Comp Biochem. Physiol.. 1977. Vol. 56B, pp. 169 to t73. Pee~lamOn Press. Printed in Great Britain
BIOCHEMICAL STUDIES OF TICK EMBRYOGENESIS. FREE AMINO ACID POOLS DURING EMBRYOGENESIS OF DERMACENTOR ANDERSONI* FOUAD N. BOCTOR'~ AND MA,~:~OC~ Y. KAMEL++ Medical Zoology Department. United States Naval Medical Research Unit Number Three (NAMRU-3), American Embassy, Cairo. Arab Republic of Egypt
(Receired 16 June 1976) Abstraet--l. During embryogenesis of the tick Dermacentor andersoni, amino acid concentrations could be separated into 3 groups. In (a) there was a slight increase on day 4 of embryogenesis followed by a sharp drop on days 8 and 12 and increase in newly-hatched larvae (day 16) (glutamic acid, aspartic acid. threonine, serine, tyrosine, phenylalanine, histidine, and methionin¢). In (b) there was a sharp drop on days 4 and 8 and increase in larvae (alanine, proline, and leucine). In (c) there was a slight increase on day 4 followed by a continuous decrease (valine, glycine + isoleucine, and lysine). 2. Total amino acid concentrations decreased during embryogenesis. No change was observed in total proteins. 3. Glutamate-pyruvate transaminase (GPT) activity significantly increased during embryogenesis but glutamate-oxalacetate transaminase (GOT) activity did not change.
INTRODUCTION
Tatchell (1969) and ~eh~i~ek & Brzost0wski (1969) demonstrated that the amino acid composition of haemolymph and saliva differs in insects and in the cattle tick, Boophilus microplus. They ruled out amino acid involvement in ionic regulation in ticks. Total free amino acids (TFAA) in argasid tick haemolymph, gut fluid, and coxal fluid were determined before and after nymphal and adult Argas (Persicargas) persicus and ,4. (P.) arboreus fed on birds (Boctor & Araman, 1971). The TFAA concentration in ha~molymph in these tick species and in exopterygote insects is lower than that in endopterygote insects (Florkin & Jeuniaux, 1964). In female A. (P.) arboreus, glutamic acid predominates in haemolymph and phenylalanine in coxal fluid. The glutamic acid and valine concentrations in the tick gut fluid and in the host blood are relatively high (Boctor, 1972). During embryogenesis, protein reserves in the egg break down to form new tissue proteins. Therefore, analysis of free amino acids during embryogenesis contributes to elucidating the process of biochemical
conversion of pre-existing proteins to tissue proteins. Information on the free amino acids pool and on changes during embryogenesis is thus vital for further biochemical investigations. However, very little is known about the biochemistry of tick embryogenesis (Diehl, 1970; Jenni, 1971; Tatchell, 1971; Boctor & Kamel, 1976). The aim of this study was to investigate changes in amino acids within the egg of Dermacentor andersoni Stiles during embryogenesis and in newly hatched larvae. The results should be helpful in the development of a tissue culture medium for propagating embryonic cells of this tick species, which is a vector of the rickettsia causing Rocky Mountain spotted fever, of the virus causing Colorado tick fever, and of the agents of several other diseases of humans and wild and domestic animals. The application and usefulness of arthropod tissue culture in studies on arboviruses and rickettsiae has been reviewed by Yunker (1971). MATERIALS AND METHODS
Tick colony
*From Research Project MF51.524.009-0055, Naval Medical Research and Development Command, National Dermacentor aadersoni specimens from Montana, U.S.A., Naval Medical Center, Bethesda, MD, U.S.A. The opinions supplied by Dr. C. E. Yunker of the Rocky Mountain and assertions contained herein are the private ones of Laboratory, were used to establish a colony in the NAMthe authors and are not to be construed as official or as RU-3 Medical Zoology Department. Larvae, nymphs, and reflecting the views of the Department of the Navy or of females were fed on white Bnskat rabbits. All nonfeeding the naval service at large. This study was assisted by Agree- tick developmental stages were maintained at 28°C and ments 03-016-1 and 03-036-N between the National Insti- 75% relative humidity. tute of Allergy and Infectious Diseases (National Institutes TFAA determination of Health) and NAMRU-3. f Present address: Biochemistry Laboratory, Cancer InTFAA were determined with the ninhydrin rea~nt stitute, National Institutes of Health, Bcthesda, MD 20014, (Rosen, 1957). Leucine was the reference standard. U.S.A. Request reprints from Medical Zoology DepartHistidine determination ment, NAMRU-3, FPO, New York 09527. + Biochemistry Laboratory, National Research Center, Histidine was determined colorimetricaUy using Paul's Dokki, Cairo. sulfanilic acid method (Macpherson, 1942). 169
170
FOL:.~,D N. BOCTORA?,iD .~IAMDOLHY. KA>IEL Table l. Changes in TFAA. total protein. GOT, and GPT during embryogenesis of Dermacentor cmdersoni ~.ge Iday',p
TF ~ -~, mg S eggs
Total protein rag, 10~)m s ~ggs
194 16.8 12.0 88 16 2
242 24.5 22.0 22.2 227
I {o t-LDO,LCiOnl 4 8 12 16 /lar,,ael
lmticidual amino acid determination
GOT unit mg t 7 19 20 21
G PT umt mg
~__0 2 30 Z 0 t Z 03 4 5 _- 0 5 _~ 0 2 5.0 : 0 3 -1) 5 0a. -__ITS not determined
The chromatography was run at the following temperatures ('C): injector (200). column (initial 70. maximum 210, programming 4 mint, detector (250). The nitrogen carrier gas was at 50 mbmin. The mg quantities of the individual amino acids were determined by the internal standard method of Gehrke et al. 11968).
