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Protein concentration in the hemolymph of the large milkweed bug, Oncopeltus fasciatus (Dallas)
Comp. Biochera. Physiol., 1966, Vol. 18, pp. 163 to 168. Pergamon Press Ltd. Printed in Great Britain
PROTEIN CONCENTRATION IN THE HEMOLYMPH OF THE LARGE MILKWEED BUG, O N C O P E L T U S F A S C I A T U S (DALLAS)* SISTER M. LORETTA TERANDO and DOROTHY FEIR Biology Department, St. Louis University, St. Louis, Missouri, 63104 (Received 1 November 1965)
A b s t r a c t - - 1 . Under all experimental conditions the protein concentration
of the hemolymph rose during the first half of the fifth stadium. During the second half of the fifth stadium the pattern is not clear. The concentration increases during the first 4 days of the adult stage and remains high until at least the fourteenth day post-molt. 2. In 1 lambda, non-pooled samples, a significant difference for mean protein concentration values was found between the following groups: (a) male and female adults, 5 days post-molt; (b) male and female adults, 10 days post-molt; and (c) the 10 per cent and 50 per cent fifth instar. 3. The concentration values reported can be used in comparative studies of protein concentration in insects of known age, sex, developmental stage and nutritional state; for studies on the hemolymph from the milkweed bug under various experimental conditions; and for comparison with other animals. INTRODUCTION A. CONSIDE1L~BLE amount of information is available on the constitution of the h e m o l y m p h of various insects (Gilbert & Schneiderman, 1961; Wyatt, 1961; Florkin & Jeuniaux, 1964). However, there are few studies which are detailed in regard to age, sex and developmental stage and which point out the statistical significance of differences between instars, sexes or individual insects. Since it is quite possible that the h e m o l y m p h constituents m a y vary u n d e r different physiological conditions such as post-molt age, instar and sex, these factors should be controlled in studies on the h e m o l y m p h . T h i s paper is a detailed study of the protein concentration of the h e m o l y m p h of the large milkweed bug, Oncopeltus f a s c i a t u s (Dallas), during the fifth stadium and during the first 14 days of the adult stage. MATERIALS AND M E T H O D S Newly molted bugs were collected during the day over periods of 8-14 hr. Male and female adults were separated on the day of molt. W a t e r and milkweed seeds were present at all times for all groups in order to insure the same nutritional * Supported by National Science Foundation Cooperative Fellowship to Sr. M. Loretta Terando and in part by U.S.P.H.S. Grant AI-04215 to D. Felt. 163
164
SISTER ~ [ . LORETTA TER.aNDO .~ND DOROTHY FEIR
state. At the time of bleeding the insects were anesthetized with COo, the antennae and legs were clipped and hemolymph was collected in capillary tubes. The amount of hemolymph taken from each bug varied from 1-5 Lambdas. The pooled samples were kept on ice in order to prevent melanization as no tyrosinase iahibitors were used because they interfered with protein determination. Samples to be filtered were passed through a Gelman membrane, pore size 5/x. Staining of this membrane showed that hemocytes were retained by it. The number of bugs used for the pooled samples ranged from 93 to 525. Non-pooled 1 lambda samples were put into solution by crushing the 1 lambda capillary, pipette with a glass pestle. All hemolymph samples were frozen at - 5 5 ° C and stored for 1 day to several weeks. Protein concentration was determined bv the Lowry method (Lowry et al., 1951). From the pooled samples a 50 iambda aliquot was taken and diluted to 10 ml with 0"7°I,,o saline. This dilution was found to lie within a convenient range on the bovine serum albumin standard curve. The 1 lambda samples were mixed with the reagents and the glass was centrifuged off before reading. One centrifugation at 3000 rev/min for 15 rain was sufficient to