Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias iuna.код для вставкиСкачать
Archives of Insect Biochemistry and Physiofogy 19:203-221 (1992) Selectivity in Storage Hexamerin Clearing Demonstrated With Hemolymph Transfusions Between Hyalophora cecropia and Actias luna Muh-liang Pan and William H. Telfer Department of Zoology, University of Tennessee, Knoxville (Map.);Department of Biology, University of Pennsylvania, Philadelphia (W. H . T.) When Hyalophora cecropia hemolymph was injected into wandering Actias h a larvae, a methionine-rich hexamerin was selectively transferred to the host’s fat body, and completely cleared from the hemolymph by the time of pupal eclosion. Donor arylphorin was30-40% removedfrom the hemolymph, and riboflavin-binding hexamerin was even less completely cleared. During the pupal-adult molt, these rates were reversed: methionine-rich hexamerin disappeared no faster than bovine serum albumin, while riboflavin-binding hexamerin was rapidly and completely cleared from the hemolymph, even though A. luna hernolymph lacks a homologue of this protein; arylphorin, again, was cleared at an intermediate rate. Selective clearing of the three hexamerins occurred at similar stages in H. cecropia, their species of origin. Developmentally programmed clearing, with selectivity at least partially conserved between genera, was also demonstrated with transfused vitellogenin: in A. luna females that were forming yolk, H . cecropia vitellogenin was cleared more rapidly than bovine serum albumin; but in younger females, and in males at all stages of metamorphosis, this M,510,000 molecule was instead an indicator of nonselective, large protein clearing. Nonselective clearing was more complete during adult development than during pupation. It also showed signs of being more effective for small than for large proteins, insensitive to carbohydrate conjugates, and unsaturated at the protein levels used. Key words: arylphorin, methionine-richhexamerin, riboflavin-bindinghexamerin, vitellogenin, fat body, storage protein Acknowledgments: Supported by a grant to W.H.T. from the National Institutesof Health (GM 32909). Received June26,1991; accepted November 12,1991. Address reprint requests to William H. Telfer, Department of Biology, University of Penneylvania, Philadelphia, PA 19104-6018. 0 1992 Wiley-Ltss, Inc. Pan and Telfer 204 INTRODUCTION Molting and metamorphosing insects respond to the nutritional needs of tissue morphogenesis by consuming a reserve of soluble hexamerins' that are deposited in the hemolymph primarily during larval feeding stages. Hexamerin depletion has been documented during larval molts [l-51, as well as during adult development (reviewed in [6-91). During the first metamorphic molt of holometabolous insects, depletion has not been demonstrated, but this may be because it has been obscured by another aspect of hexamerin physiology-a massive transfer by endocytosis from the hemolymph to a secondary storage site, the protein granules of the fat body [lo-131. The goal of the work described here was to distinguish the relative contributions of nonselective and selective processes to hexamerin utilization. There is evidence for both kinds of mechanism. On the one hand, transfer of hexamerins from the hemolymph to the fat body protein granules in pupating holometabola is widely regarded as a selective process: the transfer mechanism is thought to have relatively little effect on other hemolymph proteins, and is synchronized with the presence of an ecdysone-induced, selective binding protein . On the other hand, nonselective processes have been implicated in two ways. Fat body protein granules are in part the products of nonselective uptake, because horseradish peroxidase injected into the hemolymph is an effective marker of the process [ 101. Furthermore, hexamerin utilization during the last nymphal molt of Blattella orientalis has been suggested to entail primarily nonselective clearance, with molecular size being a primary determinant . Recent findings in Hyalqhora cecropia raise the question of selectivity in another context. Many insects produce two hexamerins that differ in composition and developmental profile; in the Lepidoptera these include ArH,+ the principal protein of pupal hernolymph, and MtH, which is stored primarily in the fat body (reviewed in [8,9]). H . cecropia, by contrast, produces four hexamerins, all of which rise to high concentrations in the hemolymph at the initiation of metamorphosis, and disappear shortly before or after adult eclosion. Two of the four have the developmental profile, fat body localization, and amino acid composition of MtH . On the basis of their behavior in native gel electrophoresis, we designate them here as MtH-F and -S~(for fast and slow). The latter has been shown in as yet unpublished sequencing studies (H. Massey, personal communication) to be the more similar of the two to the MtH described for mori [MI; MtH-F, the form whose clearing is studied here, appears to be more similar to a second MtH, whose C-terminal sequence was recently described from Manduca sexta by Corpuz et al. (clone 119 in ). The second extra storage protein in H. cecropia is RbH, a riboflavin-binding hexamerin that is stored primarily in pupal hemolymph [MI. We have been ~~~~~ 'Hexamerin is adopted here in place of the more traditional "storage protein," because it more precisely designates the particular set of storage proteins discussed