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Disc electrophoresis of septicemic and melanized plasma from greater wax moth larvae, Galleria mellonella
Disc Electrophoresis of Septicemic and Melanized Plasma from Greater Wax Moth Larvae, Galleria mellonella’ DOSALI) Department
M.
MCKINSTRY”
of Entomology,
AND
Llniversity Received
of Maryland, November
L.
STEINHAUER
College
Park,
ALLES
26,
Maryland
707d2
1969
Disc electrophoresis of fresh plasma from mature wax moth larvae. Gderia mellonellu, yielded 16 protein fractions. Septicemia resulting from infection with Pseudomonas aeruginosu reduced the amount of protein in five fractions, with maximum reduction occurring 12 hr after inoculation. The fraction showing the greatest reduction was the third most proximal migrating band, with evident change apparent as early as 2 hr after inoculation. Infected larvae began to show symptoms at 6 hr and died about 12 hr after inoculation. Blood from uninfected larvae melanized in vitro gave electropherograms that resembled those obtained from terminal ( 12 hr postinoculation) septicemic plasma Five fractions showed reduced protein. The greatest reduction again occurred in fraction 3.
Thompson, and Cantwell ( 1962). Only mature larvae, about l-month-old, with an average weight of 0.2 g/larva, were used for experimental purposes. A lyophilized culture of Pseudomonas aeruginosa, ATCC No. 10145, was subcultured on nutrient agar slants and stored at 5°C. Still nutrient broth cultures, 15 ml incubated at 37OC for 24 hr, served as the inoculum source for larval injections. Inoculum quantitation involved the pour plate method. Specifics relative to this method included the use of 9 ml Dulbecco’s phosphate-buffered saline blanks, triplicate plating, and colony counting after 24 hr incubation at 37°C. Cultures contained bctween 2.44 X lo8 and 4.90 X lo* bacteria/ ml. Thus 3 ~1 of inoculum, the dose used for larval injections, contained between 7.32 X 1W and 1.47 X 10” bacteria. Preliminarv studies indicated that this dosage woild give 100% mortality within 11 to i4 hr postinoculation (rc~ferred to as P. T. hen,after ) . Disc clectrophoresis methods were adapted from those of the Canal Industrial Corporation ( 1962). Modifications of the
Plasma electropherograms from diseased insects may show protein depletion while plasma from nondiseased larvae melanized in vitro shows similar change (Bullock, 1963). To further explore this phenomenon, we adapted high resolution disc electrophoresis for separation of plasma proteins from mature wax moth, Galleria mellonella, larvae and then compared electrophoregrams of bacteremic and in vitro melanized Pl asma. MATERIALS
AND
METHODS
Galleria mellonella larvae were reared at 34°C according to the method of Dutky, 1 Scientific Article No. A1573, Contribution NO. 4287, of the Maryland Agricultural Experiment Station, College Park, Maryland 20742. This report constitutes part of a thesis submitted by the senior author to the Graduate School of the University of Maryland in partial fulfillment of the requirements for the M. S. degree. The investigation was sup ported by N.S.F. Grant GB-2849. 2 Present address, Department of Dairy Science, University of llaryland, College Park, Maryland 20742. 123
124
RICKISSTHY
AXD
standard procedures included: (1) 5 (i.d.) X 65-mm glass tubes containing 25 mm of 6.5 acrylamide gel overlayered with 20 mm of 5% acrylamide gel, 10 mm spacer gel and 10 mm sample gel; (2) 12 tube runs of about 55 min using a constant 200 V; (3) 2 ~1 of fresh larval plasma per tube. The standard 5 X-pH 8.3 glycine buffer was used. Protein fractions, stained with amido Schwartz, were quantitated using a Canalco Model E microdensitometer. Septicemic Plasma Procedures. Three groups of 11 larvae were each injected with 3 ~1 of inoculum per individual while an additional three groups of 11 larvae used as controls each received a 3-$ injection of sterile nutrient broth. One replicate was conducted for each group. In each group hemolymph was collected from six larvae while the other five larvae were held for observation at 24 hr P. I. An electric microinjector employing a 0.25~~ tuberculin syringe and a 27 gauge needle was used for the hemocoelic inoculation of the nitrogenanesthetized larvae. The site of injection was the lateral area of the cuticle near the prolegs. Following injection the larvae were held at 34°C. Hemolymph was collected from one group of infected larvae and one group of control larvae at separate times, either 2, 6, or 12 hr P. I. Larvae were heatfixed, to prevent melanization, immediately prior to sampling. Fixation involved larval immersion in a 60°C water bath for 1 min. The larvae were then punctured in one of the prolegs and the hemolymph collected in capillary tubes. The hemolymph was centrifuged for 5 min at 11,500 rpm (13,000 g) in an International Model MB microcapillary centrifuge. The resulting plasma from each group was pooled and subjected immediately to disc electrophoresis. Six gel tubes were run for each sample. Following protein staining, fraction densities were determined using five gels from each sample. Mehized Plasmu Procedures. Hemolymph-was taken from 6 larvae and pooled
