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Common electrophoretic properties of the fat body, haemolymph, and oöcytes of adult Tenebrio molitor
J. InsectPhyriol., 1970, Vol. 16,pp. 14.43to 1453. Pergamon Press. Ptinted in Great Britain
COMMON ELECTROPHORETIC PROPERTIES OF THE FAT BODY, HAEMOLYMPH, AND OijCYTES OF ADULT TENEBRIO MOLITOR* S. M. PEMRICKt
and A. BUT2
Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221 (Received 12June
1969; mvised 27 September 1%9)
Aktrnct-Electropherograms of the haemolymph indicated the presence, at a pH of 9.8, of eighteen negatively charged proteins of which at least five (bands Nos. 4, 13, 14, 15 and 16 from the origin) were common to the fat body and oticytes. The distribution of the per cent composition of the five bands varied between unmated and mated females in the three tissues over the 4 days studied (days 3,7,8, and 10 after emergence). C ircumstantial evidence suggested a threestep process of fat body protein synthesis followed by protein release into the haemolymph and its subsequent uptake by the o&ytea. Band No. 13 had the highest average per cent composition for both mated and unmated fanales in all three tiesues reaching a maximum, on day 7 after emergence, of 56 (unmated females) or 58 (mated females) per cent of the total protein concentration in the o&ytes. Band No. 4, however, held the higheat per cent composition of the five measured haemolymph protein bands on day 3 after emergence for both mated and unmated females, and on days 8 and 10 after emergence for umnated females when in the fat body it was also tied with band No. 13 for highest per cent composition. On day 3 after emergence fat body band No. 16, in mated females, represented the highest per cent composition of total protein and waa four times higher than in unmated femalea of the same age, and similarly in the ’ okytcs, on day 3, band No. 16 was 34 per cent higher for mated than unmated females. Spectrophotometric analyses of fluctuations in the haemolymph protein concentration indicated a similar pattern for unmated and mated females from emergence to day 10 after emergence, the haemolymph protein concentration being &niflcantly greater (P< O-05) for mated females on days 4 and 9 after emergence (O-05 > P > 0*025). The mean value on day 9 was approximately 60 per cent higher for mated than unmated females. A possible relationship between the cyclic pattern of haemolymph protein concentration and the patterns of fat body protein and RNA synthesis is discussed. INTRODUCTION
adult fat body of the mealworm Tenebnh moldor is supplying a certain percentage of the necessary proteins for growth and development, there should be IF THE
* This research was supported in part by an American Cancer Society Institutional Grant. 7 Present address: Department of Biological Sciences, State University of New York at Albany, Albany, New York 12203. 1443
1444
S
M.
PEMRICK AND
A. Bun
a similarity between the protein fractions in the fat body, haemolymph, and target organ (ovaries). Such a relationship, however, would be only an indication that a pathway of protein synthesis, release, and utilization existed, for it is quite possible that different tissues, in the same hormonal environment, might,direct the synthesis of similar proteins. Sections of this pathway (fat body to haemolymph, or haemolymph to oiicytes) have been substantiated in studies of both juvenile and adult stages. When SHIGMATSU(1958) noted the protein synthetic capabilities of the larval fat body in the silkworm Bombyx wi, he also reported that this protein, which was released from the fat body during incubation procedures, was similar to the globular components of the haemolymph. Similarly, LAUFER(1960) concluded through starch gel electrophoretic studies that the fat body of the giant silk moths H’ulophora cecropia and Sam&a cynthiu was the source of several of the proteins of the blood, and PRICE and BOSMAN(1966) separated by acrylamide gel electrophoresis the haemolymph proteins of the blowfly Calliphora erythrocephaku and found them to be similar to those released from the fat body under in v&o conditions. In addition, NIELSON and MILLS (1968) compared the electrophoretic mobility of the haemolymph and o&yte proteins during yolk formation in the American cockroach Per@neta amerbna and reported the protein patterns in both tissues were not only similar, but also cyclic in nature, thereby suggesting that the haemolymph proteins were involved in ovarian development. Earlier HILL (1962) had reported the presence of a haemolymph protein fraction in the desert locust Schistocet-ca gregaria which increased during yolk deposition and after ovariectomy. Evidence for a sex difference in haemolymph protein patterns comes from a variety of sources. AJXYODI(1967) cited evidence for a specific female protein in the haemolymph of the viviparous cockroach iVuu#zoeta cinerea but was not able, however, to equate this with any of the fat body or ovarian proteins. THOMASand NATION (1966) also reported a sex-linked protein in the haemolymph of the American cockroach Pe@aneta amekuna. However, CHIPPENDALEand BECK (1967) disclosed that in pharate pupae of the European corn borer Ostrikia nubiZuZis a specific protein accumulated in the fat body of females at an earlier age and to a higher concentration than in the fat body of males, and they further suggested that subsequently this protein was transported to the ovaries where it comprised the major protein fraction. Immunoelectrophoretic techniques provided more exact evidence concerning the similarity of various protein fractions of the fat body, haemolymph, and oocytes. TELFER (1954) demonstrated immunologically identical proteins in the haemolymph and oiicytes of the silkworm Hyalophoru and later concluded that every haemolymph protein has an antigenic counterpart in the egg (TELFER, 1965). Recently COLES (1965) revealed through fluorescent immunoelectrophoretic studies two protein bands in Rhodnius eggs which shared identical mobility patterns with two specific adult bands in the haemolymph. Previous studies (PEMRICK and BUTZ, 1970a, b) have indicated that in the mealworm Tenebrio moZitor the fat body is metabolically active during the adult