in this paper. The term was coined by J. Kunkel and previously introduced in references  and . *Abbreviations used: ArH = arylphorin; BSA = bovine serum albumin; C M = carboxymethyl; DEAE = diethylaminoethyl; EDTA = ethyldiaminetetraacetic acid; HSA = human serum albumin; MtH-F and -S = methionine-rich hexamerin-fast and -slow, indicating relative electrophoretic mobilities in native gel electrophoresis; Ovn = ovalbumin; PAGE = polyacrylamide gel electrophoresis; RbH = riboflavin-binding hexamerin; Vg = vitellogenin SelectiveClearing 205 unable to detect significant levels of this protein in the hemolymph of six other species of Saturniids, in B. mori, or in M. sexta . A hexamer that binds riboflavin is now known, however, from Heliothis virescens and Galleria meZZonelZa (R. Miller and D. Silhacek, personal communication), and it may therefore be widely but erratically distributed among lepidoptera. Of particular concern in the present context is the specificity of the transfer of MtH from the hemolymph to the fat body during pupal development and, as shown below, of RbH disappearance from the hemolymph during adult development. We wanted in particular to determine how developingadults of another Saturniid, Actias Zuna, which does not contain detectable amounts of RbH in its hemolymph, deal with this protein after transfusion with H. cecropia hemolymph. To distinguish selective from nonselective clearing, we compared in this study the ability of metamorphosing H . cecropia and A . Zuna to remove a series of endogenous and foreign proteins from their hemolymph. It was possible to identify the metamorphic stages at which selective clearing of ArH, MtH, and RbH occurs, and to demonstrate that each has a unique pattern of clearing. MATERIALS AND METHODS Experimental Insects and Hemolymph Collection Protein clearing from the hemolymph during the larval-pupal molt was analyzed by transfusing H . cecropia female pupal hemolymph into A . luna larvae that had been reared in the laboratory on sweet gum leaves. The larvae were injected 0-12 h before the end of wandering and the initiation of spinning with 0.25 ml of cell-free hemolymph containing 2 mg of BSA. They were bled either at pupal eclosion, or 40 days later, when they had entered diapause. Fat bodies were dissected from these pupae, rinsed several times in physiological saline (40 mM KCl, 15 mM MgC12,5 mM CaC12,llO mM tris-succinate, pH 6.5, and 5 mM phenylthiourea), and homogenized in an equal weight of physiological saline containing two protease inhibitors-5 mM phenylmethylsulfonyl fluoride and 0.5 p,g leupeptin/ml. For studies on pharate adults, field-reared pupae of H . cecropia and A . Zuna were stimulated to terminate diapause by storage for at least 5 months at 6°C. They were then transferred as needed to 25°C and 50%relative humidity, under which conditions the pupal-adult molt is initiated within 2 weeks. Prior to apolysis, they were injected with, in the case of A . Zuna, 0.1 ml of H . cecropia pupal female hemolymph containing 2 mg of BSA and several crystals of phenylthiourea. For studies on nonselective clearing, H . cecropia pupae received 20 mg HSA, BSA, or Ovn dissolved in 0.15 M KC1. Vg was introduced into H . cecropia male pupae by injection with 0.1 ml of female pupal hemolymph containing several crystals of phenylthiourea. A 30 pl sample of hemolymph was drawn from each individual through a wing sac incision 24 h after injection, well before apolysis. A second sample was collected during the pupal-adult molt, which, from apolysis to eclosion, lasts 21 days in H.cecropia and 14 days in A . luna. The second bleeding was either at tarsal claw darkening (day 13 of this molt in H.cecropia and day 9 in A . luna) or several days later than this (day 19 in H . cecropia and day 13 in A . luna). As will be shown below, the later bleeding coincided with the end of the most rapid decline in the concentration of ArH and RbH. 206 Pan and Telfer Protein Measurements Protein concentrations in the hernolymph samples were measured by rocket immunoelectrophoresis [20,21], or by Oudin’s antiserum-agar technique . Rabbit antisera to isolated ArH, RbH, Vg, and MtH-F were utilized for the analysis of H. cecropia proteins; the production and reactions of these antisera, except for anti-MtH-F, were described in an earlier report . MtH-F was isolated from pupal male hernolymph. The high molecular weight protein fractions eluting from a Bio Gel A-1.5 column were pooled and sub- Fig. I . Crossed-rocket irnrnunoelectrophoresis showing the reactions between pupal female hemolyrnph and rabbit antisera against MtH-F and MtH-S that had been separated from each other by native-PAGE, as described in Materials and Methods. Three microliters of a 1:5 dilution of hernolymph were placed in each of the sample wells (indicated by arrows), and electrophoresed toward the right. The separated hemolyrnph proteins were then electrophoresed into agarose containing 2% rabbit antisera against MtH-F (left) or MtH-S (right). In contrast with the three reactions shown by anti-MtH-S, anti-MtH-F formed a single rocket, and could therefore be reliably used to measure hernolymph concentrations of this protein. Selective Clearing 207 jected to ion exchange chromatography (DEAE-agarose at pH 7.2, and ionic strength 32 mM) in order to remove ArH . The DEAE flow-through fractions were then chromatographed on CM-agarose, which, at pH 6.2 and 32 mM ionic strength, retains lipophorin