STEINHAUER
as previously described. However, these larvae were not infected with bacteria nor heat-fixed prior to sampling. From another 6 nonheat-fixed larvae, hemolymph was withdrawn, pooled, and stirred at 24°C fol 3 min during which time it became black (melanized) . One replicate was conducted for each group. Following electrophoresis and protein staining, fraction densities were determined using five gels from each pooled sample. RESULTS
Sixteen protein fractions, numbered from 1 to 16 in order of increasing mobility, were observed following electrophoresis of control, septicemic, and melanized plasma (Fig. 1). Fractions 12, 13, and 14 were faint, and the presence of one or more was sometimes doubtful. Several fractions, sometimes coalesced, were combined for the purpose of quantitation as follows: 1 + 2; 8 + 9; 15 + 16. Electropherograms of septicemic plasma displayed the following trends when compared to control electropherograms, during the 12 hr disease period (Fig. 1, Table 1). Fractions 1 and 2 appeared coalesced after 6 hr P. I. Fraction 3, depressed at 2 hr P. I., became greatly reduced by 12 hr P. I. Fractions 7, 8 + 9, 10, and 11 were reduced by 12 hr P. I. No changes or slight increases were noted in fractions 4, 5, 6, and 15 -I- 16. Symptoms displayed by septicemic larvae were observed. Slight behavioral irregularities, first observed at 6 hr P. I., became pronounced by 8 and 10 hr P. I. as evidenced by slower crawling and spasmodic twitching. The cuticle and hemolymph appeared darker than normal. Death, as ascertained by cessation of motion, occurred between 11 and 13 hr P. I. Mortality, as observed at 24 hr P. I., was 100% among infected larvae, and pronounced blackening was noted. Mortality among the control animals was less than 1%.
: A
E3
A
C
A
FIG. 1. Disc electropherograms of control (A), 2 hr P. I. (B). and in vitro melanized (E) plasma from mntnre Gnllcricr rndTo~rr~l/n inoculation with P.wrdomonas aeruginosa.
Electropherograms of melanized plasma displayed the following trends when compared to control electropherograms (Fig. 1, Table 1). Fraction 1 -I- 2 was reduced. Fraction 3 was greatly reduced. Fractions 8 + 9, 10, and 11 were reduced. No marked TABLE .I THE PEHCEN,~A(:E INCREASE OR DECREASE IX ‘~HK \T~~~~~~ PROTEIN FRACTIONS OF PLASMA FRO\I Galleria LARVAE 2, 6, AND 12 HR AFTER IV~~UL.~TI~N WITH Pserrdomonns acrc4ginosa, AND AFTER ~IELAXIZATION “j, Increase or decrease plasma protein
_ .After
Protein fraction number
inoculation
with
P. aerrrginosa
1+2 3 1 5 6 7 X+9 10 11 155 + 16
2 hr
6hr
12 hr.
$12 -38 -s +4 +5 -9 -15
$44 -65 -11 +7 +2 -12 -17 + 10 - 6 +4
+8 -80 +20 +13 +14 -30 -30 -30 -38 +12
0 f21 +19
in -. After melanization -60 -75 +5 f2 f12 t-13 -45 -25 -47 +20
0
A
6 11r 1’. I. (C), lar\
J<‘. 1”. 1. I-efrw
E’
1” hr P. 1. (I),, to 11r111rs Ie)‘t-
Infection appears to alter insect hemolymph proteins as dotermined by electrophoresis. Barlow ( 1962), Bullock ( 1963 ) , Watanabe ( 1967)) and Bennett ct al. (1968) h avc o 1)scrved aberrant electropherograms of hemolymph from infected insects. Protein depletion, pnrticularlv in one or two fractions, was the most common abnormality observed. These studies represented a range of hosts, pathogens, and clectrophoretic techniques. In vitro melnnization of insect htmolymph also gives altcrrd clectrophoretic~ patterns as observed by Bullock ( 1963) and Barry (1964). In both studies the decrcasca of a low mobility fraction was reported. Our results are in general agreement with the above observations since we noted protein depletion with preferential clccreasc ill clectropherograrns of septiccmic Wld plasma melanizcd in vitro. The simiIarit!. bctwecn thcsc two types of clcctrophrro-
126
MCKINSTHY
AND STEINHAUER
grams is probably a reflection of melanization during septicemia since the hemolymph and cuticle appeared darker during the terminal stage of infection. Thomson (1962) states that natural melanins are often conjugated with protein. Perhaps the depleted proteins we observed were bound to melanin which showed no electrophoretic mobility. The repeated observations of the similarities in hemolymph protein electropherograms of diseased insects and melanized insect hemolymph in vitro suggest a relationship between melanization and insect defense against pathogens. One possibility seems to be that proteins which conjugate with quinones to form melanin are capable of conjugating in some way with foreign pathogens in a nonspecific type of resistance to infection. It is presently premature to try to fit this idea to existing evidence, but the process merits further investigation. ACKNOWLEDGMENT The technical staff is gratefully
assistance of Mrs. acknowIedged.