PROPERTIES OF FAT BODY,
HAEMOLYMPH,
AND 06)cITEs
OF TENEBRIO MOLITOR
1445
stage, synthesizing RNA and protein in a cyclic pattern which appears to be related to ovarian development. It is the purpose of the present investigation to link the protein synthetic cycles of the fat body with a plausible pathway of protein release and utilization. MATERIALS
AND METHODS
Haemolymph protkn concentration Maintenance of cultures was described previously (PEMRICKand BUTZ, 1970a, b). Haemolymph samples were withdrawn from mated and unmated females at emergence and every day thereafter for 10 days. The haemolymph was collected by removing the forelegs and taking the haemolymph up into a 20 ~1 calibrated micropipette as described by MORDUE(1965b). Samples of 5 d per insect could usually be obtained in this manner. The blood was then transferred to 3.0 ml of ice-cold 5% trichloracetic acid (TCA) and centrifuged for 10 min (5000 g). The precipitate was washed (3~0 ml 5% TCA) and centrifuged for 5 min (2500g) at which time the supematant was discarded. The protein concentration was then determined from the final precipitate by the method of LOWRY et al. (1951). The optical density was recorded at 750 rryl against a water blank. Electrophoresis: Preparation of samples Samples of haemolymph (5 ~1 per insect), fat body, and oiicytes were removed from mated and unmated females at representative days after emergence. The first oiicytes were noticed around day 3. Four 4 samples of haemolymph were applied directly to the cellulose acetate membranes. Fat body tissue (four pooled samples per day studied; 3 insects per sample) was homogenized in a cold ground-glass homogenizer containing 0.5 ml of cold saline (BUTZ, 1957), centrifuged, and 4 ~1 of the supematant became the tissue extract. Oticytes (four pooled samples per day studied: 3 insects per sample) were also homogenized in cold saline and centrifuged. The clear zone of the supematant (5 ~1) was applied to the acetate strips. Control samples of &oracic muscle (pooled samples) were homogenized in cold saline and centrifuged, and 5 ~1 of the supematant became the tissue extract. Electrophoresis : procedure The samples (4 to 5 ~1 of the pooled extracts) were applied to the centre of the cellulose acetate membranes (Gelman Sepraphore, III) which had been soaked in the chamber in Gelman High Resolution Buffer (pH 9.8), and blotted lightly. The unit was then allowed to equilibrate for 10 min at which time the power supply was set at 300 V for 45 min. The membranes were then stained in Ponceau S (0.5 per cent in TC_4), washed several times (5% acetic acid) and, for quantitative measurements, the membranes were cleared (acetic acid : methanol) and scanned. In order to express in tabular form the percentage composition of a given fraction
14%
S.
M.
to the total
protein, a planimeter from the scanner.
bMRICK
AND
A. Bun
was employed to integrate the curves obtained
RESULTS
Ekctmphoretic analyses of the proteins of the haemoiym$h, fat body, and eggs Analyses of electropherograms from the haemolymph indicated the presence of eighteen negatively charged proteins (pH 98), which were numbered 1 to 18 from the origin. There did not appear to be a specific sex protein in Tenebrio, since the electrophoretic patterns of males and females were similar. Of the eighteen negatively charged proteins in the haemolymph at least five (band Nos. 4, 13, 14, 15, and 16 from the origin) were common to the fat body and eggs. It is quite possible that other proteins were common to the three tissues but in amounts too small to quantitate with any degree of accuracy. Control electropherograms of homogenized extracts of thoracic muscle failed to yield proteins with a mobility similar to band Nos. 4, 13, 14, 15, and 16. Therefore, the average per cent composition of these five bands was recorded in the fat body, haemolymph, and Mcytes for both mated and unmated females on days 3 (Table l), 7 (Table Z), 8 (Table 3), and 10 (Table 4) after emergence. Band No. 13 maintained the highest average per cent composition of total protein for both mated and unmated females in all three tissues. Day three afier emergence The pattern of per cent composition of the five bands varied between mated and unmated females in the fat body and oiicytes but was similar in the haemolymph (Table 1). Fat body band No. 16 in mated females represented the highest per cent composition of total protein and was four times higher than in unmated females. T-LB
I-DAY
Fat body
.I
Unmated md No
I
Mated
3 A~~BREMEKGKNCE
Haemolymph
I
Unmated
I
Mated
Okyter Unmated
I
Mated
PROPERTIES OF FAT BODY, HAEMOLYhXPH, AND 06CYTES OF TENEBBIO
MQLZTOR
1447
Similarly oGcyte band No. 16 was nearly 34 per cent higher for mated than unmated females. Band No. 4 in the haemolymph held the highest per cent composition of total protein for both mated and unmated females and, although in the o&y&s band No. 4 dropped behind band No. 13, it comprised 15 per cent of the total protein compared to a low level of 6 (unmated) or 7 (mated) per cent of the total protein in the fat body. Band No. 13 for both mated and unmated females accounted for a higher percentage of the total protein in the fat body than in either the haemolymph or okytes. Day seven after emergence Band No. 13 (Table 2) attained its peak concentration of 56 (unmated) and 58 (mated) per cent of the total protein in the ticytes, while maintaining relatively high concentrations in the fat body and haemolymph. Although the per cent TABLE~-DAY 7 MWEREMERGENCE Fat
body Mated
Hoemalymph Unmated
Mated
Oikytes Unmoted
Mated
composition of band No. 4 varied considerably between the fat body and oiicytes, the value for unmated females was 40 (o&y@) to 60 (fat body) per cent higher than for mated females. However, the per cent composition of band No. 14, although similar for both the fat body and o&ytes, was also higher for unmated than mated females, the value for unmated females being twice- as high as for mated females.