give a stable reading. The solutions were read at 750 m/~ with a red filter in a Spectronic 20. RESULTS In Fig. 1 values for not filtered, pooled samples are plotted. The ordinate indicates mg protein/ml of hemolymph. The abscissa is divided into two parts, the first part representing percentage of fifth stadium. Percentage of fifth stadium was used rather than days post-molt because the instars varied in the length of time required to molt to the adult. For example, fifth day post-molt for a bug with a fifth stadium of 10 days was not the same day chronologically as the fourth day post-molt of a bug with an 8 day stadium ; but both times represented 50 per cent of the stadium. The second part of the abscissa represents days after molting to the adult. During the first third of the stadium the values were 31-42 mg protein/ml hemolymph. Half-way through the stadium there was an increase to 56-58 mg protein/ml hemolymph. The 1 day post-molt adult had a protein concentration of 47 mg protein/ml hemolymph. The concentration increased and remained high for the adult until at least the fourteenth day. Values for the adult male and female protein concentrations were compared by paired sampling using a t-test. No significant difference was found. In Table 1 values for all the experimental conditions are summarized. When the filtered, pooled samples are examined, low initial values are seen in the fifth and adult stadia, with an increase midway through the fifth stadium and during the first 4 days of the adult stadium. In the seventh day post-molt adult the concentration was 73-76 mg protein/ml hemolymph, or about double the initial values for both the fifth instar and adult stages. Since about 0.1 ml of hemolymph was lost in the filtering process, it was necessary to test unfiltered samples for protein concentration. In general, the unfiltered samples have somewhat higher values, but the two periods of protein
PROTEIN
CONCENTP, ATION
IN
THE
HEMOLY%IPH
OF
THE
LARGE
MILKVCEED
165
BUG
increase over lower initial values noted in the filtered samples are also evident here. Paired sampling using a t-test indicated no significant difference between the two sets of data.
120
II0
I00 8---J,
p r
/
t
,'
i
~0
70
2:
60
.3
•
FEMALE
•
MALE
NOT NOT
FILTERED. FILTERED,
POOLED POOLED
20
E 10
20
30 OF
40 50 FIFTH
60 70 50 STADIUI!
90
i00
I
2 3 ADULT.
4
$ 6 7 DAYS POST-
8
9 I0 MOLT
11
13
FIG. 1. Comparison of male with female protein concentration using unfiltered, pooled hemolymph samples.
14
166
SISTER .~,1. LORETTA TER.~\'DO AND DOROTHY FEIR
T h e m e a n values for male and female 1 lambda, non-pooled samples follow the same trend of low initial values with an increase as development proceeds (Table 1). T h e range of values indicates the large individual variation. T h e mean TABLE 1--HEMoLYMPH PROTEINCONC'K-NTRATIONS Mg protein/ml hemolymph 1 lambda, not pooled Age (per cent of fifth stadium) 10"0 12"5 15"0 20"0 25"0 30"0 37'3 45"5 50"0 55"6 60"0 66"7 77"8 80"0 87"5 100"0 Adult (day~ post-molt) 1 2 3 4 5 6 7 8 10 14
Filtered, pooled Unfiltered, pooled Female
Male
34"9
38"1
37"3
43"1
36"4
40"0
48"9
47-0
36"4
33"9
51'0 36-2
51-0 39"1
30-1 35-1 62"7 77-0 69"9 74.7 76"0
36"3 32"7 62'2 68'9 68.8 74.0 73"1
Female
Male
31"1
32"8
31"9 35"5
40"0 39"1
41"1
42"2
50"0 56"2
53"2 58"2
47"3
50"5
54"2
50'9
57'7
57"4
47"3 46"2 54"3 65"2 94"4 81"5 83'2 96"5
47-0 48-6 64"2 65"8 75"8 78"2 66-3 75"8
95"1
70"5
Female
Male
Mean of 14 bugs Range
Mean of 14 bugs Range
35"8* 24"8-49"1
29"8 20"9-37"9
67"4* 49"7-78'9
68"5 57"2-84"9
48"1 41-0-54"6
45"2
53"6 43"1-64"8
48"3 37-9-59"6
35-3-55"4
lO0"6t 73"7-123"3 86"2t 58"3-110-8
116"65 94"3-143"7 86-8** 57-2-110"5
Significant differences: * P<0"01 ; t P<0"02; :~ P<0"001. values for the unfiltered pooled samples do, however, lie within the range of the 1 lambda, non-pooled samples. As described above, these 1 lambda samples were not collected and prepared by the same m e t h o d as the pooled samples, so this m a y explain the somewhat higher values.