The egg homogenates (i00 mg) and the other biological fluids were homogenized in 10 ml of distilled water and deproteinized with 50 ml of 10~, picric acid. The solutions were left for 1 hr and centrifuged at 6000 g ; the supernatant was passed through a Dowex 2x-8 column to remove excess picric acid (Moore & Stein. 1951). The eluant was shaken with ether and the ether layer discarded. The samples were concentrated in a rotary evaporator at 6 0 C under reduced pressure; the residue was kept at -20-'C until analysis. The free amino acia8 were analyzed by gasliquid chromatography as N-trifluoroacetyl-n-butyl derivatives according to Gehrke et aL (1968). The analyses were carried out in a Hewlett Packard gas-liquid chromatograph model 5750 equipped with a flame ionization detector and a strip chart recorder with disc integrator. The boro-silicate glass column (180cm long. 4ram i.d.) was packed with 1"~, (w/w) ethyleneglycoktdipate coated on 100~120 mesh preheated chromosorb G (Applied Science La boratories).
Transaminase determinations The glutamatc-pyruvate and glutamate-oxalacetate transaminases in e~,~, homogenates were determined according to Sigma Tech. Bull. 505 (Sigma Chemical Co., St. Louis, May 1963). R ES U L T S
Changes in TFAA (ninhydrin-reacting substances) and proteins T F A A concentrations determined by the ninhydrin method decreased continuously but no changes in
Table 2. Concentrations of individual free amino acids in the rabbit host serum and in the female haemolymph and egg homogenate of Dermacentor andersoni Rabbit host serum -'~mmo acid Alanine Valine Glycine + isoteucine
Leucine Proline Threonine Serine Cysteine Methionine Phenylalanine
Lysine
mg'100 ml
",,
rag, I00 g
'!~
43 l.l 8.2 0.5 1.2 0.8 2.4
152 3.8 28.7 1.7 42 2.8 8.4
9.9 35.3 11.8 144 19.6 6.4 26.5
377 13.1 4.4 51 7.3 3.9 9.8 -6.1 64 8.8 16 I 7.5
32.4 1030 430 I22.9 105.8 35.6 347 132 137 45.4 47.7 t433 789
33 10.4 4.3 12.4 I 0.7 3.6 3.5 13 1.4 4.6 4.8 14.5 7.9 -10.7 65
-
trace
-
03 0.7 0.9 5.1 0.4
11 2.4 3.1 17.8 1.4
16.4 17.2 23.7 43.3 20.I
present
--
present
--
present
7.92 16.96
2.9 6.3
106.0 64.3
I2 14
Histidine
Egg homogenate
",,
trace
Aspartic acid Glutamic acid Tyrosine Ornithinc
Haemolymph
rag.' t00 ml
42 4.9
Table 3. Changes in free individual amino acids during embryonic development of D. andersoni by gas-liquid chromatography (3 batches were used) I)a}
0
.1
R
Amino a,ad
,~g .g eggs
%,
l~g,g eggs
",,
Alanine Valine Glycine +
324 Z 50" 1030 ~ 199
330 10.39
t65 ± 8 1360 4- 198
isoleucine Leucine Proline Threonine Seriae Cysteine
43(} 1229 1058 356 347 132 137 454 477
± 20 ± 343 ~ 315 +_ 46 + 192 ± 98 _+ 34 ~ 79 -'. 151
433 I2.40 10.67 3.59 3.50 1.33 I 38 4.58 4.81
585 ± 28 689 _ 69 835 _ 164 435 _ 96 425 + 62 trace I85 ± 3S 610 +_. 177 532 _~ 112
1433 __. 253 789 _+ 2t0 1060 = 370 643 -.- 360 9909
14.46 7.93 1069 6.48
Methionine Phenylalanine
Aspartic acid Glutamic acid + glutamine Tyrosine Lysine
Histidine Total
* Standard deviation.