S. M. PSMRICK ANDA. Bun
1448
Fat body band No. 16 was 35 per cent higher for mated than unmated females; however, okyte band No. 16 was nearly 2.5 times higher for unmated than mated females. Day kght after emergence Band No. 13 represented a much lower per cent of the total protein in the fat body, haemolymph, and oijcytes than was recorded on day 7 (Table 3). However, as noted on day 7, band No. 13 comprised a higher percentage of the total protein TABLE Fot body MOtOd
~-DAY
I
8m
BMBRGENCE
Hoemolymph
T-
1Unmated
Mated
OOcytes
Jnmoted -
Mated
30
in the oiicytes than in either the fat body or haemolymph. Band No. 4 was tied with band No. 13 in the fat body of unmated females for highest per cent composition, and in the haemolymph band No. 4 was 2.5 times higher for unmated than mated females having attained in unmated females the highest per cent composition of total protein among the five bands studied. Day ten after emergence Oikyte band No. 13 (Table 4) was more than twice as high for mated as for unmated females. However, oiicyte band No. 14 was more than twice as high for unmated over mated females. As on day 8 (Table 3) band No. 4 for unmated females comprised approximately 20 per cent of the total protein of the fat body and 27 per cent of the total protein of the haemolymph, which again representedthe highest per cent composition of the five bands in the haemolymph. Band No. 14 was absent from the fat body of unmated females and was present in only neghgible amounts among mated females (O-69 per cent). Band No. 16 was 18.1 per cent of the total fat body protein of unmated females as compared to 13.4 per cent of the fat body protein in mated females. However, in the haemoIymph of mated
PROPERTIESOF FAT BODY, HAEZMOLYMPH,AND O&!YTES OF TENEBRIO
MOLITOR
1449
females band No. 16 was more than three times higher (17.72 per cent) than for urznuzted (4.90) females. In the okytes of both mated and unmated females band No. 16 comprised .a similar percentage of the soluble protein.
r TABLE ~-DAY
I
Fot
“22
40 .E + g = 30 z 6
413
body
Hoemolymph
d
I
v
i
.
10 AFTEREMZRGENCE
Unmoted
Mahd
Jnmate
Mated
413
20
z ::
& n. to
2
0
I!
I.6 -
z0
l-6-
? g
1.4-
Z E G
1.2-
j
I.O-
I:: 1
i
‘;o
I
0.6 -
:I A
0
0.60.4-
1
‘E I
I I2
I
3 I
41
51
6 1
71
6 ’
9 ’
I
IO ‘1
Doys after emergence
FIG. 1.
Haemolymph
protein
concentration Tenebrio
molitor
of unmated L.
and mated adults of
1450
S. M.
hMRICK
AND
A. Bun
Haemoljmph protek concentration The mean protein concentration (o.d. at 750 w) together with the standard deviations were recorded for mated and unmated females from emergence to day 10 after emergence (Fig. 1). The values for muted females were significantly higher (PC 0.05) than the values for unmated females on days 4 and 9 after emergence (0.05 > P>O*OZS). From emergence to day 2 there was a steady increase in the measured protein concentration; however, the value on day 2 did not differ significantly (P> O-OS) between mated and unmated females. Low levels of protein concentration were recorded at emergence and ,on days 3, 5, 7, and 10 after emergence, the mean values on days S and 10 being slightly lower for unmated females, and slightly higher for mated females, than on emergence. On day 9 the mean recorded value for mated females was approximately 60 per cent higher than for unmated females. DISCUSSION
The present electrophoretic studies on cellulose polyacetate membranes indicated that the fat body of Ten&k may be the source of several of the proteins of ovarian development. The absence of band Nos. 4, 13, 14, 15, and 16 from muscle extmcts co&med the hypothesis that these protein fractions were not common to all tissues in a similar hormonal environment. Certain interesting patterns evolved by comparing, as a function of age, the fluctuations in average per cent composition of the five fractions in the fat body, haemolymph, and o&ytes. Band No. 4 was equally distributed between mated and unmated females on day 3 after emergence representing a mere 5 per cent of the total fat body protein, 20 per cent of the haemolymph protein, and 15 per cent of the oiicyte protein. Subsequently band No. 4 was present in greater per cent concentrations among unmated than mated females, being localized, however, in either the fat body on day 7 (Table 2) or the fat body and haemolymph on days 8 (Table 3) and 10 after emergence (Table 4). This suggested that the fat body was at least one source of band No. 4, and that mating altered its synthesisand/or release. The cyclic pattern of significant differences between mated and unmated females in the rate of fat body protein and RNA synthesis (PEMRICK and BUTZ,1970a, b) coincided with the variations in band No. 4. By these calculations day 8 after emergence was suggested as the beginning of the second synthetic cycle and, therefore, similar physiologically to day 2 after emergence. Note that band No. 4 was equally distributed between mated and unmated females on day 8 (physiologically similar to day 2) in the fat body (20 per cent of total protein), on day 3 in the haemolymph (20 per cent of total protein), and on day 10 in the oiicytes (11 per cent of the total protein), indicating a plausible pathway of synthesis (fat body), release (haemolymph), and utilization (oocytes). However, oijcyte band No. 4 dropped from 15 per cent of the total protein on day 3 after emergence to have regained only partially this level by day 10 after emergence. There were two possibilities: (1) band No. 4 was converted to one of the other four oiicyte proteins being considered; (2) band No. 4
PROPERTIES OF FATBODY,IUEMOLYMPH,AND
06CYl-ES OF TEA’BBRIO
MOLITOR
1451
incorporated into the structural proteins of the oocytes which were not released during the extra&on procedure. There was also considerable evidence that the fat body was one source of band NO. 16. On days 3 and 8 after emergence, band No. 16 was present in higher per cent concentrations for mated than unmated females in the fat body and okytes. By day 7 after emergence, okyte band No. 16 was actually lower for mated than unmated females. This suggested a cyclic pattern of synthesis and/or release and a conversion of okyte band No. 16 to another protein fraction. Substantiating MORIXJE’S (1965a) statement that mating accelerated metabolism in Teneti was the observation that on day 10 after emergence band No. 16 occupied the same percentage composition in the fat body of unmated females as in the haemolymph of mated females. The present results may have implicated the fat body as the source of several of the ovarian proteins but they have not maintained that only the fat body was supplying protein for ovarian development. Since ROTH and PORTER(1964) noted that in Aedes the midgut possessed the necessary cytological machinery for protein synthesis, it is quite possible that in Tenebrio as in Pcriplwtsta (MILLS et al., 1966) both the fat body and midgut were involved in supplying proteins for ovarian development. Band No, 13 demonstrated the greatest increase in the oticytes over the timespan studied. The fat body appeared to be synthesizing at least a portion of band No. 13, since in the fat body band No. 13 increased from 24 to 37 per cent of the total protein from days 3 to 7 after emergence, while at the same time increasing from 17 to 38 per cent in the haemolymph. This did not, however, account for the nearly threefold increase in okyte band No. 13 during the same time period. Since band Nos. 13 to 16 possessed similar mobiies, it was quite possible that: (1) band Nos. 13 and 14, and band Nos. 15 and 16 were similar or (2) band Nos. 13 to 16 were four fractions of the same protein. In Table 4 (day 10 after emergence) the total percentage composition of okyte band Nos. 13 and 14 for unmated females approximated the percentage composition of &cyte band No. 13 for mated females. If fraction Nos. 13 to 16 were sub-units of one protein, the relative percentage of each sub-unit fluctuated with development (Tables 1 to 4). The total amount of band Nos. 13 to 16, however, were similar in some tissues on different days. For example, among mated females, the total amount of haemolymph band Nos. 13 .to 16 measured only 6 per cent from day 8 to 10 after emergence, and the total amount of o&yte band Nos. 13 to 16, for the same time-period, increased a mere 0.03 per cent. However, in another tissue, such as the fat body from mated females 8 and 10 days after emergence, the percentage of total protein represented by band Nos. 13 to 16 differed by as much as 64 per cent. To discuss the possible implications of these interrelationships would be beyond the scope of the present paper. Tables l-4 listed the per cent composition of the five protein fractions studied. This was only a relative measurement and was not necessarily equivalent to the actual protein concentration of each fraction.