Maxine
Blicken-
REFERENCES BARLOW, J. S.
1962. An effect of parasitism on hemolymph electropherograms. J. Insect Pathol., 4, 274-275. BARRY, C. 1964. Hemolymph proteins in the molting cycle of insects. Ph.D. Thesis. Univ. of Maryland, College Park, Md. 140 pp. BENNETT, G. A., SHOTWELL, 0. L., HALL, H. H., AND HEARN, W. R. 1968. Nemolymph proteins of healthy and diseased larvae of the Japanese beetle Popillia japonica. J. Invertebr. Puthol., 11, 112-118. BULLOCK, H. R. 1963. A study of hemolymph proteins of insects in relation to melanization and natural defense against microorganisms. Ph.D. Thesis. Univ. of Maryland, College Park, Md. 160 pp. CANAL INDUSTRIAL CORPORATION. 1962. Canalco Model 12 disc electrophoresis instructions. Rockville, Maryland. 8 pp. DUTKY, S. R., THOMPSON, J. V., AND CANTWELL, G. E. 1962. A technique for mass rearing the greater wax moth (Lepidoptera: Galleriidae). Proc. Entomol. Sot., Wash., 64, 5&58. THOMSON, R. H. 1962. Melanins. In “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds. ),” pp. 727-753. Academic Press, New York. WATANABE, H. 1967. Electrophoretic separation of the hemolymph proteins in the fall webworm, Hyphantria cunea, infected with a nuclear-polyhedrosis virus. .l. Invertebr. Pathol., 9, 570-571.
M.
MCKINSTRY”
of Entomology,
AND
Llniversity Received
of Maryland, November
L.
STEINHAUER
College
Park,
ALLES
26,
Maryland
707d2
1969
Disc electrophoresis of fresh plasma from mature wax moth larvae. Gderia mellonellu, yielded 16 protein fractions. Septicemia resulting from infection with Pseudomonas aeruginosu reduced the amount of protein in five fractions, with maximum reduction occurring 12 hr after inoculation. The fraction showing the greatest reduction was the third most proximal migrating band, with evident change apparent as early as 2 hr after inoculation. Infected larvae began to show symptoms at 6 hr and died about 12 hr after inoculation. Blood from uninfected larvae melanized in vitro gave electropherograms that resembled those obtained from terminal ( 12 hr postinoculation) septicemic plasma Five fractions showed reduced protein. The greatest reduction again occurred in fraction 3.