and RbH. The CM flow-through fractions contained a mixture of MtH-F and -S, which were then separated in nativePAGE slab gels (0.3 x 12 x 17 cm, without lane dividers) containing 4.5% acrylamide, 5 mM Tris, 38 mM glycine, pH 8.3. The two bands were visualized by staining for 3 min with 1-anilino-8-naphthalene sulfonate in pH 7.2 phosphate-buffered saline , cut out of the slab, and electroeluted from the gel in dialysis tubing containing 89 mM Tris, 89 mM boric acid, pH 8.3, and 2 mM EDTA. Eluted protein was freed from the side of the dialysis bag by reversing the current for 2 min. Immunization of a rabbit with MtH-F isolated in this manner yielded a monospecific antiserum, which produced a single reaction in crossed-rocket immunoelectrophoresis (Fig. l),as well as in Oudin tubes. Immunization of a second rabbit with the MtH-S band resulted in a weaker and more complex antiserum, which reacted with MtH-F and lipophorin, as well as with MtH-S (Fig. l),and was therefore not useful in these experiments. Antisera to BSA, HSA, and Ovn were purchased from Organon Teknika Corp. (Durham, NC), and were in all cases monospecific, as judged by the single line or zone of precipitation they produced in Ouchterlony plates, Oudin tubes, and immunoelectrophoresis. In the intergenus tests, H. cecropia proteins were measured in A . luna with antibodies that had been rendered species-specificby absorption with A . Zuna pupal hemolymph or fat body extracts . These antibodies continued to produce strong reactions with the corresponding H . cecropia standards and hemolymph, and did not form detectable reactions with A . luna proteins. In cases where isolated proteins were available, protein concentrations were obtained in units of mg/ml by reference to standard curves. ArH, RbH, and Vg were isolated for this purpose from H . cecropia pupal hemolymph by ion exchange chromatography [3,23]. In other cases serial dilutions of pupal hemolymph were used to generate the standard curves; concentrations were then derived in relative units, as percentages of the pupal standards, In either case, the results are presented here as percentage changes, so that units of concentration have been cancelled arithmetically. The amount of each protein circulating in the hemolymph was calculated by multiplying its concentration times the volume of the hemolymph at the relevant stage. Hemolymph volumes were obtained by injecting the insects with 2 mg of BSA in 0.1 m10.15 Kc1 and measuring the concentration of this protein in hemolymph samples taken 6 h later. The volumes are expressed in Table 1 as pep centages of body weight (100% X mug); the experimentalinsects were weighed prior to bleeding, and hemolymph volumes estimated by reference to Table 1. Calculations and Interpretation Protein clearing between the two stages at which the hemolymph was Sampled is expressed as a percent of the initial amount in the hemolymph: (amount in hemolymph at stage 1 - amount at stage 2) x 100%/ (amount at stage 1). The calculation assumes that clearing is not offset by new secretion. This is surely correct for the foreign proteins derived from vertebrates, and for the 208 Pan and Telfer TABLE 1. Hemolymph Volumes of H . cecropia and A. h a " Stage n H. cecropia male Pupa Pharate adult, day 13 Pharate adult, day 19 4 6 H. cecropia female Pupa Pharate adult, day 19 Hemolymphvol t SD (% of body weight) 6 63 t 11 37 -t 4 29 f 4 3 5 59 & 6 26 f 5 A. luna male Pupa, day 1 Pupa, day 40 Pupa, month 7 Pharate adult, day 9 Pharate adult, day 13 * 49 5 51 f 3 55 t 2 25 27 A. luna female Pupa, day 1 Pupa, day 40 Pupa, month 7 Pharate adult, day 9 Pharate adult, day 13 t 2 +5 45 f 1 47 f 2 60 f 7 26 k 3 23 f 4 5 5 4 4 4 *Calculatedfrom the concentration of 2 mg BSA 6 h after injection into the hemolymph. intergenus transfusions. Concerning endogenous hexamerins, however, secretion-replacementcould in principle lead to underestimations. Significant levels of hexamerin synthesis do not generally occur during metamorphosis [24-311, and this is confirmed in H. cecropia by a lack of detectable amino acid incorporation into either ArH or RbH during pharate adult development . The behavior of ArH in transfusion experiments further indicated that secretionreplacement is not significant because, as shown below, during adult development this protein is cleared from the hemolymph at least as effectively in its own species as in a host that cannot synthesize it. BSA, and in some cases HSA, Ovn, and Vg, were used as indicators of nonselective hernolymph clearing. When a hexamerin was cleared significantly more rapidly than BSA ( P < 0.005, as defined in Table 2), we interpreted it as having been selectively cleared. With P > 0,005, selectivity, if present, could not be detected over the background of nonselective processes. TABLE 2. Probability ( P )That the Protein Clearing Values for Developing Adult A. Zunu Shown in Figures 4 and 6 Are Variations of a Mean Clearing Value for BSA' Male Protein ArH, A. luna ArH, H.cecropia MtH-F, H. cecropiu RbH, H . cecropia Vg, H . cecropia Day 9 (0.509) (0.02) (0.207) <0.001* (0.15) Female Day 13 0.001* 0.004' 0.909 0.002* 0.951 Day 9 Day 13 0.299 (0.177) 0.002* (0.148) (0.018) <0.001* (0.002) 0.002* 0.016 0.002* tThe probabilities shown in this table were calculated from a t-test using the Statworks program of Data Metrics, Inc. Parentheses indicate that BSA clearing was greater than that of the test protein. *A probability consistent