subcutaneously in four different sites on the back with an emulsion of the gel homogenate (ca. 250 pg protein) with 1 ml of RIBI adjuvant (RIBI Immunochem Research Inc., Hamilton, MT). After 5 weeks, a booster injec- tion was administered following the same procedure, but using half the amount of protein. Blood was collected through an ear vein 1 week later, the serum separated from the clot, centrifuged, and stored in aliquots at - 70°C.

Specificity of the antiserum was tested by immunoblotting. Samples of hemolymph, fat body, and egg extracts were subjected to SDS-PAGE, and the proteins electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated with C. festinatus vitellin antiserum, and goat anti-rabbit Ig G alkaline phosphatase conjugate was used as the detection agent.

RESULTS Changes in Hemolymph Proteins and Fat Body Development

Proteins were found to accumuiate in the hemolymph of queenless workers during several weeks after adult eclosion. In 7-week-old insects, protein concentration in the hemolymph (as determined by total Coomassie staining) increased about 15-fold in the minor workers and 20-fold in the soldiers, compared to the concentrations found a t eclosion. However, in 7-week-old queenright workers, total protein titer in the hemolymph increased only 2-fold in minor workers and 6-fold in soldiers (Table 1).

When hemolymph from 1-2-week-old queenright and queenless C. fistinatus workers was analyzed by SDS-PAGE, a similar electrophoretic pattern was found in both groups of insects. However, important differences were found several weeks after adult eclosion. A polypeptide with an apparent M, = 185,000 constituted a major component in the hemolymph of queenless workers. Con- versely, that protein was either undetectable with Coomassie blue staining, or present in trace amounts in the hemolymph of most queenright workers, particularly the minor workers, about 6 weeks after emergence. Another hemolymph protein with an apparent M, - 73,000 was also found to accumu-

TABLE 1. Total Hemolymph Proteins of C. festinatus Workers, as Determined by Coomassie Staining*

Queenright Queenless Minor Minor

Age Soldiers workers Soldiers workers

Day 1 2.6 k 0.6 3.0 k 0.5 7 weeks 15.5 t 2.9 5.9 * 0.Y 54.4 k 4.4 45.7 -t 2.0

*Data represent means +- S.E. expressed in arbitrary units corresponding to the peak areas of proteins in 1 pl hemolymph (n = 6 for soldiers; n = 8 for minor workers).


soldiers minor workers

Fig. 1. SDS-PACE (6-15% gradient) of hemolymph from 10-week-old soldiers and minorworkers. Q -t , queenright workers; Q - , queenless workers. Approximately0.15 PI hemolymph was analyzed per sample, except forthe soldiers Q + , in which 0.5 JLI hemolymph was used. Arrows indicate two polypeptides of -185 kD and -73 kD present in queenless workers. 5 , molecular weight standards, including myosin (200 kD), p-galactosidase (116 kD), phosphorylase B (97 kD), bovine serum albumin (66 kD), ovalbumin (45 kD), carbonic anhydrase (31 kD), soybean tryp- sin inhibitor (21 kD), and lysoryme (14 kD).

late in the hemolymph of queenless workers and to be absent from the hemolymph of queenright insects (Fig. 1).

The percentage of total Coomassie staining in the 185 kD polypeptide (vitellogenin, as will be shown below) with respect to total hemolymph proteins was determined at different times after adult eclosion, in workers of both queenright and queenless condition. This polypeptide constituted about 3% in newly emerged workers and increased up to 10-15% in both 2-week-old queenright and queenless insects. However, 7 and 10 weeks after emergence, the 185 kD polypeptide constituted about 30% in queenless workers, while in queenright workers it was found to be below 4% (Table 2). The percentage of the 73 kD polypeptide could not be accurately determined since it migrated very closely to other hemolymph proteins.

