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Schizogregarine infection in grasshoppers
127
NOTES
preparations from normal insects was 11.85 ; from immune insects, the mean percentage was 12.15. No statistically significant difference was found between the two percentages (P > 0.01). Thus it is concluded that polyphenoloxidase occurs in an equal proportion of cells in the hemolymph of normal and immune insects. The method, however, does not determine enzyme quantitatively. It is therefore conceivable that a reduction in total amount of enzyme may occur after immunization. This is not indicated since the intensity of hemocyte staining appears unaltered in the
Schizogregarine
Infection
Schizogregarine (neogregarine) infection in the order Orthoptera seems to be rare although cephaline gregarines are common inhabitants of the gut of orthopteroid insects (F. M. Semans,Ohio J. Science, 43, 221-234, 1943). Thus the following observation of schizogregarine parasitism of grasshoppersis recorded. The infection was observed in a collection of 427 grasshoppersmade in the field near Lethbridge, Alberta in 1960. Though there was no evidence of disease or of excessive mortality in the field, an unusually high proportion (23 percent) of the insects died during air transit to Belleville, Ontario. Consequently the dead insects were examined microbiologically, the survivors were isolated in a cage, and observations made on mortality and on the degree of infection. The collection consisted of 12 percent Melanoplus bivittatus and 88 percent M. bilituratus; about 75 percent of the insects were adults and the rest were half-grown to mature nymphs. Schizogregarine infection occurred in 11 percent of the population but was confined largely to adults, as only 2 percent of nymphs had diagnosable infection in the final instar. About 15 percent of M.
hemolymph of the immunized larvae. The total hemocyte count decreases after active immunization (J. M. Stephens, J. Insect Pathol., 5, 152-156, 1962). It is possible that the decrease is confined to one specific cell type and that an inhibitor prevents melanization but these points remain to be investigated. JUNE
STEPHENS
CHADWICK
Department of Microbiology and Immunolog?~ Queen’s University Kingston, Ontario Accepted iVovenlber 5, 1965
in
Grasshoppers
bilituratus and 10 percent of M. bivittatus adults were infected and sex did not significantly influence the rate of infection. Schizogregarine infection was not the principal cause of mortality in the population. Most deaths were caused by infection with a white strain of the bacterium, Serratia marcescens which has been reported as a pathogen of Canadian grasshoppers (G. E. Bucher. J. Insect Pathol., 1, 391-405, 1959). The schizogregarines developed in the fat body, which became filled with a mass of spherical cysts each of which contained several spores or developing sporeblasts (Figs. l-4). Cysts commonly contained from four to eight spores, but a few were found with nine or ten spores. It appears that the development of the zygote in this specieswould produce a maximum of 16 spores/cyst and that a lessernumber would result from failure of some zygotic divisions. Spores were about 8 p in length, fusiform with sharp ends, and had smooth walls resistant to stain when mature. The taxonomic position of this schizogregarine cannot be fixed with certainty as: early stagesin schizogony were not observed. The number and structure of the spores and the infection of the fat body indicate a rela-
FIGS. 1-4. Smears of infected fat body of grasshoppers showing schizogregarine cysts containing various numbers of spores. Mature spores are 8 u long and all figures are at the same magnification. Fig. 1. Unstained wet mount. Figs. 2-4. Smears fixed and stained with Giemsa; the small dots and rods in the background are bacteria (including Serratia mavcescens) that are slightly below the focal plane of the picture and thus are not in sharp focus; the large number of bacteria shows the heavy bacterial infection that was common when the grasshoppers died. Fig. 2. Four cysts each containing five or six spores; the group of black cells (indicated by the arrow) may be gamont stages of the schizogregarine. Fig. 3. A cyst containing 11 developing sporeblasts that take the stain. Fig. 4. A cyst containing the common number of eight mature spores that resist staining. 128
129
NOTES
tionship to the genus Lipotropha Keilin Weiser, J. Protozool., 2, 6-12, 1955).
(J.