with the protein's being selectively cleared is indicated. Selective Clearing 209 RESULTS Protein Clearing During the Larval-Pupal Molt in A. Zuna Tojo et al.  found that MtH-F and -S accumulate in the fat body of H . cecropia as their concentrations decline in the hemolymph during pupation. To test the selectivity of this apparent transfer, and also to determine whether molecular recognition signals for transfer are conserved between genera, wandering stage A . Zuna larvae were transfused with H . cecropia proteins and BSA as described in Materials and Methods, and assayed after pupal eclosion. In A . Zuna hosts bled either on the day of eclosion or 40 days later, selective clearing of donor MtH-F was decisively evident. More than 99% of this protein had disappeared from the hemolymph by the day of eclosion in every pupa tested, while ArH, RbH, Vg, and BSA were at the most 37% cleared (Fig. 2, upper panel). The differences between the latter four were too small to appear significant in t-tests. In diapausing individuals bled 40 days after eclosion (Fig. 2, lower panel), the clearing values of proteins other than MtH-F were somewhat lower than in insects bled on day 1, and even included some negative values, which were not seen in any other experiment described below. Negative clearing presumably resulted from dehydration in this set of insects, so that hemolymph volumes were smaller than indicated in Table 1. Complementary to the results on protein clearing from the hemolymph was the finding that preferential amounts of donor MtH-F had accumulated in the fat bodies of the hosts (Fig. 3). Concentration in pupal fat body extracts, expressed as a percentage of concentration in donor hemolymph, was in all cases over 20 times higher for MtH-F than for Vg and BSA, around 10 times higher than for RbH, and nearly three times higher than for ArH ( P < 0.001 in all four pairings with MtH-F). ArH content of these extracts was significantly higher than that of RbH, Vg, or BSA (P < 0.001 in each pairing), but RbH content was not significantly higher than that of Vg or BSA ( P = 0.013 and 0.043, respectively).ArH was therefore selectively accumulated at an intermediatelevel during the molt, while RbH was too low to be judged selective in these tests. The loss of Vg and BSA from the hemolymph (Fig. 2, upper panel) indicated that nonselective clearing occurs during the 1 week larval-pupal molt. The amount was small, however, relative to that reported for injected proteins in the last nymphal molt in B. orientaZis , or to that described below for the pupal-adult molt in A . luna and H . cecropia. Nonselective Clearing During the Pupal-Adult Molt in A. Zuna The clearing of BSA was so pronounced during adult development in A . Zuna that it suggested that nonselective processes would interfere with the detection of selective clearing. From 45-55% of injected BSA disappeared from the hemolymph during the first 9 days of the molt, and over 70% during the first 13 days (Fig. 4).Comparable amounts of H . cecropia Vg were cleared in males, which presumably lack a selective mechanism for endocytosing this protein. Selective Clearing of Vg in A. Zuna Donor Vg disappeared from the hemolymph of females faster than BSA (Fig. 4), and in t-tests this difference appeared significant, particularly by day 13 (Table 2). That A . Zuna ovaries are able to concentrate H . cecropia Vg was con- 21 0 Pan and Telfer n W ArH a MtH-F w3H er W -I 0 0 8 vg BSA PUPAL HEMOLYMPH Fig. 2. Clearing of H. cecropia hemolymph proteins and BSAfrom the hemolymph of A. luna during the larval-pupal molt. Larvae within 12 h of the end of wandering were injected with 0.25 ml pupal female hemolymph containing 2 mg BSA and bled either on the day of pupal ecdysis (upper panel) or 40 days later (lower panel). Error bars, in this and subsequent figures, are standard deviations; they are not shown for MtH-F because this hexarnerin was no longer detectable at the time of bleeding. Results are from 7 males and I1 females on day 1, and from 3 males and 4 females on day40. firmed by testing for the latter in solubilized yolk from mature eggs that had been dissected from day 13 hosts . These preparations contained substantial concentrations of donor Vg-about six times the concentration of donor Vg in the hemolymph of day 9 females, and around 200 times that in day 13 females. Lipophorin, a second yolk precursor, also exhibited nonselective lev- Selective Clearing 21 1 18 15 12 9 6 ArH 3 0 a MtH-F I T MALE w a va DAY 40 BSA FEMALE PUPAL FAT BODY EXTRACT Fig. 3. Concentrations of H. cecropia hemolymph proteins and BSA in extracts of fat body taken from the A. luna hosts described in Figure 2. Protein concentrations in the extracts are expressed as a percent of their concentrations in the pupal hemolymph used for injection; n = 6 for both males and females on day 1 and 3 for both males and females on day 40. els of clearing when injected into A . 