Important changes were also found in the development of the perivisceral fat body in both queenright and queenless workers. While no difference was observed in 1-2-week-old insects between the above two groups, about 6 weeks after eclosion most queenright workers, particularly the minor workers, contained small amounts of perivisceral fat body. They showed virtually no abdom- inal fat body 10 weeks after emergence, as if this tissue had undergone a dramatic regression. Ten-week-old queenright soldiers also had poorly developed perivisceral fat body containing small lobes. Conversely, queenless work-


TABLE 2. Percentage of Vitellogenin With Respect to Total Hemolymph Proteins in C. festinatus Workers, as Determined by Coomassie Staining*

Queenright Queenless Minor Minor

Age So 1 die r s workers Soldiers workers

Day 1 2.8 t 1.2 2.9 * 0.8 2 weeks 11.1 k 1.7 12.2 & 2.4 15.1 t 2.5 10.4 * 2.8 7 weeks 3.3 5 1.0 0.8 * 0.6 28.4 * 2.3 29.4 k 2.0

10 weeks 2.9 t 0.5 1.2 & 0.7 28.7 f 3.9 29.6 * 2.6

*Data represent means k S.E. (n = 6 for soldiers; n = 8-10 for minor workers).

ers showed a continued increase in the perivisceral fat body mass after adult emergence (Fig. 2). SDS-PAGE of fat body extracts showed that proteins accumulate in the fat body of queenless workers for at least 10 weeks after emergence. A 185 kD polypeptide and a polypeptide with an apparent M, - 80,000 were particularly abundant in the fat body of those insects, while in most queenright workers, the perivisceral fat body was virtually depleted of proteins (Fig. 3).

Immunology Crude egg extracts were prepared from newly laid eggs collected from C.

festinatus queens and analyzed by SDS-PAGE. Mature eggs contained a single major protein band which had the same electrophoretic mobility as the 185 kD polypeptide present as a major component in the hemolymph and fat body of queenless workers (Fig. 4A). In order to confirm that this protein corres- ponded to vitellogenin, polyclonal antibodies were raised against vitellin. The specificity of the antiserum was tested against hemolymph and fat body extract prepared from queenless workers, and against egg extract prepared from newly

Fig. 2. workers, 10 weeks after adult eclosion.

Perivisceral fat bodies from queenright (below) and queenless (above) C. festinatus


A B

Fig. 3. Hemolymph (H) and fat body (F) soluble proteins analyzed by SDS-PAGE. A: 2-week-old minorworkers. 5: 10-week-old minor workers. Q- , queenless; Q + , queenright. Fat bodysam- ples correspond to extracts of whole individual perivisceral fat bodies, except for the 10-week-old queenless worker, in which only half of the extract was used to avoid overloading the sample. Approximately 0.15 kl hernolymph was analyzed per sample, except for the 2-week-old worker, in which 0.5 pI hernolymph was used. Arrows indicate two polypeptides of -185 kD and -80 kD which accumulate in the fat body of queenless workers.

laid eggs collected from queens, using SDS-PAGE and immunoblotting. The vitellin antiserum specifically recognized a polypeptide with an apparent M, = 185,000 present in the hemolymph, fat body, and mature eggs (Fig. 48). From these results we concluded that the 185 kD polypeptide corresponds to C. festinatus vitellogenin. The antibodies also reacted with a fat body polypeptide that migrated just ahead of vitellogenin on an SDS gel.

Hemolymph was collected from 5th-instar larvae, pupae, and pharate adults, and analyzed by SDS-PAGE and immunoblotting to determine the time of appearance of vitellogenin in C. festinatus workers. Hemolymph from a C. festinatus queen was also analyzed and used as a positive control. The vitellin antiserum recognized a polypeptide in the hemolymph of larvae, pupae and pharate adult workers which had a similar electrophoretic mobility to the queen vitellogenin. Therefore, vitellogenin appears to be present in workers already in the last (5th) larval instar and during the pupal stage, although it was detected only in small amounts (Fig. 5). Before adult emergence vitellogenin concentration in the hemolymph was always below 1 pg/pl, both in soldiers and minor workers. Vitellogenin was also found in male pupae in concentrations similar to those found in worker pupae, but it was virtually absent from the hemolymph of adult males (data not shown). The antiserum to C. festinatus vitellin was also tested against hemolymph from Apis mellifem queens for possible immu- nological similarities between vitellogenins of two hymenopteran species; how-


A

Fig. 4. A: SDS-PAGE (6-15% gradient) of fat body (FB) and hemolymph (H) from a queenless soldier, and egg extract (E) prepared from a C. festinatus queen. Molecular weight standards are shown on the left. B: lmmunoblot of vitellogenin (Vg) from the same samples shown in A. Vitellin antiserum was used diluted 1 :20,000.