was
1452
S. ivl.
PEMRICK
AND
A. BUTZ
Fig. 1 reflected a consistently higher haemolymph protein concentration for mated than unmated females. This evidence supported our previous conclusion (-RICK and BUTZ, 1970a, b) that fat bodies of mated females synthesized and/or released protein at a more rapid rate than unmated females, such that incorporation studies, at a pulse time of 3 hr, indicated more label in the fat body of unmated than mated females. The following similarities were evident in a comparision of the cyclic patterns of fat body protein and RNA synthesis (hMRICK and BUTZ, 1970a, b) with the cyclic pattern of haemolymph protein concentration (Fig. 1): (1) an increase to day 2, which was common to mated and unmated females; (2) an overlap in significant differences between mated and unmated females; and (3) a similar drop for mated and unmated females on day 7. Therefore, the same control mechanisms were regulating, directly or indirectly, fat body protein synthesis and haemolymph protein concentration. It would, however, be premature to speculate on the extent to which the neurosecretory cells or the corpora allata control fat body protein synthesis in the mealworm, Tenebriomolitor. Mating may result in a nervous stimulus which alters the postulated (MORDUE,1965b) synergistic activity of the neurosecretory cells and the corpora allata by regulating the release of the allotropic hormone. REFERENCES K. G. (1967) The nature of haemolymph proteins in relation to the ovarian cycle in the viviparous cockroach, Nuuphoetu cakea: J. Insect Physiol. 13, 1189-1195. Bvn A. (1957) Effects of sodium, potassium, and calcium ions on the isolated heart of the mealworm, Tenebrio molitor L. J. N. Y. ent. Sot. 65, 22-31. CHIPPENDALE G. M. and BECK S. D. (1967) Fat body proteins of OS&& nubilalis during diapause and prepupal differentiation. J. Insect Physiol. 13, 995-1006. COLES G. C. (1965) Haemolymph proteins and yolk formation in Rhodn& prolixus SM. J. exp. Biol. 43, 425-432. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. J. Intact Physiol. 8, 609-619. LAUFERH. (1960) Blood proteins in insect development. Ann. N. Y. Acud. Sci. 89,490-515. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L., and RANDW R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193.2651275. MILLS R. R., G~SLADB, F. C. and COUCHE. F. (1966) Studies on vitellogenesis in the American cockroach. J. Insect Physiol. 12, 767-779. MORDUEW. (19653 Studies on o&yte production and associated histological changes in the neuro-endocrine system in Tenebrio molitor L. J. Insect Physiol. 11, 493-504. MORDUB W. (l%Sb) Neuro-endocrine factors in the control of oijcyte production in Tenebrio molitor L. J. Insect Physiol. 11, 617-629. NIELSON D. J. and MIU R. R. (1968) Changes in electrophoretic properties of haemolymph and terminal oacyte proteins during vitellogenesis in the American cockroach. J. Insect Physiol. 14, 163-170. PEMRICK S. M. and Bvn A. (1970a) Protein synthesis of the fat body of adult Tenebrio molitor. J. Insect Physiol. 16, 643-651. PEMRICK S. M. and BUTZ A. (1970b) RNA synthesis of the fat body of adult Tenebrio molitor. 3. Insect Physiol. 16, 1171-1177. PRICKB. M. and BOSMANT. (1966) The electrophoretic separation of proteins isolated from the larva of the blowfly, Calliphora erythrocephala. 3. Insect Physiol. 12, 741-745. ADIYODI
PROPERTIES OFFATBODY,HAEMOLYMPH,
AND 06CYTPS
OF TENEBRIO
MOLlTOR
1453
ROTH T. F. and PORTBRK. R. (1964) Yolk protein uptake in the oiicytes of the mosquito A&s aegypi L. g. Cell Bs’ol. 20, 313-332. SHIGMATSU H. (1958) Synthesis of blood protein by the fat body in the silkworm, Bombyx mori L. Nature, L+ond.182, 880-882. TeLFw W. H. (1954) Immunological studies of insect metamorphosis-II. Tbe role of a sex-linked blood protein in egg formation by the cecropia silkworm. g. gea. Physiol. 37,
539-558. TELFERW. H. (1965) The mechanism and control of yolk formation.
A. Rev. Ent. 10, 161-184. THOMAS K. K. and NATION J. L. (1966) Control of a sex-limited haemolymph protein by corpora allata during ovarian development in Periplaneta americana L. Biol. Bull., Woods Hole 130, 254-265.