2una males and, as would be expected, a low level of selective clearing in females (results not shown). For the present study, the significance of these results is their demonstration that selective clearing, if it occurs on a large enough scale, can be successfully detected against the high level of nonselective clearing that occurs in developing adults. Hexamerin Clearing During the Pupal-Adult Molt in A. luna During the pupal-adult molt, hexamerin clearing occurred with a new and different set of selectivities. In place of MtH-F, RbH was now the most rapidly cleared of the transfused H . cecropia proteins (Fig. 4). In the first 9 days of the molt 93%of this protein disappeared from the hemolymph of males, and 88% from that of females; it was undetectable, or reduced to trace levels in all individuals of both sexes by day 13. The differences between RbH and BSA clear- 21 2 Pan and Telfer T MALE 40 n W K ArH 20 a MtH-F w 6 w vs 100 0SA 8 40 20 PUPAL-DAY9 PUPAL-DAY13 DAYS-DAY1 3 PERIOD OF CLEARING Fig. 4. Clearing of injected proteins from A. luna hemolymph during the pupal-adult molt. Chilled pupae were injected with 0.1 ml H. cecropia female pupal hernolymph containing 2 mg of BSA, and bled on either days 9 or 13 of the molt. Donor proteins were assayed with H. cecropia antibodies absorbed with A. luna pupal hemolymph. Day 9-13 values were calculated from the average concentrations on days 9 and 13. For males, n = 5 on day 9 and 3 on day 13; for females, n = 4 on day9 and 3 on day 13. ing were highly significant at day 9 (P < 0.001 in both sexes, Table 2), and only slightly less so on day 13 ( P = 0.002), when BSA clearing had become more complete. In view of the absence of an endogenous RbH from A . luna hemolymph, this evidence of a robust selective clearing was surprising. Closer timing of RbH clearing was sought by sampling the hemolymph of five injected males periodically during early and mid-adult development. In all five insects the concentration of RbH began to drop rapidly on the fourth day of the molt (Fig. 5); by day 10 it had disappeared entirely from the hernolymph of three individuals, and was falling toward this level in the other two. An additional experiment indicated that the ability to effect a rapid rate of Selective Clearing 21 3 140 120 100 80 60 40 20 0 0 2 4 6 8 10 12 14 ADULT DEVELOPMENT, DAY Fig. 5. Time-course of the concentration changes of H . cecropia RbH in the hemolymph of five developing adult A. luna males. TC, tarsal claw darkening; E, eclosion. Comparisons of pupal and pharate adult hemolymph volumes (Table 1) suggest that the rise in concentration during the first few days of the molt is due to blood volume reduction. RbH clearing extends into late development. Three A . luna males were injected with 0.1 ml H . cecropia pupal female hemolymph on day 8 of the pupal-adult molt in this experiment. Five days later, an average of 99.9% of the injected RbH had disappeared from the hernolymph, compared with 78% of donor ArH and only 15%of donor Vg. ArH transfused into A . luna pupae was less readily cleared; in females, only 66% had disappeared from the hemolymph by day 9, well under the values for BSA (Fig. 4). In males, however, selectivity compared with BSA clearing was detectable on day 13 ( P = 0.004, Table 2). Finally, MtH-F, the most completely cleared of the three hexamerins during the larval-pupal molt, was the least effectively cleared during the pupal-adult molt (Fig. 4). In neither sex was the percentage cleared significantly different from than that of BSA (Table 2). The Clearing of Endogenous ArH in A. Zuna In A . luna, endogenous ArH was more effectively cleared than that transfused from H . cecropia. At tarsal claw darkening, 51% of endogenous pupal ArH had been cleared from the hemolymph in males, and 62% in females (Fig. 6), compared with the figures of 34% and 37% for transfused ArH (Fig. 4) ( P = 0.040 and 0.048 for males and females, respectively). While not highly significant, the differences are consistent enough to suggest a genus-level difference between the receptor recognition features of this protein. By day 13, nearly all endogenous ArH had disappeared from the hemolymph (Fig. 6, Table 2). The clearing of endogenous MtH-F during adult development was not examined, because the concentration of this protein in pupal hemolymph was already extremely low. The concentration of endogenous MtH-F in A . luna pupae was 214 Pan and Telfer 1 loo ao MALE FEMALE 60 40 20 0 P-DAY9 P-DAY 13 DAYS-DAY1 3 PERIOD OF CLEARING Fig. 6. Clearing of endogenous ArH from the hemolymph of pharate adult A. h a . On both days 9 and 13, n = 4 for males and 3 for females. less than 1%of its concentration in the H.cecropia hemolymph that had been used for transfusion. Endogenous Hexamerin Clearing in H.cecropia In W.cecropia the concentrations of endogenous ArH and RbH declined in synchrony with each other from the high levels characteristic of pupal hemolymph to nearly zero in the adult (Fig. 7). Measurements of clearing in males indicated that 64% of ArH and 54% of RbH disappeared from the hemolymph during the pupa to day 13 interval, and over 99% of both hexamerins had disappeared by day 19 (Fig. 8). BSA was cleared at a similar rate during the first 13 days of the molt, but lagged significantly behind hexamerin clearing during the day 13-19 period, so that selective clearing of the two hexamerins now becomes evident (P < 0.001, Table 3). When comparison was made with Vg rather than with BSA, by contrast, selectivity of endogenous hexamerin clearing became apparent early in the molt. Only 14%of injected Vg had been cleared by day 13 (Fig. 8), and this was substantially less than the percent clearing figures for the two hexamerins (P < 0.001 in both cases, Table 3). Since Vg and the hexarnerins have similar molecular weights, a recognition signal other than size must account for the difference. Characterization of Nonselective Clearing in H . cecropia Males Molecular size-dependence