A

Fig. 5. A: SDS-PAGE of hemolymph from C. festinatus 5th instar larvae (lane l ) , pupae (lane 2), pharate adults (lane 3), and queen (lane 4). Approximately 1 IJ.I hemolymph was analyzed per sample except for the queen (-0.15 PI). Lane 5: Hemolymph from A. rnelfifera queen. Vg, vitellogenin. 6: lmmunoblot of vitellogenin from the same samples shown in A. Vitellin antiserum was used diluted 1:15,000.


ever, our antibodies failed to recognize the honey bee vitellogenin (Fig. 5B, lane 5).

Subunit Composition and Molecular Weight Two-dimensional electrophoresis was performed using nondenaturing con-

ditions in the first dimension and SDS-PAGE in the second dimension in order to determine the subunit structure of C. festinatus vitellogenin and vitellin. Vitellin constituted the only major protein of newly laid eggs of C. festinatus queens. A protein with similar electrophoretic mobility to that of vitellin was detected both in the hemolymph and fat body of workers (Fig. 6A). When these proteins were subjected to SDS-PAGE in the second dimension, both vitellogenin and vitellin gave a single type of apoprotein with the same electrophoretic mobility and an apparent M, = 185,000 (Fig. 6B).

Vitellogenin and vitellin were subjected to pore limiting gradient electrophoresis (4-20% acrylamide) in order to estimate the molecular weight of the native molecules. Vitellin had a M, -460,000. Although vitellogenin migrated very close to another major hemolymph protein, its molecular weight was not apparently different from that of vitellin (Fig. 7). Under non-pore-limiting conditions, vitellin was found to migrate slightly ahead of vitellogenin.

DISCUSSION

C. festinatus vitellogenin is the first vitellogenin identified in an ant species. Two other vitellogenic proteins identified in Hymenoptera are from A . mellifera [lo] and an unidentified sphecid wasp [9]; they differ from the rest of insect vitellogenins in that the native molecule is composed of a single type of apoprotein. The results of the present study with C. festinatus are in agreement with the above finding. In those two studies, however, the authors concluded that both the honey bee vitellogenin and the wasp vitellin are composed of only one subunit, based on the molecular weight estimate of the native molecules (about 300 kD in the case of the honey bee, and 200 kD in the wasp) and that of the constituent polypeptides (180 kD in both cases), The molecular weight estimate we obtained for C. festinatus vitellogenin and vitellin was about 460,000 and, in SDS-polyacrylamide gel electrophoresis, both molecules gave a single type of polypeptide with an apparent M, = 185,000. These results suggest that vitellogenin and vitellin in this species each consists of two apoproteins of 185 kD. Recently, it has been indicated that vitellogenin and vitellin of the primitive hymenopteran species, Afhal ia rmae are composed by a large (160-170 kD) and a small (48-50 kD) polypeptide [14]. However, these researchers did not use any protease inhibitor for sample preparation, and thus it cannot be ruled out that the small polypeptide resulted from proteoly- sis [15].

Using native-PAGE under non-pore-limiting conditions, C. festinatus vitellin migrates slightly ahead of vitellogenin. Small differences in mobility between vitellogenin and vitellin have also been reported in other insect species, and have been associated to differences in charge, which can result from a difference in amino acid composition or in lipid content between both molecules [16].

The antiserum obtained against C. festinatus vitellin specifically recognized


H E FB

Fig. 6. A: Native PAGE (5% acrylamide) of hemolymph (H), egg (E), and fat body (FB) soluble proteins. Vg, vitellogenin. B: Two-dimensional electrophoresis. Protein bands were excised from native gel shown in A, and layered on top of an SDS-gel (6-15% gradient), as represented in the figure (see text for details). Arrows indicate vitellogenin and vitellin subunits. Molecular weight standards are shown on both sides of the gel.

a single polypeptide of about 185 kD in the hemolymph of both queen and workers, when analyzed by SDS-PAGE. The antiserum also recognized native vitellogenin and vitellin (data not shown). In spite of using a mixture of protease inhibitors during sample preparation, the antiserum reacted with a polypeptide with an apparent M, -170,000 present in fat body homogenate.