COMMON ELECTROPHORETIC PROPERTIES OF THE FAT BODY, HAEMOLYMPH, AND OijCYTES OF ADULT TENEBRIO MOLITOR* S. M. PEMRICKt
and A. BUT2
Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221 (Received 12June
1969; mvised 27 September 1%9)
Aktrnct-Electropherograms of the haemolymph indicated the presence, at a pH of 9.8, of eighteen negatively charged proteins of which at least five (bands Nos. 4, 13, 14, 15 and 16 from the origin) were common to the fat body and oticytes. The distribution of the per cent composition of the five bands varied between unmated and mated females in the three tissues over the 4 days studied (days 3,7,8, and 10 after emergence). C ircumstantial evidence suggested a threestep process of fat body protein synthesis followed by protein release into the haemolymph and its subsequent uptake by the o&ytea. Band No. 13 had the highest average per cent composition for both mated and unmated fanales in all three tiesues reaching a maximum, on day 7 after emergence, of 56 (unmated females) or 58 (mated females) per cent of the total protein concentration in the o&ytes. Band No. 4, however, held the higheat per cent composition of the five measured haemolymph protein bands on day 3 after emergence for both mated and unmated females, and on days 8 and 10 after emergence for umnated females when in the fat body it was also tied with band No. 13 for highest per cent composition. On day 3 after emergence fat body band No. 16, in mated females, represented the highest per cent composition of total protein and waa four times higher than in unmated femalea of the same age, and similarly in the ’ okytcs, on day 3, band No. 16 was 34 per cent higher for mated than unmated females. Spectrophotometric analyses of fluctuations in the haemolymph protein concentration indicated a similar pattern for unmated and mated females from emergence to day 10 after emergence, the haemolymph protein concentration being &niflcantly greater (P< O-05) for mated females on days 4 and 9 after emergence (O-05 > P > 0*025). The mean value on day 9 was approximately 60 per cent higher for mated than unmated females. A possible relationship between the cyclic pattern of haemolymph protein concentration and the patterns of fat body protein and RNA synthesis is discussed. INTRODUCTION
adult fat body of the mealworm Tenebnh moldor is supplying a certain percentage of the necessary proteins for growth and development, there should be IF THE
* This research was supported in part by an American Cancer Society Institutional Grant. 7 Present address: Department of Biological Sciences, State University of New York at Albany, Albany, New York 12203. 1443
1444
S
M.
PEMRICK AND
A. Bun
a similarity between the protein fractions in the fat body, haemolymph, and target organ (ovaries). Such a relationship, however, would be only an indication that a pathway of protein synthesis, release, and utilization existed, for it is quite possible that different tissues, in the same hormonal environment, might,direct the synthesis of similar proteins. Sections of this pathway (fat body to haemolymph, or haemolymph to oiicytes) have been substantiated in studies of both juvenile and adult stages. When SHIGMATSU(1958) noted the protein synthetic capabilities of the larval fat body in the silkworm Bombyx wi, he also reported that this protein, which was released from the fat body during incubation procedures, was similar to the globular components of the haemolymph. Similarly, LAUFER(1960) concluded through starch gel electrophoretic studies that the fat body of the giant silk moths H’ulophora cecropia and Sam&a cynthiu was the source of several of the proteins of the blood, and PRICE and BOSMAN(1966) separated by acrylamide gel electrophoresis the haemolymph proteins of the blowfly Calliphora erythrocephaku and found them to be similar to those released from the fat body under in v&o conditions. In addition, NIELSON and MILLS (1968) compared the electrophoretic mobility of the haemolymph and o&yte proteins during yolk formation in the American cockroach Per@neta amerbna and reported the protein patterns in both tissues were not only similar, but also cyclic in nature, thereby suggesting that the haemolymph proteins were involved in ovarian development. Earlier HILL (1962) had reported the presence of a haemolymph protein fraction in the desert locust Schistocet-ca gregaria which increased during yolk deposition and after ovariectomy. Evidence for a sex difference in haemolymph protein patterns comes from a variety of sources. AJXYODI(1967) cited evidence for a specific female protein in the haemolymph of the viviparous cockroach iVuu#zoeta cinerea but was not able, however, to equate this with any of the fat body or ovarian proteins. THOMASand NATION (1966) also reported a sex-linked protein in the haemolymph of the American cockroach Pe@aneta amekuna. However, CHIPPENDALEand BECK (1967) disclosed that in pharate pupae of the European corn borer Ostrikia nubiZuZis a specific protein accumulated in the fat body of females at an earlier age and to a higher concentration than in the fat body of males, and they further suggested that subsequently this protein was transported to the ovaries where it comprised the major protein fraction. Immunoelectrophoretic techniques provided more exact evidence concerning the similarity of various protein fractions of the fat body, haemolymph, and oocytes. TELFER (1954) demonstrated immunologically identical proteins in the haemolymph and oiicytes of the silkworm Hyalophoru and later concluded that every haemolymph protein has an antigenic counterpart in the egg (TELFER, 1965). Recently COLES (1965) revealed through fluorescent immunoelectrophoretic studies two protein bands in Rhodnius eggs which shared identical mobility patterns with two specific adult bands in the haemolymph. Previous studies (PEMRICK and BUTZ, 1970a, b) have indicated that in the mealworm Tenebrio moZitor the fat body is metabolically active during the adult