of nonselective clearing was suggested by the finding that only 14% of injected Vg was cleared between the pupal and day 13 stages, relative to 62% for BSA (P = 0.004). HSA and Ovn, two other proteins in the size range of BSA, showed an even greater difference (Fig. 8; P < 0.001). Vg clearing continued to be significantly less than that of the three vertebrate proteins between days 13 and 19. Selective Clearing "" 215 I ArH I A 60 0 40 20 0 RbH 4 w c E 0 I U n -1 1 3 5 7 9 11 13 15 17 19 21 23 ADULT DEVELOPMENT, DAY Fig. 7. Time-course of the changes in concentration of ArH and RbH in the hemolymph of developing adult H. cecropia. Except where they overlap, the points represent concentrations in single individuals.TC, tarsal claw darkening; E, eclosion. The very different carbohydrate conjugate compositions of Ovn and HSA had no detectable effect (Fig. 8, Table 3). An indication of whether nonselective clearing is saturated at the protein levels used was investigated by reducing the Ovn dose from 20 to 2 mg per pupa. The percentage of injected Ovn removed from the hernolymph was the same for the two doses. Comparison between two doses of BSA during adult development in A. luna females yielded similar results (results not shown), Finally, greater amounts of HSA than BSA were consistently cleared from the hemolymph (Fig. 8), despite the fact that these are presumably very similar molecules. We have no explanation of the difference. 21 6 Pan and Telfer 100 ArH n 8o 2 60 LLI RbH rl vs W BSA A 0 $ 40 HSA 20 ~vn-20 ~vn-2 0 PUPAL-DAY13 PUPAL-DAY19 DAY1 3-DAY19 PERIOD OF CLEARING Fig. 8. Clearing of proteins from the hemolymph during the pupal-adult molt of H. cecropia males. ArH and RbH are endogenous hexamerins; the clearing for these can be considered minimum values, because some replacement by secretion is not entirely ruled out. Vg was injected into male pupae as 0.1 ml female pupal hemolymph. BSA, HSA, and Ovn-20 were injected into pupae with a dose of 20 mg in 0.1 mlO.15 M KCI;Ovn-2 designates injection with l/lOth this dose. Day 13-19 values were calculated from the average concentrations on days 13 and 19. On day 13, n = 16 (ArH, RbH), 3 (BSA, VG, Ovn), and 5 (HSA). On day 19, n = 18 (ArH, RbH), 5 (Vg, HSA, Ovn-2),3 (BSA), and 6 (Ovn-20). DISCUSSION The order of effectiveness of donor hexamerin clearing during adult development (RbH>ArH>MtH) was the opposite from that observed during pupation (MtH>ArH>RbH). This contrast can only be explained by changes in a set of mechanisms that can distinguish between the three hexamerins. Despite their common structural features, the different hexamerins of H . cecropia show very little antigenic cross reaction [12,20], an indication of extensive differences between the molecular configurations of their surfaces. Among these differences are features allowing their individual recognition by the cells that dispose of them. It is noteworthy that the clearing of donor MtH during pupation, and RbH during adult development, are both sufficiently robust to make them more conspicuous against the background of nonselective clearing than the effects of yolk formation on the clearing of Vg. Following the models of vitellogenesis [33,34] and of fat body protein granule formation [10,13,14], we suppose that the cells responsible for selective clearing of the hexamerins during adult development carry appropriate receptors on their surfaces, and internalize the bound proteins by endocytosis. The model has the added advantage of explaining the clearing of BSA, HSA, and Ovn,for solute diffusion into the fluid-filled cavities of endocytoticinpocketings [20,35], and nonspecific adsorption to endocytotic cell surfaces, are plausible explanations of nonselective clearing. Most insect cells are to some degree endocytotic. Locke and Collins  Selective Clearing 21 7 TABLE 3. Significanceof the Differences Between Average Clearing Values of Proteins During Adult Development in H. cecropia Males libH Pupal stage to day 13 ArH .06 WH Ovn Ovn Vg BSA HSA (20 mg) (2 mg) C.001 .8 <.001 .33 ,004 ,04 <.001 <.001 .006 <.001 .048 .001 <.001 .006 .001 .067 .189 .525 <.001 <.001 <.001 <.001 <.001 c.001 .001 <.001 .001 002 <.001 ,001 ,566 ,039 "g BSA HSA Ovn (20 mg) Pupal stage to day 19 ArH 0.18 RbH vg BSA HSA Ovn (20 mg) .01 c.001 <.001 <.001 <.001 .139 * .025 .03 showed that horseradish peroxidase injected into larvae of Culpodes ethlius was incorporated into multivesicular bodies by endocytotic routes in a wide variety of tissues. And Tobe and Loughton  found that labeled hemolymph proteins injected into 5th instar nymphs of Locustu migrutovia were detectable by autoradiography in every tissue examined. Three insect tissues, however, are especially differentiated for rapid endocytosis. In addition to vitellogenic oocytes and fat body cells, each of which passes through a special phase of endocytosis during its differentiation, pericardial cells are highly active endocytotic cells throughout much of the insect's life [15,38-401. They have been presumed to be important primarily in nonselective clearing of hemolymph proteins; whether they also engage in selective endocytosis has not been shown. The clearing of MtH from the hemolymph and its simultaneous accumulation in the fat body during the larval-pupal molt has been described in several other famiIies of lepidoptera [5,41,42]. The experiments described here confirm that fat body endocytosis entails a high degree of selectivity. The