PROTEIN
C O N C E N T R A T I O N I N THE H E M O L Y ~ I P H OF THE LARGE M I L K W E E D B U G
167
DISCUSSION It is interesting to compare these results with those from studies on other insects. Nowesielski & Patton (1965) reported that hemolymph from the last two instars and newly molted adult of the house cricket, Acheta domesticus L., has a high protein concentration. In the less than 1 day old adult female the concentration is 69 mg/ml. The level drops shortly after the final molt and by the tenth day of adult life it has dropped by about 20 per cent (to 56 mg in the female). Siakotos (1960) found the protein concentration of the hemolymph of the American cockroach, Periplaneta americana L., decreased by two-thirds on the fourth day after the last nymphal molt, but by the eighth day it was back to the initial level. In the adult, the concentration decreased by the fourth day after molt to the adult stage. The decline continued until, by the eighth day, the protein concentration had decreased to one-half the level at the time of molt. Studies on moths by Laufer (1960) and Teller & Williams (1953) indicate an increase in protein in the last larval instar during the time of increased prothoracic hormone activity. However, as the adult developed the protein concentration decreased. The data on the milkweed bug indicate a rise in protein concentration about half-way through the fifth stadium, paralleling the rise found in other insects. Probably this reflects increased protein synthesis under the influence of ecdysone. .Mthough the information available is limited, the increasing protein concentration in the adult seems unique to the milkweed bug. It may reflect continued deposition of endocuticle (Dingle, 1965) or perhaps a type of protein storage, since the bugs continue to feed and maintain their weight during the times studied. The filtered samples are probably nearest the true value of the protein concentration of the hemolymph, since by this method the hemocytes were removed. However, considering the large numbers of insects which are needed in order to get a large enough sample to filter, the slight error which might be introduced by the presence of hemocytes is negligible when the saving in time and specimens is considered. Determinations on 1 lambda samples are possible and this is the only method used which permits estimation of range.
REFERENCES DINGLE H. (1965) The relation between age and flight activity in the milkweed bug, Oncopeltus. 07. exp. Biol. 42, 269-283. FLOmCIN M. & JEVNIAtrXCH. (1964) Hemob~mph: Composition. In The Physiology of the Insecta (Edited by ROCmSTEINM.), Vol. III, Chapter 2. Academic Press, New York. GILBERT L. I. & SCm~qZIDE~.'~tANH. A. (1961) Some biochemical aspects of insect metamorphosis. Am. Zool. 1, 11-51. L^trt~R H. (1960) Blood proteins in insect development. Ann. N. Y. Acad. Sd. 89, 490-515. Low~v O. H., ROSEBROUGHN. J., FAaR A. L. & RANDALLR. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. NowosmtsKZ J. W. & PATTONR. L. (1965) Variation in the hemolymph protein, amino acid, and lipid levels in adult house crickets, Acheta domesticus L., of different ages. J. Insect Physiol. 11, 263-270.
168
SISTER .-M. LORETTA TER,~NDO .~_X;D DOROTHY FEIR
SL~d-~OTOSA. N. (1960) T h e conjugated plasma proteins of the American cockroach. J. gem Physiol. 43, 1015-1030. TELFER W. H. & WILLXa~xlSC. M. (1953) Qualitative and quantitative description of the blood antigens of the Cecropia silkworm. J. gen. Physiol. 36, 389--413. WYATT G. R. (1961) T h e biochemistry of insect hemol.vmph..4. R~'. Ent. 6, 75-102.