PROPERTIES OF FAT BODY,
HAEMOLYMPH,
AND 06)cITEs
OF TENEBRIO MOLITOR
1445
stage, synthesizing RNA and protein in a cyclic pattern which appears to be related to ovarian development. It is the purpose of the present investigation to link the protein synthetic cycles of the fat body with a plausible pathway of protein release and utilization. MATERIALS
AND METHODS
Haemolymph protkn concentration Maintenance of cultures was described previously (PEMRICKand BUTZ, 1970a, b). Haemolymph samples were withdrawn from mated and unmated females at emergence and every day thereafter for 10 days. The haemolymph was collected by removing the forelegs and taking the haemolymph up into a 20 ~1 calibrated micropipette as described by MORDUE(1965b). Samples of 5 d per insect could usually be obtained in this manner. The blood was then transferred to 3.0 ml of ice-cold 5% trichloracetic acid (TCA) and centrifuged for 10 min (5000 g). The precipitate was washed (3~0 ml 5% TCA) and centrifuged for 5 min (2500g) at which time the supematant was discarded. The protein concentration was then determined from the final precipitate by the method of LOWRY et al. (1951). The optical density was recorded at 750 rryl against a water blank. Electrophoresis: Preparation of samples Samples of haemolymph (5 ~1 per insect), fat body, and oiicytes were removed from mated and unmated females at representative days after emergence. The first oiicytes were noticed around day 3. Four 4 samples of haemolymph were applied directly to the cellulose acetate membranes. Fat body tissue (four pooled samples per day studied; 3 insects per sample) was homogenized in a cold ground-glass homogenizer containing 0.5 ml of cold saline (BUTZ, 1957), centrifuged, and 4 ~1 of the supematant became the tissue extract. Oticytes (four pooled samples per day studied: 3 insects per sample) were also homogenized in cold saline and centrifuged. The clear zone of the supematant (5 ~1) was applied to the acetate strips. Control samples of &oracic muscle (pooled samples) were homogenized in cold saline and centrifuged, and 5 ~1 of the supematant became the tissue extract. Electrophoresis : procedure The samples (4 to 5 ~1 of the pooled extracts) were applied to the centre of the cellulose acetate membranes (Gelman Sepraphore, III) which had been soaked in the chamber in Gelman High Resolution Buffer (pH 9.8), and blotted lightly. The unit was then allowed to equilibrate for 10 min at which time the power supply was set at 300 V for 45 min. The membranes were then stained in Ponceau S (0.5 per cent in TC_4), washed several times (5% acetic acid) and, for quantitative measurements, the membranes were cleared (acetic acid : methanol) and scanned. In order to express in tabular form the percentage composition of a given fraction
14%
S.
M.
to the total
protein, a planimeter from the scanner.
bMRICK
AND
A. Bun
was employed to integrate the curves obtained
RESULTS
Ekctmphoretic analyses of the proteins of the haemoiym$h, fat body, and eggs Analyses of electropherograms from the haemolymph indicated the presence of eighteen negatively charged proteins (pH 98), which were numbered 1 to 18 from the origin. There did not appear to be a specific sex protein in Tenebrio, since the electrophoretic patterns of males and females were similar. Of the eighteen negatively charged proteins in the haemolymph at least five (band Nos. 4, 13, 14, 15, and 16 from the origin) were common to the fat body and eggs. It is quite possible that other proteins were common to the three tissues but in amounts too small to quantitate with any degree of accuracy. Control electropherograms of homogenized extracts of thoracic muscle failed to yield proteins with a mobility similar to band Nos. 4, 13, 14, 15, and 16. Therefore, the average per cent composition of these five bands was recorded in the fat body, haemolymph, and Mcytes for both mated and unmated females on days 3 (Table l), 7 (Table Z), 8 (Table 3), and 10 (Table 4) after emergence. Band No. 13 maintained the highest average per cent composition of total protein for both mated and unmated females in all three tissues. Day three afier emergence The pattern of per cent composition of the five bands varied between mated and unmated females in the fat body and oiicytes but was similar in the haemolymph (Table 1). Fat body band No. 16 in mated females represented the highest per cent composition of total protein and was four times higher than in unmated females. T-LB
I-DAY
Fat body
.I
Unmated md No
I
Mated
3 A~~BREMEKGKNCE
Haemolymph
I
Unmated
I
Mated
Okyter Unmated
I
Mated
PROPERTIES OF FAT BODY, HAEMOLYhXPH, AND 06CYTES OF TENEBBIO
MQLZTOR
1447
Similarly oGcyte band No. 16 was nearly 34 per cent higher for mated than unmated females. Band No. 4 in the haemolymph held the highest per cent composition of total protein for both mated and unmated females and, although in the o&y&s band No. 4 dropped behind band No. 13, it comprised 15 per cent of the total protein compared to a low level of 6 (unmated) or 7 (mated) per cent of the total protein in the fat body. Band No. 13 for both mated and unmated females accounted for a higher percentage of the total protein in the fat body than in either the haemolymph or okytes. Day seven after emergence Band No. 13 (Table 2) attained its peak concentration of 56 (unmated) and 58 (mated) per cent of the total protein in the ticytes, while maintaining relatively high concentrations in the fat body and haemolymph. Although the per cent TABLE~-DAY 7 MWEREMERGENCE Fat
body Mated
Hoemalymph Unmated
Mated
Oikytes Unmoted
Mated
composition of band No. 4 varied considerably between the fat body and oiicytes, the value for unmated females was 40 (o&y@) to 60 (fat body) per cent higher than for mated females. However, the per cent composition of band No. 14, although similar for both the fat body and o&ytes, was also higher for unmated than mated females, the value for unmated females being twice- as high as for mated females.
