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Fat body depletion, a debilitating result of milky disease in Japanese beetle larvae
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Fat Body Depletion,
34,
92-94
(1979)
a Debilitating Result of Milky Disease Japanese Beetle Larvae
Biochemical investigations of the hemolymph of Japanese beetle larvae infected with Bacillus popilliae, causative agent of milky disease, indicate minor changes in organic acids and free amino acids; some are reduced during the course of the disease while others become more abundant (L. A. Bulla, Jr., R. A. Rhodes, and G. St. Julian, Annu. Rev. Microbial. 29, 183, 1975). This information has not been translated into media for in vitro sporulation of B. popilliae and does not explain the cause of larval death from milky disease, although a drastic reduction in a lipoglycoprotein was noted. Milky larvae can live for weeks or even months and continue feeding but they do not metamorphose through pupation, for the incidence of such infection among adult beetles is very low (W. E. Fleming, Tech. Bull. No. 1383, p. 49, ARS, USDA, 1968). During the dissection of larvae for other experimental purposes we observed that the fat body cells of milky diseased larvae appeared to be severely depleted. The trophocyte (mesodermal) cells that compose the fat body are suspended in the hemocoel by connective tissue and are constantly bathed by hemolymph. They accumulate, store, resynthesize, and release products of digestion. Trophocyte cells increase in size and number as a healthy larva matures and become loaded with inclusions of fat, protein, and glycogen as seen in Figures la, b. In contrast, every milky diseased larva examined contained a depleted fat body in which trophocyte cells appeared thin and granular and were devoid of spherical lipid globules as shown in Figures lc, d. Since depletion of the fat body has not previously been considered a debilitating result of milky disease, we attempted to
determine if actual differences in fat body weight between healthy and diseased larvae could be measured. Healthy larvae were selected as being not milky by visual test. Some of these were found to contain B. popilliae when dissected and were then excluded. A few larvae of this group may have contained subnormal fat bodies due to starvation in the field or other undetectable causes. Milky larvae were selected for their obvious milky appearance. They represented a range from early sporulation of B. popifliae in the hemolymph to advanced (very milky) stages of the disease. Larvae were washed and inactivated in hot water (45-50°C) and pinned down on a paraffin block in a clean Petri dish. The integument was slit on a mid-dorsal line and pinned out, and the hemolymph was gently washed away with distilled water. To determine the dry weight of the fat body, trophocyte cells were collected by suction in a plastic hypodermic syringe with a smooth blunted 20-gauge needle. Water was added to facilitate collection and transfer to a tared aluminum foil dish. The larval remains and diluted milky hemolymph were combined in a second tared dish, and both samples were heated to dryness at 110°C for 18 to 20 hr. Table 1 includes data from 28 healthy and 24 milky diseased larvae. Obviously, the healthy larvae contain more fat body reserves than milky larvae; mean values differ by a factor of 4.3 (dried fat body) and 3.4 (percentage dried fat body). Beard (reviewed by W. E. Fleming, 1968, lot. cit.) noted that milky disease per se did not necessarily kill host larvae, but that they eventually died because of an inability to proceed into the next metamorphic phase. We propose that depletion of fat body reserves during milky disease is the 92
0022.2011/79/040092-03$01.00/0
in
NOTES
93
94
NOTES TABLE COMPARISON
OF FAT
BODIES
OF HEALTHY
Larval weight (mg)
Healthy larvae (28 insects) Milky diseased larvae (24 insects)
AND
1 MILKY
Dried fat body b-s)
DISEASED
JAPANESE
BEETLE
Dried residue” (mg)
LARVAE
Percentage” dried fat body
Mean
SE”
Mean
SE
Mean
SE
Mean
SE
249
5.9
22.6”
1.5
32.4
1.5
39.6”
2.1
239
6.4
5.3
1.6
36.7
1.6
11.5
2.3
‘{ Includes insect integument, gut, rectal sac, and hemolymph. * Percentage dried fat body = (dried fat body/dried fat body + dried residue) x 100. ( Standard error of the mean. ” Difference between means significant at the 1% level; otherwise not significant at the 5% level.