PROTEIN CONCENTRATION IN THE HEMOLYMPH OF THE LARGE MILKWEED BUG, O N C O P E L T U S F A S C I A T U S (DALLAS)* SISTER M. LORETTA TERANDO and DOROTHY FEIR Biology Department, St. Louis University, St. Louis, Missouri, 63104 (Received 1 November 1965)
A b s t r a c t - - 1 . Under all experimental conditions the protein concentration
of the hemolymph rose during the first half of the fifth stadium. During the second half of the fifth stadium the pattern is not clear. The concentration increases during the first 4 days of the adult stage and remains high until at least the fourteenth day post-molt. 2. In 1 lambda, non-pooled samples, a significant difference for mean protein concentration values was found between the following groups: (a) male and female adults, 5 days post-molt; (b) male and female adults, 10 days post-molt; and (c) the 10 per cent and 50 per cent fifth instar. 3. The concentration values reported can be used in comparative studies of protein concentration in insects of known age, sex, developmental stage and nutritional state; for studies on the hemolymph from the milkweed bug under various experimental conditions; and for comparison with other animals. INTRODUCTION A. CONSIDE1L~BLE amount of information is available on the constitution of the h e m o l y m p h of various insects (Gilbert & Schneiderman, 1961; Wyatt, 1961; Florkin & Jeuniaux, 1964). However, there are few studies which are detailed in regard to age, sex and developmental stage and which point out the statistical significance of differences between instars, sexes or individual insects. Since it is quite possible that the h e m o l y m p h constituents m a y vary u n d e r different physiological conditions such as post-molt age, instar and sex, these factors should be controlled in studies on the h e m o l y m p h . T h i s paper is a detailed study of the protein concentration of the h e m o l y m p h of the large milkweed bug, Oncopeltus f a s c i a t u s (Dallas), during the fifth stadium and during the first 14 days of the adult stage. MATERIALS AND M E T H O D S Newly molted bugs were collected during the day over periods of 8-14 hr. Male and female adults were separated on the day of molt. W a t e r and milkweed seeds were present at all times for all groups in order to insure the same nutritional * Supported by National Science Foundation Cooperative Fellowship to Sr. M. Loretta Terando and in part by U.S.P.H.S. Grant AI-04215 to D. Felt. 163
164
SISTER ~ [ . LORETTA TER.aNDO .~ND DOROTHY FEIR
state. At the time of bleeding the insects were anesthetized with COo, the antennae and legs were clipped and hemolymph was collected in capillary tubes. The amount of hemolymph taken from each bug varied from 1-5 Lambdas. The pooled samples were kept on ice in order to prevent melanization as no tyrosinase iahibitors were used because they interfered with protein determination. Samples to be filtered were passed through a Gelman membrane, pore size 5/x. Staining of this membrane showed that hemocytes were retained by it. The number of bugs used for the pooled samples ranged from 93 to 525. Non-pooled 1 lambda samples were put into solution by crushing the 1 lambda capillary, pipette with a glass pestle. All hemolymph samples were frozen at - 5 5 ° C and stored for 1 day to several weeks. Protein concentration was determined bv the Lowry method (Lowry et al., 1951). From the pooled samples a 50 iambda aliquot was taken and diluted to 10 ml with 0"7°I,,o saline. This dilution was found to lie within a convenient range on the bovine serum albumin standard curve. The 1 lambda samples were mixed with the reagents and the glass was centrifuged off before reading. One centrifugation at 3000 rev/min for 15 rain was sufficient to give a stable reading. The solutions were read at 750 m/~ with a red filter in a Spectronic 20. RESULTS In Fig. 1 values for