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Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias iuna.

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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 [14].
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 [15].
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 [12]. 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 [17]).
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 [4] and [8].
*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 [19]. 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 [3].
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 [20].
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 [3]. 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 [22], 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 [21]. 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 [32].
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. [12] 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 [15], 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 [20]. 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 [36]
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 [37] 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 [43]. 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 [45].
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
[15]. 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.
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