Thompson, and Cantwell ( 1962). Only mature larvae, about l-month-old, with an average weight of 0.2 g/larva, were used for experimental purposes. A lyophilized culture of Pseudomonas aeruginosa, ATCC No. 10145, was subcultured on nutrient agar slants and stored at 5°C. Still nutrient broth cultures, 15 ml incubated at 37OC for 24 hr, served as the inoculum source for larval injections. Inoculum quantitation involved the pour plate method. Specifics relative to this method included the use of 9 ml Dulbecco’s phosphate-buffered saline blanks, triplicate plating, and colony counting after 24 hr incubation at 37°C. Cultures contained bctween 2.44 X lo8 and 4.90 X lo* bacteria/ ml. Thus 3 ~1 of inoculum, the dose used for larval injections, contained between 7.32 X 1W and 1.47 X 10” bacteria. Preliminarv studies indicated that this dosage woild give 100% mortality within 11 to i4 hr postinoculation (rc~ferred to as P. T. hen,after ) . Disc clectrophoresis methods were adapted from those of the Canal Industrial Corporation ( 1962). Modifications of the
Plasma electropherograms from diseased insects may show protein depletion while plasma from nondiseased larvae melanized in vitro shows similar change (Bullock, 1963). To further explore this phenomenon, we adapted high resolution disc electrophoresis for separation of plasma proteins from mature wax moth, Galleria mellonella, larvae and then compared electrophoregrams of bacteremic and in vitro melanized Pl asma. MATERIALS
AND
METHODS
Galleria mellonella larvae were reared at 34°C according to the method of Dutky, 1 Scientific Article No. A1573, Contribution NO. 4287, of the Maryland Agricultural Experiment Station, College Park, Maryland 20742. This report constitutes part of a thesis submitted by the senior author to the Graduate School of the University of Maryland in partial fulfillment of the requirements for the M. S. degree. The investigation was sup ported by N.S.F. Grant GB-2849. 2 Present address, Department of Dairy Science, University of llaryland, College Park, Maryland 20742. 123
124
RICKISSTHY
AXD
standard procedures included: (1) 5 (i.d.) X 65-mm glass tubes containing 25 mm of 6.5 acrylamide gel overlayered with 20 mm of 5% acrylamide gel, 10 mm spacer gel and 10 mm sample gel; (2) 12 tube runs of about 55 min using a constant 200 V; (3) 2 ~1 of fresh larval plasma per tube. The standard 5 X-pH 8.3 glycine buffer was used. Protein fractions, stained with amido Schwartz, were quantitated using a Canalco Model E microdensitometer. Septicemic Plasma Procedures. Three groups of 11 larvae were each injected with 3 ~1 of inoculum per individual while an additional three groups of 11 larvae used as controls each received a 3-$ injection of sterile nutrient broth. One replicate was conducted for each group. In each group hemolymph was collected from six larvae while the other five larvae were held for observation at 24 hr P. I. An electric microinjector employing a 0.25~~ tuberculin syringe and a 27 gauge needle was used for the hemocoelic inoculation of the nitrogenanesthetized larvae. The site of injection was the lateral area of the cuticle near the prolegs. Following injection the larvae were held at 34°C. Hemolymph was collected from one group of infected larvae and one group of control larvae at separate times, either 2, 6, or 12 hr P. I. Larvae were heatfixed, to prevent melanization, immediately prior to sampling. Fixation involved larval immersion in a 60°C water bath for 1 min. The larvae were then punctured in one of the prolegs and the hemolymph collected in capillary tubes. The hemolymph was centrifuged for 5 min at 11,500 rpm (13,000 g) in an International Model MB microcapillary centrifuge. The resulting plasma from each group was pooled and subjected immediately to disc electrophoresis. Six gel tubes were run for each sample. Following protein staining, fraction densities were determined using five gels from each sample. Mehized Plasmu Procedures. Hemolymph-was taken from 6 larvae and pooled
STEINHAUER
as previously described. However, these larvae were not infected with bacteria nor heat-fixed prior to sampling. From another 6 nonheat-fixed larvae, hemolymph was withdrawn, pooled, and stirred at 24°C fol 3 min during which time it became black (melanized) . One replicate was conducted for each group. Following electrophoresis and protein staining, fraction densities were determined using five gels from each pooled sample. RESULTS
Sixteen protein fractions, numbered from 1 to 16 in order of increasing mobility, were observed following electrophoresis of control, septicemic, and melanized plasma (Fig. 1). Fractions 12, 13, and 14 were faint, and the presence of one or more was sometimes doubtful. Several fractions, sometimes coalesced, were combined for the purpose of quantitation as follows: 1 + 2; 8 + 9; 15 + 16. Electropherograms of septicemic plasma displayed the following trends when compared to control electropherograms, during the 12 hr disease period (Fig. 1, Table 1). Fractions 1 and 2 appeared coalesced after 6 hr P. I. Fraction 3, depressed at 2 hr P. I., became greatly reduced by 12 hr P. I. Fractions 7, 8 + 9, 10, and 11 were reduced by 12 hr P. I. No changes or slight increases were noted in fractions 4, 5, 6, and 15 -I- 16. Symptoms displayed by septicemic larvae were observed. Slight behavioral irregularities, first observed at 6 hr P. I., became pronounced by 8 and 10 hr P. I. as evidenced by slower crawling and spasmodic twitching. The cuticle and hemolymph appeared darker than normal. Death, as ascertained by cessation of motion, occurred between 11 and 13 hr P. I. Mortality, as observed at 24 hr P. I., was 100% among infected larvae, and pronounced blackening was noted. Mortality among the control animals was less than 1%.
: A
E3
A
C
A
FIG. 1. Disc electropherograms of control (A), 2 hr P. I. (B). and in vitro melanized (E) plasma from mntnre Gnllcricr rndTo~rr~l/n inoculation with P.wrdomonas aeruginosa.