switch to selective RbH clearing seen during adult development requires either that a tissue other than the fat body initiate endocytotic activity, or that fat body cells expose to the hemolymph a new set of membrane receptors, The experiments also demonstrate that the molecular features underlying selectivity of MtH, RbH, and Vg clearing are, to an experimentally useful degree, shared by Hyufophoru and Actius. Cross reactions in adsorptive endocytosis were earlier seen in the uptake of H.cecropia Vg by Antherueu polyphernus ovaries . On the other hand, endogenous ArH tended to be more effectively cleared in both H.cecropia and A . lunu than in transfused hosts, suggesting that binding specificities, though similar, have evolved to a detectable degree within the Saturniidae. Concerning the fates of the hexamerins that disappear from the hemolymph 218 Pan and Telfer during adult development, the best information now available comes from experiments with Diptera. Hydrolysis to the level of free amino acids, which are then reincorporated into adult tissue proteins, was demonstrated in Calliphora vicinae by injecting labeled calliphorin, the major hexamerin of most Diptera, into mature larvae [MI. Label stayed primarily in intact calliphorin during pupation, but during the pupal-adult molt, when hexamers disappear from the insect, it was redistributed to newly synthesized proteins in a wide variety of tissues. In addition, some calliphorin originating from the hemolymph becomes localized without degradation in cuticle, apparently by selectivetransfer across the epidermis . BSA and Vg were relatively unaffected by nonselective clearing during the larval-pupal molt, but during the pupal-adult molt they, in addition to HSA and Ovn, were 70-95% cleared from the hemolymph. In W.cecropia males, Vg was much more resistant to clearing than BSA, HSA, and Ovn, suggesting that high molecular weight confers protection, as it does in B. orientalis nymphs . There was a tendency for this protection to disappear between days 13 and 19, as it does during molting in B. orientalis. Either a hypothetical filter separating the hemolymph from endocytotic cells becomes more permeable at this time, or a new set of cells that are not so tightly screened from the hemolymph initiates endocytotic activity. Two additional features of the foreign protein clearing mechanism are its poor ability to discriminate between HSA and Ovn, and its proportionate response to a 10-fold difference in the amount of Ovn and BSA injected. Clearing by fluid phase endocytosiswould exhibit exactly these traits. While adsorptive endocytosis is not ruled out, it would require binding sites that do not discriminate between heavily glycosylated and nonglycosylated proteins, and that show no indication of saturability by the injected foreign protein. These are preliminary conclusions that will require exploring with an expanded panel of foreign proteins. The most surprising result of the transfusion experiments was the more rapid clearing of RbH in pharate adults of A . luna, which lacks an endogenous form of this protein in its hernolymph, than of H. cecropia, which possesses it. The occurrence of RbH in G. mellonella, H . 'uirescens, and H . cecropia suggests a wide distribution of this storage hexamer among moths, but its absence from many other species suggests that its hernolymph storage function is readily lost during evolution. A . Zuna's efficient clearing mechanism may therefore be a physiological relic inherited from an ancestor that had not yet lost RbH from its hemolymph. Questions such as these emphasize the great potential of hexamerin studies for generating new insights into the role of hemolymph proteins in insect metamorphosis. LITERATURE CITED 1. Duhamel R, Kunkel J: A molting rhythm for serum proteins of the cockroach, Blutta orientalis. Comp Biochem Physiol60B, 333 (1978). 2. Kramer S, Mundall E, Law J: Purification and properties of manducin, an amino acid stor age protein of the haemolymph of larval and pupal Munducu sexta. Insect Biochem 10, 279 (1980). Selective Clearing 219 3. Telfer W, Keim P, Law J: Arylphorin, a new protein from Hyalaphoru cecropia: comparisons with calliphorin and manducin. Insect Biochem 13,601 (1983). 4. Karpells S, Leonard D, Kunkel J: Cyclic fluctuations in arylphorin, the principal serum stor age protein of Lymantria dispur, indicate multiple roles in development. Insect Biochem 20, 73 (1989). 5. Bean D, Silhacek D: Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth, Galleria mellonella. Arch Insect Biochem Physiol 10, 333 (1989). 6. Roberts D, Brock H: The major serum proteins of Dipteran larvae. Experientia 37,103 (1981). 7. Scheller K: The Larval Serum Proteins of Insects. Thieme-Stratton, New York, 190 pp. (1983). 8. Kanost M, Kawooya J, Law J, Ryan R, Van Heusden M, Ziegler R Insect Hemolymph Proteins. Adv Insect Physiol22,299 (1990). 9. Telfer W, Kunkle J: The function and evolution of insect storage hexamers. Annu Rev Entomol 36, 205 (1991). 10. Locke M, Collins J: The structure and formation of protein granules in the fat body of an insect. J Cell BiolZ6, 857 (1965). 11. Chippendale C , Kilbi B: Relationship between the proteins of the haemolymph and fat body during development of Pieris brassicue. J Insect Physiol15,905 (1969). 12. Tojo S, Betchaku T, Ziccardi V, Wyatt G: Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia. J Cell Biol78,823 (1978). 