S. M. PSMRICK ANDA. Bun
1448
Fat body band No. 16 was 35 per cent higher for mated than unmated females; however, okyte band No. 16 was nearly 2.5 times higher for unmated than mated females. Day kght after emergence Band No. 13 represented a much lower per cent of the total protein in the fat body, haemolymph, and oijcytes than was recorded on day 7 (Table 3). However, as noted on day 7, band No. 13 comprised a higher percentage of the total protein TABLE Fot body MOtOd
~-DAY
I
8m
BMBRGENCE
Hoemolymph
T-
1Unmated
Mated
OOcytes
Jnmoted -
Mated
30
in the oiicytes than in either the fat body or haemolymph. Band No. 4 was tied with band No. 13 in the fat body of unmated females for highest per cent composition, and in the haemolymph band No. 4 was 2.5 times higher for unmated than mated females having attained in unmated females the highest per cent composition of total protein among the five bands studied. Day ten after emergence Oikyte band No. 13 (Table 4) was more than twice as high for mated as for unmated females. However, oiicyte band No. 14 was more than twice as high for unmated over mated females. As on day 8 (Table 3) band No. 4 for unmated females comprised approximately 20 per cent of the total protein of the fat body and 27 per cent of the total protein of the haemolymph, which again representedthe highest per cent composition of the five bands in the haemolymph. Band No. 14 was absent from the fat body of unmated females and was present in only neghgible amounts among mated females (O-69 per cent). Band No. 16 was 18.1 per cent of the total fat body protein of unmated females as compared to 13.4 per cent of the fat body protein in mated females. However, in the haemoIymph of mated
PROPERTIESOF FAT BODY, HAEZMOLYMPH,AND O&!YTES OF TENEBRIO
MOLITOR
1449
females band No. 16 was more than three times higher (17.72 per cent) than for urznuzted (4.90) females. In the okytes of both mated and unmated females band No. 16 comprised .a similar percentage of the soluble protein.
r TABLE ~-DAY
I
Fot
“22
40 .E + g = 30 z 6
413
body
Hoemolymph
d
I
v
i
.
10 AFTEREMZRGENCE
Unmoted
Mahd
Jnmate
Mated
413
20
z ::
& n. to
2
0
I!
I.6 -
z0
l-6-
? g
1.4-
Z E G
1.2-
j
I.O-
I:: 1
i
‘;o
I
0.6 -
:I A
0
0.60.4-
1
‘E I
I I2
I
3 I
41
51
6 1
71
6 ’
9 ’
I
IO ‘1
Doys after emergence
FIG. 1.
Haemolymph
protein
concentration Tenebrio
molitor
of unmated L.
and mated adults of
1450
S. M.
hMRICK
AND
A. Bun
Haemoljmph protek concentration The mean protein concentration (o.d. at 750 w) together with the standard deviations were recorded for mated and unmated females from emergence to day 10 after emergence (Fig. 1). The values for muted females were significantly higher (PC 0.05) than the values for unmated females on days 4 and 9 after emergence (0.05 > P>O*OZS). From emergence to day 2 there was a steady increase in the measured protein concentration; however, the value on day 2 did not differ significantly (P> O-OS) between mated and unmated females. Low levels of protein concentration were recorded at emergence and ,on days 3, 5, 7, and 10 after emergence, the mean values on days S and 10 being slightly lower for unmated females, and slightly higher for mated females, than on emergence. On day 9 the mean recorded value for mated females was approximately 60 per cent higher than for unmated females. DISCUSSION
The present electrophoretic studies on cellulose polyacetate membranes indicated that the fat body of Ten&k may be the source of several of the proteins of ovarian development. The absence of band Nos. 4, 13, 14, 15, and 16 from muscle extmcts co&med the hypothesis that these protein fractions were not common to all tissues in a similar hormonal environment. Certain interesting patterns evolved by comparing, as a function of age, the fluctuations in average per cent composition of the five fractions in the fat body, haemolymph, and o&ytes. Band No. 4 was equally distributed between mated and unmated females on day 3 after emergence representing a mere 5 per cent of the total fat body protein, 20 per cent of the haemolymph protein, and 15 per cent of the oiicyte protein. Subsequently band No. 4 was present in greater per cent concentrations among unmated than mated females, being localized, however, in either the fat body on day 7 (Table 2) or the fat body and haemolymph on days 8 (Table 3) and 10 after emergence (Table 4). This suggested that the fat body was at least one source of band No. 4, and that mating altered its synthesisand/or release. The cyclic pattern of significant differences between mated and unmated females in the rate of fat body protein and RNA synthesis (PEMRICK and BUTZ,1970a, b) coincided with the variations in band No. 4. By these calculations day 8 after emergence was suggested as the beginning of the second synthetic cycle and, therefore, similar physiologically to day 2 after emergence. Note that band No. 4 was equally distributed between mated and unmated females on day 8 (physiologically similar to day 2) in the fat body (20 per cent of total protein), on day 3 in the haemolymph (20 per cent of total protein), and on day 10 in the oiicytes (11 per cent of the total protein), indicating a plausible pathway of synthesis (fat body), release (haemolymph), and utilization (oocytes). However, oijcyte band No. 4 dropped from 15 per cent of the total protein on day 3 after emergence to have regained only partially this level by day 10 after emergence. There were two possibilities: (1) band No. 4 was converted to one of the other four oiicyte proteins being considered; (2) band No. 4
PROPERTIES OF FATBODY,IUEMOLYMPH,AND
06CYl-ES OF TEA’BBRIO
MOLITOR
1451
incorporated into the structural proteins of the oocytes which were not released during the extra&on procedure. There was also considerable evidence that the fat body was one source of band NO. 16. On days 3 and 8 after emergence, band No. 16 was present in higher per cent concentrations for mated than unmated females in the fat body and okytes. By day 7 after emergence, okyte band No. 16 was actually lower for mated than unmated females. This suggested a cyclic pattern of synthesis and/or release and a conversion of okyte band No. 16 to another protein fraction. Substantiating MORIXJE’S (1965a) statement that mating accelerated metabolism in Teneti was the observation that on day 10 after emergence band No. 16 occupied the same percentage composition in the fat body of unmated females as in the haemolymph of mated females. The present results may have implicated the fat body as the source of several of the ovarian proteins but they have not maintained that only the fat body was supplying protein for ovarian development. Since ROTH and PORTER(1964) noted that in Aedes the midgut possessed the necessary cytological machinery for protein synthesis, it is quite possible that in Tenebrio as in Pcriplwtsta (MILLS et al., 1966) both the fat body and midgut were involved in supplying proteins for ovarian development. Band No, 13 demonstrated the greatest increase in the oticytes over the timespan studied. The fat body appeared to be synthesizing at least a portion of band No. 13, since in the fat body band No. 13 increased from 24 to 37 per cent of the total protein from days 3 to 7 after emergence, while at the same time increasing from 17 to 38 per cent in the haemolymph. This did not, however, account for the nearly threefold increase in okyte band No. 13 during the same time period. Since band Nos. 13 to 16 possessed similar mobiies, it was quite possible that: (1) band Nos. 13 and 14, and band Nos. 15 and 16 were similar or (2) band Nos. 13 to 16 were four fractions of the same protein. In Table 4 (day 10 after emergence) the total percentage composition of okyte band Nos. 13 and 14 for unmated females approximated the percentage composition of &cyte band No. 13 for mated females. If fraction Nos. 13 to 16 were sub-units of one protein, the relative percentage of each sub-unit fluctuated with development (Tables 1 to 4). The total amount of band Nos. 13 to 16, however, were similar in some tissues on different days. For example, among mated females, the total amount of haemolymph band Nos. 13 .to 16 measured only 6 per cent from day 8 to 10 after emergence, and the total amount of o&yte band Nos. 13 to 16, for the same time-period, increased a mere 0.03 per cent. However, in another tissue, such as the fat body from mated females 8 and 10 days after emergence, the percentage of total protein represented by band Nos. 13 to 16 differed by as much as 64 per cent. To discuss the possible implications of these interrelationships would be beyond the scope of the present paper. Tables l-4 listed the per cent composition of the five protein fractions studied. This was only a relative measurement and was not necessarily equivalent to the actual protein concentration of each fraction.