2890 1185 945 732 11493
+ 376 = 543 _ 343 _~ 125
12
16
#g.s eggs
",,
,~gjg eggs
'!.i
~g 8 tar',ae
%,
1.43 11.83
93 ~ 18 462 ~ 164
151 784
126 + 12 302 4- 30
30-1 7.28
305 - 34 215 _.- 19
3.94 2.78
5.09 5.99 7.26 3.78 3.69 -0.91 5.30 4.62
194 + I46 362 ± 90 116 ± 34 114 + 32 141 ~ 99 trace 120 _+ 67 255 4- 112 336 ± 123
3.29 6.14 1.94 1.93 2.39 -2.03 4.32 57
228 + 3..I. 252 a. l l 8 376 + 176 229 _ 62 115 ± 29 trace 55 _4- 19 102 ± 18 208 ± 63
5.50 6.08 9.07 550 2.77 I 1.32 2.46 502
21I ___ 46 399 --. 203 796 ± 30.1 367 s 120 335 -2 67 tra~ 270 - 2.13 226 - 86 455 z 249
2.73 5.16 10.3 4.75 4 33 -3.44 2.92 5sg
25.14 10.31 822 6.36
2355 272 420 652 58924
+ 220 + 89 .-%.I01 ± 156
39.96 4.61 7. L2 1106
973 + 4.4.6 ± 274 +. 262 + 4140
198 212 77 93
23.48 10.76 6.61 6.32
1972 1067 328 463 7722
_- 715 ± 454 -_' 169 _- 135
25.53 13.81 424 5.99
Biochemical studies of tick emb~'ogenesis
17
15
15
1"5
I-0
t0
1,0,
iO
0.5
5
0-5;
5
1"5
TYROSIN~
L
I
///
I
.it
=
PROLINE
I
I
I
1
4"0 "GLUTAMIC+t,,~ GLUTAMINE t3 bJ i • 3'0 E 2"0
BIOCHEMICAL STUDIES OF TICK EMBRYOGENESIS. FREE AMINO ACID POOLS DURING EMBRYOGENESIS OF DERMACENTOR ANDERSONI* FOUAD N. BOCTOR'~ AND MA,~:~OC~ Y. KAMEL++ Medical Zoology Department. United States Naval Medical Research Unit Number Three (NAMRU-3), American Embassy, Cairo. Arab Republic of Egypt
(Receired 16 June 1976) Abstraet--l. During embryogenesis of the tick Dermacentor andersoni, amino acid concentrations could be separated into 3 groups. In (a) there was a slight increase on day 4 of embryogenesis followed by a sharp drop on days 8 and 12 and increase in newly-hatched larvae (day 16) (glutamic acid, aspartic acid. threonine, serine, tyrosine, phenylalanine, histidine, and methionin¢). In (b) there was a sharp drop on days 4 and 8 and increase in larvae (alanine, proline, and leucine). In (c) there was a slight increase on day 4 followed by a continuous decrease (valine, glycine + isoleucine, and lysine). 2. Total amino acid concentrations decreased during embryogenesis. No change was observed in total proteins. 3. Glutamate-pyruvate transaminase (GPT) activity significantly increased during embryogenesis but glutamate-oxalacetate transaminase (GOT) activity did not change.