not filtered, pooled samples are plotted. The ordinate indicates mg protein/ml of hemolymph. The abscissa is divided into two parts, the first part representing percentage of fifth stadium. Percentage of fifth stadium was used rather than days post-molt because the instars varied in the length of time required to molt to the adult. For example, fifth day post-molt for a bug with a fifth stadium of 10 days was not the same day chronologically as the fourth day post-molt of a bug with an 8 day stadium ; but both times represented 50 per cent of the stadium. The second part of the abscissa represents days after molting to the adult. During the first third of the stadium the values were 31-42 mg protein/ml hemolymph. Half-way through the stadium there was an increase to 56-58 mg protein/ml hemolymph. The 1 day post-molt adult had a protein concentration of 47 mg protein/ml hemolymph. The concentration increased and remained high for the adult until at least the fourteenth day. Values for the adult male and female protein concentrations were compared by paired sampling using a t-test. No significant difference was found. In Table 1 values for all the experimental conditions are summarized. When the filtered, pooled samples are examined, low initial values are seen in the fifth and adult stadia, with an increase midway through the fifth stadium and during the first 4 days of the adult stadium. In the seventh day post-molt adult the concentration was 73-76 mg protein/ml hemolymph, or about double the initial values for both the fifth instar and adult stages. Since about 0.1 ml of hemolymph was lost in the filtering process, it was necessary to test unfiltered samples for protein concentration. In general, the unfiltered samples have somewhat higher values, but the two periods of protein
PROTEIN
CONCENTP, ATION
IN
THE
HEMOLY%IPH
OF
THE
LARGE
MILKVCEED
165
BUG
increase over lower initial values noted in the filtered samples are also evident here. Paired sampling using a t-test indicated no significant difference between the two sets of data.
120
II0
I00 8---J,
p r
/
t
,'
i
~0
70
2:
60
.3
•
FEMALE
•
MALE
NOT NOT
FILTERED. FILTERED,
POOLED POOLED
20
E 10
20
30 OF
40 50 FIFTH
60 70 50 STADIUI!
90
i00
I
2 3 ADULT.
4
$ 6 7 DAYS POST-
8
9 I0 MOLT
11
13
FIG. 1. Comparison of male with female protein concentration using unfiltered, pooled hemolymph samples.
14
166
SISTER .~,1. LORETTA TER.~\'DO AND DOROTHY FEIR
T h e m e a n values for male and female 1 lambda, non-pooled samples follow the same trend of low initial values with an increase as development proceeds (Table 1). T h e range of values indicates the large individual variation. T h e mean TABLE 1--HEMoLYMPH PROTEINCONC'K-NTRATIONS Mg protein/ml hemolymph 1 lambda, not pooled Age (per cent of fifth stadium) 10"0 12"5 15"0 20"0 25"0 30"0 37'3 45"5 50"0 55"6 60"0 66"7 77"8 80"0 87"5 100"0 Adult (day~ post-molt) 1 2 3 4 5 6 7 8 10 14
Filtered, pooled Unfiltered, pooled Female
Male
34"9
38"1
37"3
43"1
36"4
40"0
48"9
47-0
36"4
33"9
51'0 36-2
51-0 39"1
30-1 35-1 62"7 77-0 69"9 74.7 76"0
36"3 32"7 62'2 68'9 68.8 74.0 73"1
Female
Male
31"1
32"8
31"9 35"5
40"0 39"1
41"1
42"2
50"0 56"2
53"2 58"2
47"3
50"5
54"2
50'9
57'7
57"4
47"3 46"2 54"3 65"2 94"4 81"5 83'2 96"5
47-0 48-6 64"2 65"8 75"8 78"2 66-3 75"8
95"1
70"5
Female
Male
Mean of 14 bugs Range
Mean of 14 bugs Range
35"8* 24"8-49"1
29"8 20"9-37"9
67"4* 49"7-78'9
68"5 57"2-84"9
48"1 41-0-54"6
45"2
53"6 43"1-64"8
48"3 37-9-59"6
35-3-55"4
lO0"6t 73"7-123"3 86"2t 58"3-110-8
116"65 94"3-143"7 86-8** 57-2-110"5
Significant differences: * P<0"01 ; t P<0"02; :~ P<0"001. values for the unfiltered pooled samples do, however, lie within the range of the 1 lambda, non-pooled samples. As described above, these 1 lambda samples were not collected and prepared by the same m e t h o d as the pooled samples, so this m a y explain the somewhat higher values.