Electropherograms of melanized plasma displayed the following trends when compared to control electropherograms (Fig. 1, Table 1). Fraction 1 -I- 2 was reduced. Fraction 3 was greatly reduced. Fractions 8 + 9, 10, and 11 were reduced. No marked TABLE .I THE PEHCEN,~A(:E INCREASE OR DECREASE IX ‘~HK \T~~~~~~ PROTEIN FRACTIONS OF PLASMA FRO\I Galleria LARVAE 2, 6, AND 12 HR AFTER IV~~UL.~TI~N WITH Pserrdomonns acrc4ginosa, AND AFTER ~IELAXIZATION “j, Increase or decrease plasma protein
_ .After
Protein fraction number
inoculation
with
P. aerrrginosa
1+2 3 1 5 6 7 X+9 10 11 155 + 16
2 hr
6hr
12 hr.
$12 -38 -s +4 +5 -9 -15
$44 -65 -11 +7 +2 -12 -17 + 10 - 6 +4
+8 -80 +20 +13 +14 -30 -30 -30 -38 +12
0 f21 +19
in -. After melanization -60 -75 +5 f2 f12 t-13 -45 -25 -47 +20
0
A
6 11r 1’. I. (C), lar\
J<‘. 1”. 1. I-efrw
E’
1” hr P. 1. (I),, to 11r111rs Ie)‘t-
Infection appears to alter insect hemolymph proteins as dotermined by electrophoresis. Barlow ( 1962), Bullock ( 1963 ) , Watanabe ( 1967)) and Bennett ct al. (1968) h avc o 1)scrved aberrant electropherograms of hemolymph from infected insects. Protein depletion, pnrticularlv in one or two fractions, was the most common abnormality observed. These studies represented a range of hosts, pathogens, and clectrophoretic techniques. In vitro melnnization of insect htmolymph also gives altcrrd clectrophoretic~ patterns as observed by Bullock ( 1963) and Barry (1964). In both studies the decrcasca of a low mobility fraction was reported. Our results are in general agreement with the above observations since we noted protein depletion with preferential clccreasc ill clectropherograrns of septiccmic Wld plasma melanizcd in vitro. The simiIarit!. bctwecn thcsc two types of clcctrophrro-
126
MCKINSTHY
AND STEINHAUER
grams is probably a reflection of melanization during septicemia since the hemolymph and cuticle appeared darker during the terminal stage of infection. Thomson (1962) states that natural melanins are often conjugated with protein. Perhaps the depleted proteins we observed were bound to melanin which showed no electrophoretic mobility. The repeated observations of the similarities in hemolymph protein electropherograms of diseased insects and melanized insect hemolymph in vitro suggest a relationship between melanization and insect defense against pathogens. One possibility seems to be that proteins which conjugate with quinones to form melanin are capable of conjugating in some way with foreign pathogens in a nonspecific type of resistance to infection. It is presently premature to try to fit this idea to existing evidence, but the process merits further investigation. ACKNOWLEDGMENT The technical staff is gratefully
assistance of Mrs. acknowIedged.
Maxine
Blicken-
REFERENCES BARLOW, J. S.
1962. An effect of parasitism on hemolymph electropherograms. J. Insect Pathol., 4, 274-275. BARRY, C. 1964. Hemolymph proteins in the molting cycle of insects. Ph.D. Thesis. Univ. of Maryland, College Park, Md. 140 pp. BENNETT, G. A., SHOTWELL, 0. L., HALL, H. H., AND HEARN, W. R. 1968. Nemolymph proteins of healthy and diseased larvae of the Japanese beetle Popillia japonica. J. Invertebr. Puthol., 11, 112-118. BULLOCK, H. R. 1963. A study of hemolymph proteins of insects in relation to melanization and natural defense against microorganisms. Ph.D. Thesis. Univ. of Maryland, College Park, Md. 160 pp. CANAL INDUSTRIAL CORPORATION. 1962. Canalco Model 12 disc electrophoresis instructions. Rockville, Maryland. 8 pp. DUTKY, S. R., THOMPSON, J. V., AND CANTWELL, G. E. 1962. A technique for mass rearing the greater wax moth (Lepidoptera: Galleriidae). Proc. Entomol. Sot., Wash., 64, 5&58. THOMSON, R. H. 1962. Melanins. In “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds. ),” pp. 727-753. Academic Press, New York. WATANABE, H. 1967. Electrophoretic separation of the hemolymph proteins in the fall webworm, Hyphantria cunea, infected with a nuclear-polyhedrosis virus. .l. Invertebr. Pathol., 9, 570-571.