INTRODUCTION
Tatchell (1969) and ~eh~i~ek & Brzost0wski (1969) demonstrated that the amino acid composition of haemolymph and saliva differs in insects and in the cattle tick, Boophilus microplus. They ruled out amino acid involvement in ionic regulation in ticks. Total free amino acids (TFAA) in argasid tick haemolymph, gut fluid, and coxal fluid were determined before and after nymphal and adult Argas (Persicargas) persicus and ,4. (P.) arboreus fed on birds (Boctor & Araman, 1971). The TFAA concentration in ha~molymph in these tick species and in exopterygote insects is lower than that in endopterygote insects (Florkin & Jeuniaux, 1964). In female A. (P.) arboreus, glutamic acid predominates in haemolymph and phenylalanine in coxal fluid. The glutamic acid and valine concentrations in the tick gut fluid and in the host blood are relatively high (Boctor, 1972). During embryogenesis, protein reserves in the egg break down to form new tissue proteins. Therefore, analysis of free amino acids during embryogenesis contributes to elucidating the process of biochemical
conversion of pre-existing proteins to tissue proteins. Information on the free amino acids pool and on changes during embryogenesis is thus vital for further biochemical investigations. However, very little is known about the biochemistry of tick embryogenesis (Diehl, 1970; Jenni, 1971; Tatchell, 1971; Boctor & Kamel, 1976). The aim of this study was to investigate changes in amino acids within the egg of Dermacentor andersoni Stiles during embryogenesis and in newly hatched larvae. The results should be helpful in the development of a tissue culture medium for propagating embryonic cells of this tick species, which is a vector of the rickettsia causing Rocky Mountain spotted fever, of the virus causing Colorado tick fever, and of the agents of several other diseases of humans and wild and domestic animals. The application and usefulness of arthropod tissue culture in studies on arboviruses and rickettsiae has been reviewed by Yunker (1971). MATERIALS AND METHODS
Tick colony
*From Research Project MF51.524.009-0055, Naval Medical Research and Development Command, National Dermacentor aadersoni specimens from Montana, U.S.A., Naval Medical Center, Bethesda, MD, U.S.A. The opinions supplied by Dr. C. E. Yunker of the Rocky Mountain and assertions contained herein are the private ones of Laboratory, were used to establish a colony in the NAMthe authors and are not to be construed as official or as RU-3 Medical Zoology Department. Larvae, nymphs, and reflecting the views of the Department of the Navy or of females were fed on white Bnskat rabbits. All nonfeeding the naval service at large. This study was assisted by Agree- tick developmental stages were maintained at 28°C and ments 03-016-1 and 03-036-N between the National Insti- 75% relative humidity. tute of Allergy and Infectious Diseases (National Institutes TFAA determination of Health) and NAMRU-3. f Present address: Biochemistry Laboratory, Cancer InTFAA were determined with the ninhydrin rea~nt stitute, National Institutes of Health, Bcthesda, MD 20014, (Rosen, 1957). Leucine was the reference standard. U.S.A. Request reprints from Medical Zoology DepartHistidine determination ment, NAMRU-3, FPO, New York 09527. + Biochemistry Laboratory, National Research Center, Histidine was determined colorimetricaUy using Paul's Dokki, Cairo. sulfanilic acid method (Macpherson, 1942). 169
170
FOL:.~,D N. BOCTORA?,iD .~IAMDOLHY. KA>IEL Table l. Changes in TFAA. total protein. GOT, and GPT during embryogenesis of Dermacentor cmdersoni ~.ge Iday',p
TF ~ -~, mg S eggs
Total protein rag, 10~)m s ~ggs
194 16.8 12.0 88 16 2
242 24.5 22.0 22.2 227
I {o t-LDO,LCiOnl 4 8 12 16 /lar,,ael
lmticidual amino acid determination
GOT unit mg t 7 19 20 21
G PT umt mg
~__0 2 30 Z 0 t Z 03 4 5 _- 0 5 _~ 0 2 5.0 : 0 3 -1) 5 0a. -__ITS not determined
The chromatography was run at the following temperatures ('C): injector (200). column (initial 70. maximum 210, programming 4 mint, detector (250). The nitrogen carrier gas was at 50 mbmin. The mg quantities of the individual amino acids were determined by the internal standard method of Gehrke et al. 11968).
The egg homogenates (i00 mg) and the other biological fluids were homogenized in 10 ml of distilled water and deproteinized with 50 ml of 10~, picric acid. The solutions were left for 1 hr and centrifuged at 6000 g ; the supernatant was passed through a Dowex 2x-8 column to remove excess picric acid (Moore & Stein. 1951). The eluant was shaken with ether and the ether layer discarded. The samples were concentrated in a rotary evaporator at 6 0 C under reduced pressure; the residue was kept at -20-'C until analysis. The free amino acia8 were analyzed by gasliquid chromatography as N-trifluoroacetyl-n-butyl derivatives according to Gehrke et aL (1968). The analyses were carried out in a Hewlett Packard gas-liquid chromatograph model 5750 equipped with a flame ionization detector and a strip chart recorder with disc integrator. The boro-silicate glass column (180cm long. 4ram i.d.) was packed with 1"~, (w/w) ethyleneglycoktdipate coated on 100~120 mesh preheated chromosorb G (Applied Science La boratories).