Fig. 7. Native pore limiting PAGE (4-20% gradient) of egg (E), hernolymph (H), and fat body (FB) soluble proteins. Electrophoresis was performed for 2,000 volt-hours at 4°C. Molecular weight standards are shown o n the left.

Although it cannot be ruled out that this product derived from vitellogenin as a result of some proteolytic activity, another possible explanation is that the 170 kD polypeptide is a precursor of vitellogenin apoprotein, previous to the glycosylation step. In the mosquito Aedes aegypti, monoclonal antibodies specific to vitellogenin apoproteins also precipitated two vitellogenin precursors, one of 220 kD, and another of 235 kD; the latter one has been suggested to be derived from cotranslational glycosylation [17]. C. festinatus vitellin antiserum tested against hemolymph from A. mellifera queens did not cross-react with the honey bee vitellogenin. This suggests that even though the size of the apoproteins of C. festinatus and A. mellifera vitellogenins is very similar, the antigenic determinants are not conserved. On the other hand, immunologi- cal similarities among vitellogenins of different species have been found between Maizduca sexta and HyuIophora cecropia [MI.

In A. melliferu queens, vitellogenin has been reported to first appear in the hemolymph of pharate adults [ll], and in honey bee workers, vitellogenin is already detected in newly emerged individuals [8]. By SDS-PAGE and immunoblotting, we were able to detect a polypeptide, with the same apparent molecular weight as the vitellogenin apoprotein, in the hemolymph of 5th-instar larva, pupa, and pharate adult workers, that cross-reacted with vitellin antiserum. That polypeptide was also present in the hemolymph of male pupae, but it was hardly detectable with Coomassie staining in adult male hemolymph. In A. melliferu, vitellogenin also has been found in males, but only traces of the protein were detected in the hemolymph of insects older than 2 weeks [19].

The influence of isolation from the parent colony on hemolymph proteins and fat body development in workers has not been reported in any other ant


species. The results of the present study demonstrate the existence of dramatic changes occurring in workers removed from the rest of the colony, and thus, from the influence of the queen. Several weeks after adult eclosion, total protein content in the hemolymph of queenless workers was much higher than in queenright individuals of the same age. Additionally, certain proteins, such as vitellogenin, preferentially accumulated with respect to other proteins. Whereas the percentage of vitellogenin in the hemolymph of queenless workers increased from about 3% (day 1 after emergence) to 30% in 7-week-old insects, in queenright workers, vitellogenin percentage in the hemolymph remained unchanged or below the values found in newly emerged adults. An accumulation of vitellogenin in the hemolymph of queenless workers, as compared to queenright individuals, has also been reported in the honey bee A . rnelliferu [8,20].

Total protein concentration in the hemolymph of queenless workers increased about 15-fold in minor workers, and 20-fold in soldiers during the first 7 weeks of adult life, while the concentration of vitellogenin increased up to 100-fold [21], from the mean titer found in newly emerged workers. These results indicate that vitellogenin is regulated independently from other hemolymph proteins.

The accumulation of proteins in the hemolymph and fat body of queenless workers, together with the large increase in the perivisceral fat body mass, indicate that protein synthesis by the fat body is stimulated in workers iso- lated from the colony, and specifically the synthesis of certain proteins including vitellogenin. In queenright workers, particularly the minor workers, we observed the opposite situation. About 6 weeks after adult eclosion, vitellogenin is virtually absent from the hemolymph, and the fat body of those insects has usually regressed and contains low amounts of protein. Additionally, no vitellogenin uptake into the oocytes appears to occur in those individuals [21].

The present results demonstrate that young queenright workers, both soldiers and minor workers, can synthesize vitellogenin. They also indicate that vitellogenin synthesis is inhibited in workers of queenright colonies later in adult life. This is in agreement with some results reported in A . mellifevu, in which vitellogenin synthesis in queenless workers was found to be twice as high as in queenright workers [ll]. The mechanisms of that inhibition, however, remain unknown. Results with the bumble bee, Bombus terrestris, sup- port a role of juvenile hormone in the control of worker oogenesis [1,2], but further research will be required to elucidate the factors involved in the regulation of reproduction in social Hymenoptera.