13. Locke J, McDermid H, Brac T, Atkinson €3: Developmental changes in the synthesis of haemolymph polypeptides and their sequestration by the prepupal fat body in Callpodes ethlius Stoll (Lepidoptera: Hesperiidae). Insect Biochem 12,431 (1982). 14. Natori S: Structure of storage protein receptor in Sarcophuga. Biol Chem Hoppe-Seyler 368, 572 (1987). 15. Duhamel R, Kunkel J: Molting cycle regulation of hemolymph protein clearance in cockroaches: possible size-dependent mechanism. J Insect Physiol33,155 (1987). 16. Sakurai H, Fujii T, Izumi S, Tomino S: Structure and expression of gene coding for sex-specific storage protein of Bombyx mori. J Biol Chem 263,7876 (1988). 17. Corpuz L, Choi H, Muthukrishnan S, Krarner K Sequences of two cDNAs and expression of the genes encoding methionine-rich storage proteins of Manducu sexta. Insect Biochem 21,265 (1991). 18. Telfer W, Massey H: A storage hexamer from Hyulophora that binds riboflavin and resembles the apoprotein of hemocyanin. In: Molecular Entomology. Law J, ed. Alan R Liss, New York, p. 305-314 (1987). 19. Telfer W, Canaday D: Storage proteins of saturniid moths: interspecific similarities and contrasts. Biol Chem Hoppe-Seyler 368,571 (1987). 20. Telfer W, Pan M: Adsorptive endocytosis of vitellogenin, lipophorin, and microvitellogenin during yolk formation in Hyalopkoru. Arch Insect Biochem Physiol9,339 (1988). 220 Pan and Telfer 21. Pan M, Telfer W The use of rocket immunoelectrophoresis in the study of insect haemolymph proteins. Bull Inst Zoo1 Academia Sinica 15,109 (1990). 22. Hartman B, Udenfriend S: A method for immediate visualization of proteins in acrylamide gels and its use for preparation of antibodies to enzymes. Anal Biochem 30,391 (1969). 23. Kulakosky P, Telfer W: Selective endocytosis, in vitro, by ovarian follicle from Hyulophoru cecropia. Insect Biochem 17,845 (1987). 24. Izumi S, Tojo S, Tomino S: Translation of fat body mRNA from the silkworm, Bornbyx rnori. Insect Biochem 10,429 (1980). 25. Lepesant J, Levine M, Garen A, Lepesant-KejzlarvoaJ, Rat L, Somme-Martin G: Developmentally regulated gene expression in Drosophilu larval fat bodies. J Mol Appl Gen 1, 371 (1982). 26. Mintzas A, Chrysanthis G, Christodoulou C, Marmaras V: Translation of the mRNAs coding for the haemolymph proteins of Cerutitis cupitutu in a cell-free system. Comparison of the translatable mRNA levels to the respective biosynthetic levels of the protein in the fat body during development. Dev Biol95,492 (1983). 27. Sat0 J, Roberts D: Synthesis of larval serum proteins 1 and 2 of Drosophilu melunoguster by third instar fat body. Insect Biochem 23, 1 (1983). 28. Schenkel H, SchellerK Stage- and tissue-specificexpression of the genes encoding calliphorin, the major larval serum protein of Culliphoru vicinu. Wilhelm Roux's Arch Dev Biol 195, 290 (1986). 29. Sekeris C, Scheller K: Calliphorin, a major protein of the blowfly: correlation between the amount of protein, its biosynthesis, and the titer of translatable calliphorin-mRNA during development. Dev Biol59,lZ (1977). 30. Tamura H, Tahara T, Kuroiwa S, Obinata M, Natori S: Differential expression of two abundant messenger RNAs during development of Surcophugu peregrinu. Dev Biol99, 145 (1983). 31. Webb B, Riddiford L: Synthesis of two storage proteins during larval development of the tobacco hornworm, Marzducu sextu. Dev Biol230,671 (1988). 32. Kulakosky P, Telfer W: Lipophorin as a yolk precursor in Hyulophoru cecropia: uptake kinetics and competition with vitellogenin. Arch Insect Biochem Physiol24,269 (1990). 33. Telfer W, Heubner E, Smith D: The cell biology of vitellogenic follicles in Hyulophoru and Rhodnius. In: Insect Ultrastructure. King R, Akai H, eds. Plenum Press, New York, Vol. 1, pp 118-149 (1982). 34. Raikhel A, Dhadialla T Accumulation of yolk proteins in insect oocytes. Annu Rev Entomol 36,217 (1992). 35. Kulakosky P, Telfer W: Kinetics of yolk precursor uptake in Hyulophoru cecropia: stimulation of microvitellin endocytosis by vitellogenin. Insect Biochem 19,367 (1990). 36. Locke M, Collins J:Protein uptake in multivesicular bodies in the molt-intermolt cycle of an insect. Science 155,467 (1967). 37. Tobe S, Loughton 8: An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust. J Insect Physiol25, 1331 (1969). Selective Clearing 221 38. Bowers B: Coated vesicles in the pericardial cells of the aphid (Myzus persicae Sulz). Protoplasma 59,351 (1964). 39. Crossley A: The ultrastructure and function of pericardial cells and other necrophyles in Calliphoru erythrocephala. Tissue Cell 4,529 (1972). 40. Gochoco C, Kunkel J, Nordin J: Experimental modifications of an insect vitellin affect its structure and uptake by oocytes. Arch Insect Biochem Physiol8, 179 (1988). 41. Tojo S, Nagata M, Jbbayashi M Storage proteins in the silkworm, Bornbyx rnori. Insect Biochem 10,289 (1980). 42. Ryan R, Keirn P, Wells M, Law J: Purification and properties of a predominantly femalespecific protein from the hemolymph of the larva of the tobacco hornworm, Manduca sexta. J Biol Chem 260,782 (1985). 43. Telfer W: The selective accumulation of blood proteins by the growing oocytes of saturniid moths. Biol Bull 118,338 (1960). 44. Levenbook L, Bauer A: The fate of larval storage proteins during adult development of Calliphoru vicina. Insect Biochem 14,77 (1984). 45. Konig M, Agrawal 0, Schenkel H, Scheller K Incorporation of calliphorin into the cuticle of the developing blowfly, Calliphoru vicina. Wilhelm Roux’s Arch Dev Biol195,296 (1986).