Transaminase determinations The glutamatc-pyruvate and glutamate-oxalacetate transaminases in e~,~, homogenates were determined according to Sigma Tech. Bull. 505 (Sigma Chemical Co., St. Louis, May 1963). R ES U L T S
Changes in TFAA (ninhydrin-reacting substances) and proteins T F A A concentrations determined by the ninhydrin method decreased continuously but no changes in
Table 2. Concentrations of individual free amino acids in the rabbit host serum and in the female haemolymph and egg homogenate of Dermacentor andersoni Rabbit host serum -'~mmo acid Alanine Valine Glycine + isoteucine
Leucine Proline Threonine Serine Cysteine Methionine Phenylalanine
Lysine
mg'100 ml
",,
rag, I00 g
'!~
43 l.l 8.2 0.5 1.2 0.8 2.4
152 3.8 28.7 1.7 42 2.8 8.4
9.9 35.3 11.8 144 19.6 6.4 26.5
377 13.1 4.4 51 7.3 3.9 9.8 -6.1 64 8.8 16 I 7.5
32.4 1030 430 I22.9 105.8 35.6 347 132 137 45.4 47.7 t433 789
33 10.4 4.3 12.4 I 0.7 3.6 3.5 13 1.4 4.6 4.8 14.5 7.9 -10.7 65
-
trace
-
03 0.7 0.9 5.1 0.4
11 2.4 3.1 17.8 1.4
16.4 17.2 23.7 43.3 20.I
present
--
present
--
present
7.92 16.96
2.9 6.3
106.0 64.3
I2 14
Histidine
Egg homogenate
",,
trace
Aspartic acid Glutamic acid Tyrosine Ornithinc
Haemolymph
rag.' t00 ml
42 4.9
Table 3. Changes in free individual amino acids during embryonic development of D. andersoni by gas-liquid chromatography (3 batches were used) I)a}
0
.1
R
Amino a,ad
,~g .g eggs
%,
l~g,g eggs
",,
Alanine Valine Glycine +
324 Z 50" 1030 ~ 199
330 10.39
t65 ± 8 1360 4- 198
isoleucine Leucine Proline Threonine Seriae Cysteine
43(} 1229 1058 356 347 132 137 454 477
± 20 ± 343 ~ 315 +_ 46 + 192 ± 98 _+ 34 ~ 79 -'. 151
433 I2.40 10.67 3.59 3.50 1.33 I 38 4.58 4.81
585 ± 28 689 _ 69 835 _ 164 435 _ 96 425 + 62 trace I85 ± 3S 610 +_. 177 532 _~ 112
1433 __. 253 789 _+ 2t0 1060 = 370 643 -.- 360 9909
14.46 7.93 1069 6.48
Methionine Phenylalanine
Aspartic acid Glutamic acid + glutamine Tyrosine Lysine
Histidine Total
* Standard deviation.
2890 1185 945 732 11493
+ 376 = 543 _ 343 _~ 125
12
16
#g.s eggs
",,
,~gjg eggs
'!.i
~g 8 tar',ae
%,
1.43 11.83
93 ~ 18 462 ~ 164
151 784
126 + 12 302 4- 30
30-1 7.28
305 - 34 215 _.- 19
3.94 2.78
5.09 5.99 7.26 3.78 3.69 -0.91 5.30 4.62
194 + I46 362 ± 90 116 ± 34 114 + 32 141 ~ 99 trace 120 _+ 67 255 4- 112 336 ± 123
3.29 6.14 1.94 1.93 2.39 -2.03 4.32 57
228 + 3..I. 252 a. l l 8 376 + 176 229 _ 62 115 ± 29 trace 55 _4- 19 102 ± 18 208 ± 63
5.50 6.08 9.07 550 2.77 I 1.32 2.46 502
21I ___ 46 399 --. 203 796 ± 30.1 367 s 120 335 -2 67 tra~ 270 - 2.13 226 - 86 455 z 249
2.73 5.16 10.3 4.75 4 33 -3.44 2.92 5sg
25.14 10.31 822 6.36
2355 272 420 652 58924
+ 220 + 89 .-%.I01 ± 156
39.96 4.61 7. L2 1106
973 + 4.4.6 ± 274 +. 262 + 4140
198 212 77 93
23.48 10.76 6.61 6.32
1972 1067 328 463 7722
_- 715 ± 454 -_' 169 _- 135
25.53 13.81 424 5.99
Biochemical studies of tick emb~'ogenesis
17
15
15
1"5
I-0
t0
1,0,
iO
0.5
5
0-5;
5
1"5
TYROSIN~
L
I
///
I
.it
=
PROLINE
I
I
I
1
4"0 "GLUTAMIC+t,,~ GLUTAMINE t3 bJ i • 3'0 E 2"0