underlying reason why milky third-instar larvae cannot enter pupation. They lack the energy-rich lipid reserves necessary to physiologically trigger this next step in metamorphosis. In early instars, diseased larvae are likewise inhibited from molting. Milky disease develops slowly and causes depletion of much of the stored reserves, particularly lipids. Larvae continue feeding even in advanced stages of the disease in an attempt to replenish fat body reserves in order to enter pupation, but this does not occur in nature because infected adult beetles are practically unknown. Since they are inert, spores in the hemolymph should not inhibit replenishment of the fat body, but there is a con-
tinuing low-level vegetative proliferation that must interfere. Chemical changes in the depleted fat body cells of milky diseased larvae should be examined for substances that might contribute to sporulation of B. popilliae in laboratory culture. KEY WORDS: Fat body depletion; milky disease, Japanese beetle larvae. E. S. SHARPE R. W. DETROY U.S. Department of Agriculture Science and Education Administration Agricultural Research Northern Regional Research Center 1815 North University Street Peoria. Illinois 61604 Recaiwd
July 5, 1978
OF INVERTEBRATE
PATHOLOGY
Fat Body Depletion,
34,
92-94
(1979)
a Debilitating Result of Milky Disease Japanese Beetle Larvae
Biochemical investigations of the hemolymph of Japanese beetle larvae infected with Bacillus popilliae, causative agent of milky disease, indicate minor changes in organic acids and free amino acids; some are reduced during the course of the disease while others become more abundant (L. A. Bulla, Jr., R. A. Rhodes, and G. St. Julian, Annu. Rev. Microbial. 29, 183, 1975). This information has not been translated into media for in vitro sporulation of B. popilliae and does not explain the cause of larval death from milky disease, although a drastic reduction in a lipoglycoprotein was noted. Milky larvae can live for weeks or even months and continue feeding but they do not metamorphose through pupation, for the incidence of such infection among adult beetles is very low (W. E. Fleming, Tech. Bull. No. 1383, p. 49, ARS, USDA, 1968). During the dissection of larvae for other experimental purposes we observed that the fat body cells of milky diseased larvae appeared to be severely depleted. The trophocyte (mesodermal) cells that compose the fat body are suspended in the hemocoel by connective tissue and are constantly bathed by hemolymph. They accumulate, store, resynthesize, and release products of digestion. Trophocyte cells increase in size and number as a healthy larva matures and become loaded with inclusions of fat, protein, and glycogen as seen in Figures la, b. In contrast, every milky diseased larva examined contained a depleted fat body in which trophocyte cells appeared thin and granular and were devoid of spherical lipid globules as shown in Figures lc, d. Since depletion of the fat body has not previously been considered a debilitating result of milky disease, we attempted to
determine if actual differences in fat body weight between healthy and diseased larvae could be measured. Healthy larvae were selected as being not milky by visual test. Some of these were found to contain B. popilliae when dissected and were then excluded. A few larvae of this group may have contained subnormal fat bodies due to starvation in the field or other undetectable causes. Milky larvae were selected for their obvious milky appearance. They represented a range from early sporulation of B. popifliae in the hemolymph to advanced (very milky) stages of the disease. Larvae were washed and inactivated in hot water (45-50°C) and pinned down on a paraffin block in a clean Petri dish. The integument was slit on a mid-dorsal line and pinned out, and the hemolymph was gently washed away with distilled water. To determine the dry weight of the fat body, trophocyte cells were collected by suction in a plastic hypodermic syringe with a smooth blunted 20-gauge needle. Water was added to facilitate collection and transfer to a tared aluminum foil dish. The larval remains and diluted milky hemolymph were combined in a second tared dish, and both samples were heated to dryness at 110°C for 18 to 20 hr. Table 1 includes data from 28 healthy and 24 milky diseased larvae. Obviously, the healthy larvae contain more fat body reserves than milky larvae; mean values differ by a factor of 4.3 (dried fat body) and 3.4 (percentage dried fat body). Beard (reviewed by W. E. Fleming, 1968, lot. cit.) noted that milky disease per se did not necessarily kill host larvae, but that they eventually died because of an inability to proceed into the next metamorphic phase. We propose that depletion of fat body reserves during milky disease is the 92
0022.2011/79/040092-03$01.00/0
in
NOTES
93
94
NOTES TABLE COMPARISON
OF FAT
BODIES
OF HEALTHY
Larval weight (mg)
Healthy larvae (28 insects) Milky diseased larvae (24 insects)
AND
1 MILKY
Dried fat body b-s)
DISEASED
JAPANESE
BEETLE
Dried residue” (mg)
LARVAE
Percentage” dried fat body
Mean
SE”
Mean
SE
Mean
SE
Mean
SE
249
5.9
22.6”
1.5
32.4
1.5
39.6”
2.1
239
6.4
5.3
1.6
36.7
1.6
11.5
2.3
‘{ Includes insect integument, gut, rectal sac, and hemolymph. * Percentage dried fat body = (dried fat body/dried fat body + dried residue) x 100. ( Standard error of the mean. ” Difference between means significant at the 1% level; otherwise not significant at the 5% level.
underlying reason why milky third-instar larvae cannot enter pupation. They lack the energy-rich lipid reserves necessary to physiologically trigger this next step in metamorphosis. In early instars, diseased larvae are likewise inhibited from molting. Milky disease develops slowly and causes depletion of much of the stored reserves, particularly lipids. Larvae continue feeding even in advanced stages of the disease in an attempt to replenish fat body reserves in order to enter pupation, but this does not occur in nature because infected adult beetles are practically unknown. Since they are inert, spores in the hemolymph should not inhibit replenishment of the fat body, but there is a con-
tinuing low-level vegetative proliferation that must interfere. Chemical changes in the depleted fat body cells of milky diseased larvae should be examined for substances that might contribute to sporulation of B. popilliae in laboratory culture. KEY WORDS: Fat body depletion; milky disease, Japanese beetle larvae. E. S. SHARPE R. W. DETROY U.S. Department of Agriculture Science and Education Administration Agricultural Research Northern Regional Research Center 1815 North University Street Peoria. Illinois 61604 Recaiwd
July 5, 1978