LITERATURE CITED

1. Roseler P-F: Juvenile hormone control of oogenesis in bumblebee workers, Borrtbus terrestris. J Insect PhysiolZ3,985 (1977).

2. Roseler P-F, Riiseler I: Studies on the regulation of juvenile hormone titre in bumblebee workers, Bombus terrestris. J Insect Physiol24, 707 (1978).

3 . Roseler P-F, Roseler I, van Honk CGJ: Evidence b r inhibition of corpora d a t a activity in workers of Bombus terrestris by a pheromone from the queen’s mandibular glands. Experientia 37, 348 (1981).

4. van Doorn A: Investigations into the regulation of dominance behaviour and of the division of labour in bumblebee colonies (Bombus terrestris). Neth J Zoo1 37,255 (1987).


5. Hildebrandt HH, Kaatz €3-H: Impact of queen pheromone on the physiological status of worker honey bees (Apis melliferu L.). Proc 11th Int Congr IUSSI, India, pp 740,741 (1990).

6. Riiseler P-F, Riiseler I, Strambi A, Augier R Influence of insect hormones on the establish- ment of dominance hierarchies among foundresses of the paper wasp, Polistes gullicus. Behav Ecol Sociobiol75, 133 (1984).

7. Strambi A: Physiology and reproduction in social wasps. In: Social Insects. An Evolutionary Approach to Castes and Reproduction. Engels W, ed. Springer-Verlag, Berlin, pp 59-75 (1990).

8. Engels W: Occurrence and significance of vitellogenins in female castes of social Hymenop- tera. Amer Zoo1 14, 1229 (1974).

9. Harnish DG, White BN: Insect vitellins: Identification, purification, and characterization from eight orders. J Exp Zoo1 220, 1 (1982).

10. Wheeler DE, Kawooya JK: Purification and Characterization of honey bee vitellogenin. Arch Insect Biochem PhysiolZ4, 253 (1990).

11. Kaatz H, Dittrich F: Hormonal and pheromonal regulation of vitellogenin synthesis in honey bees. In: Molecular Insect Science. Hagedorn HH, Hildebrand JG, Kidwell MG, Law JH, eds. Plenum Press, New York, pp 322 (1990).

12. Laemmli U K Cleavage of structural proteins during the assembly of the head of bacterio- phage T4. Nature 227,680 (1970).

13. Boulard C, Lecroisey A: Specific antisera produced by direct immunization with slices of polyacrylamide gel containing small amounts of protein. J Immunol Methods 50,221 (1982).

14. Hatakeyama M, Sawa M, Oishi K: Ovarian development and vitellogenesis in the sawfly, Athuliu rosue ruficornis Jakovlev (Hymenoptera, Tenthredinidae). Invert Reprod Dev 17, 237 (1990).

15. Shapiro JP, Law JH, Wells MA: Lipid transport in insects. Annu Rev Entomol33, 297 (1988). 16. lmboden H, Law JH: Heterogeneity of vitellins and vitellogenins of the tobacco hornworm,

Munducu sextu L. Time course of vitellogenin appearance in the haemolymph of the adult female. Insect Biochem 23, 151 (1983).

17. Bose SG, Raikel AS: Mosquito vitellogenin subunits originate from a common precursor. Biochem Biophys Res Commun 155,436 (1988).

18. Osir EO, Wells MA, Law JH: Studies on vitellogenin from the tobacco hornworm, Manducu sextu. Arch Insect Biochem Physiol3,217 (1986).

19. Trenczek T, Zillikens A, Engels W: Developmental patterns of vitellogenin haemolymph titre and rate of synthesis in adult drone honey bees (Apis melliferd. J Insect Physiol35,475 (1989).

20. Rutz W, Luscher M: The occurrence of vitellogenin in workers and queens of Apis melliferu and the possibility of its transmission to the queen. J Insect Physiol20,897 (1974).

21. Martinez T, Wheeler D: Effect of the queen, brood and worker caste on haemolymph vitellogenin titre in Cumponotusfestiriatus workers. J Insect Physiol, in press (1991).

identification of vitellogenin in the ant, camponotus festinatus: changes in hemolymph proteins and...

Documents