PROTEIN
C O N C E N T R A T I O N I N THE H E M O L Y ~ I P H OF THE LARGE M I L K W E E D B U G
167
DISCUSSION It is interesting to compare these results with those from studies on other insects. Nowesielski & Patton (1965) reported that hemolymph from the last two instars and newly molted adult of the house cricket, Acheta domesticus L., has a high protein concentration. In the less than 1 day old adult female the concentration is 69 mg/ml. The level drops shortly after the final molt and by the tenth day of adult life it has dropped by about 20 per cent (to 56 mg in the female). Siakotos (1960) found the protein concentration of the hemolymph of the American cockroach, Periplaneta americana L., decreased by two-thirds on the fourth day after the last nymphal molt, but by the eighth day it was back to the initial level. In the adult, the concentration decreased by the fourth day after molt to the adult stage. The decline continued until, by the eighth day, the protein concentration had decreased to one-half the level at the time of molt. Studies on moths by Laufer (1960) and Teller & Williams (1953) indicate an increase in protein in the last larval instar during the time of increased prothoracic hormone activity. However, as the adult developed the protein concentration decreased. The data on the milkweed bug indicate a rise in protein concentration about half-way through the fifth stadium, paralleling the rise found in other insects. Probably this reflects increased protein synthesis under the influence of ecdysone. .Mthough the information available is limited, the increasing protein concentration in the adult seems unique to the milkweed bug. It may reflect continued deposition of endocuticle (Dingle, 1965) or perhaps a type of protein storage, since the bugs continue to feed and maintain their weight during the times studied. The filtered samples are probably nearest the true value of the protein concentration of the hemolymph, since by this method the hemocytes were removed. However, considering the large numbers of insects which are needed in order to get a large enough sample to filter, the slight error which might be introduced by the presence of hemocytes is negligible when the saving in time and specimens is considered. Determinations on 1 lambda samples are possible and this is the only method used which permits estimation of range.
REFERENCES DINGLE H. (1965) The relation between age and flight activity in the milkweed bug, Oncopeltus. 07. exp. Biol. 42, 269-283. FLOmCIN M. & JEVNIAtrXCH. (1964) Hemob~mph: Composition. In The Physiology of the Insecta (Edited by ROCmSTEINM.), Vol. III, Chapter 2. Academic Press, New York. GILBERT L. I. & SCm~qZIDE~.'~tANH. A. (1961) Some biochemical aspects of insect metamorphosis. Am. Zool. 1, 11-51. L^trt~R H. (1960) Blood proteins in insect development. Ann. N. Y. Acad. Sd. 89, 490-515. Low~v O. H., ROSEBROUGHN. J., FAaR A. L. & RANDALLR. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. NowosmtsKZ J. W. & PATTONR. L. (1965) Variation in the hemolymph protein, amino acid, and lipid levels in adult house crickets, Acheta domesticus L., of different ages. J. Insect Physiol. 11, 263-270.
168
SISTER .-M. LORETTA TER,~NDO .~_X;D DOROTHY FEIR
SL~d-~OTOSA. N. (1960) T h e conjugated plasma proteins of the American cockroach. J. gem Physiol. 43, 1015-1030. TELFER W. H. & WILLXa~xlSC. M. (1953) Qualitative and quantitative description of the blood antigens of the Cecropia silkworm. J. gen. Physiol. 36, 389--413. WYATT G. R. (1961) T h e biochemistry of insect hemol.vmph..4. R~'. Ent. 6, 75-102.