was
1452
S. ivl.
PEMRICK
AND
A. BUTZ
Fig. 1 reflected a consistently higher haemolymph protein concentration for mated than unmated females. This evidence supported our previous conclusion (-RICK and BUTZ, 1970a, b) that fat bodies of mated females synthesized and/or released protein at a more rapid rate than unmated females, such that incorporation studies, at a pulse time of 3 hr, indicated more label in the fat body of unmated than mated females. The following similarities were evident in a comparision of the cyclic patterns of fat body protein and RNA synthesis (hMRICK and BUTZ, 1970a, b) with the cyclic pattern of haemolymph protein concentration (Fig. 1): (1) an increase to day 2, which was common to mated and unmated females; (2) an overlap in significant differences between mated and unmated females; and (3) a similar drop for mated and unmated females on day 7. Therefore, the same control mechanisms were regulating, directly or indirectly, fat body protein synthesis and haemolymph protein concentration. It would, however, be premature to speculate on the extent to which the neurosecretory cells or the corpora allata control fat body protein synthesis in the mealworm, Tenebriomolitor. Mating may result in a nervous stimulus which alters the postulated (MORDUE,1965b) synergistic activity of the neurosecretory cells and the corpora allata by regulating the release of the allotropic hormone. REFERENCES K. G. (1967) The nature of haemolymph proteins in relation to the ovarian cycle in the viviparous cockroach, Nuuphoetu cakea: J. Insect Physiol. 13, 1189-1195. Bvn A. (1957) Effects of sodium, potassium, and calcium ions on the isolated heart of the mealworm, Tenebrio molitor L. J. N. Y. ent. Sot. 65, 22-31. CHIPPENDALE G. M. and BECK S. D. (1967) Fat body proteins of OS&& nubilalis during diapause and prepupal differentiation. J. Insect Physiol. 13, 995-1006. COLES G. C. (1965) Haemolymph proteins and yolk formation in Rhodn& prolixus SM. J. exp. Biol. 43, 425-432. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. J. Intact Physiol. 8, 609-619. LAUFERH. (1960) Blood proteins in insect development. Ann. N. Y. Acud. Sci. 89,490-515. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L., and RANDW R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193.2651275. MILLS R. R., G~SLADB, F. C. and COUCHE. F. (1966) Studies on vitellogenesis in the American cockroach. J. Insect Physiol. 12, 767-779. MORDUEW. (19653 Studies on o&yte production and associated histological changes in the neuro-endocrine system in Tenebrio molitor L. J. Insect Physiol. 11, 493-504. MORDUB W. (l%Sb) Neuro-endocrine factors in the control of oijcyte production in Tenebrio molitor L. J. Insect Physiol. 11, 617-629. NIELSON D. J. and MIU R. R. (1968) Changes in electrophoretic properties of haemolymph and terminal oacyte proteins during vitellogenesis in the American cockroach. J. Insect Physiol. 14, 163-170. PEMRICK S. M. and Bvn A. (1970a) Protein synthesis of the fat body of adult Tenebrio molitor. J. Insect Physiol. 16, 643-651. PEMRICK S. M. and BUTZ A. (1970b) RNA synthesis of the fat body of adult Tenebrio molitor. 3. Insect Physiol. 16, 1171-1177. PRICKB. M. and BOSMANT. (1966) The electrophoretic separation of proteins isolated from the larva of the blowfly, Calliphora erythrocephala. 3. Insect Physiol. 12, 741-745. ADIYODI
PROPERTIES OFFATBODY,HAEMOLYMPH,
AND 06CYTPS
OF TENEBRIO
MOLlTOR
1453
ROTH T. F. and PORTBRK. R. (1964) Yolk protein uptake in the oiicytes of the mosquito A&s aegypi L. g. Cell Bs’ol. 20, 313-332. SHIGMATSU H. (1958) Synthesis of blood protein by the fat body in the silkworm, Bombyx mori L. Nature, L+ond.182, 880-882. TeLFw W. H. (1954) Immunological studies of insect metamorphosis-II. Tbe role of a sex-linked blood protein in egg formation by the cecropia silkworm. g. gea. Physiol. 37,
539-558. TELFERW. H. (1965) The mechanism and control of yolk formation.
A. Rev. Ent. 10, 161-184. THOMAS K. K. and NATION J. L. (1966) Control of a sex-limited haemolymph protein by corpora allata during ovarian development in Periplaneta americana L. Biol. Bull., Woods Hole 130, 254-265.