At death the typically infected grasshopper resembled one that had been starved ; the gut lacked food particles and the fat body contained few fat globules, so that the nuclei of the fat cells were visible scattered through the tissue, much of which was filled with schizogregarine cysts. Bacteria rapidly invaded the hemocoel of dead insects and decomposed them. Invasion by the pathogen S. marcescens killed most of the schizogregarine-infected insects prematurely, and in such cases the dead insects had typical symptoms of bacterial disease, which included large numbers of bacteria in the body cavity (Figs. 3, 4) and extensive destruction of the tissues. An attempt was made to transmit the pathogen so that early stages of its development could be studied. Purified spores were obtained by centrifuging them from a suspension of infected insects macerated in water. Spores on lettuce leaves were fed to a healthy population of M. bivittatus bred in the laboratory. Several infected insects were ground up with alfalfa meal and fed to the same population in the dry food. The experimental insects, consisting of 65 nymphs in instars three to five and of 40 young adults,
The
Potential Combination
Role
of with
Virus
were examined at intervals for 30 days, but none developed schizogregarine infection. The failure to transmit the disease to halfgrown nymphs and to adults may indicate that only very young nymphs are susceptible to invasion. If this be so, then the disease in any given individual must progress slowly, as diagnosable infections occurred only in adults or mature nymphs. Moreover. some infected adults in the field collection did not die until 20 days after their arrival at the laboratory. Schizogregarine infection appears to be estremely rare in grasshoppers, for it was not encountered a second time, though many field collections of grasshoppers from western Canada have been examined. E. A. Steinhaus and G. Marsh (Hilgardia, 33, 349-490: 1962) recorded schizogregarine infection in adult grasshoppers from Montana, Melanoplus bilituratus (M. mexicanus), and an unidentified species. This appears to be the only previous report of schizogregarine infection in the family Acrididae. GORDON E. BUCHER Research Institute, Canada Department of Agriculture, Belleville, Ontario, Canada Accepted December
a Feeding the of
Nuclear
Stimulant
8, 1965
Used
in
Polyhedrosis
Heliothisl
Larvae of the corn earworm, Heliothis zea, are highly responsive to a water-soluble feeding stimulant or arrestant extracted from corn silks and kernals by Starks et al. (Ann. Entomol. Sot. Am., 58, 74-76). In this study laboratory tests were conducted: (a) to determine if the corn extract would elicit an increased feeding response by
H. zea when applied to cotton foliage and (b) to determine if a mixture of stimulant and inclusion bodies of the nuclear polyhedrosis virus (PIB’s) would increase the insecticidal efficiency of the virus. A lyophilized preparation of the feeding stimulant was prepared by Starks et al. (op. cit.).’ Six parts of corn extract were mixed with one part
1 Publication Number 1341, Mississippi Agricultural Experiment Station in cooperation with Entomology Research Division, U.S. Department of Agriculture.
2 Corn extract preparations of the feeding stimulant were provided by K. J. Starks, Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, Tifton, Georgia.
NOTES
preparations from normal insects was 11.85 ; from immune insects, the mean percentage was 12.15. No statistically significant difference was found between the two percentages (P > 0.01). Thus it is concluded that polyphenoloxidase occurs in an equal proportion of cells in the hemolymph of normal and immune insects. The method, however, does not determine enzyme quantitatively. It is therefore conceivable that a reduction in total amount of enzyme may occur after immunization. This is not indicated since the intensity of hemocyte staining appears unaltered in the
Schizogregarine
Infection
Schizogregarine (neogregarine) infection in the order Orthoptera seems to be rare although cephaline gregarines are common inhabitants of the gut of orthopteroid insects (F. M. Semans,Ohio J. Science, 43, 221-234, 1943). Thus the following observation of schizogregarine parasitism of grasshoppersis recorded. The infection was observed in a collection of 427 grasshoppersmade in the field near Lethbridge, Alberta in 1960. Though there was no evidence of disease or of excessive mortality in the field, an unusually high proportion (23 percent) of the insects died during air transit to Belleville, Ontario. Consequently the dead insects were examined microbiologically, the survivors were isolated in a cage, and observations made on mortality and on the degree of infection. The collection consisted of 12 percent Melanoplus bivittatus and 88 percent M. bilituratus; about 75 percent of the insects were adults and the rest were half-grown to mature nymphs. Schizogregarine infection occurred in 11 percent of the population but was confined largely to adults, as only 2 percent of nymphs had diagnosable infection in the final instar. About 15 percent of M.
hemolymph of the immunized larvae. The total hemocyte count decreases after active immunization (J. M. Stephens, J. Insect Pathol., 5, 152-156, 1962). It is possible that the decrease is confined to one specific cell type and that an inhibitor prevents melanization but these points remain to be investigated. JUNE
STEPHENS
CHADWICK
Department of Microbiology and Immunolog?~ Queen’s University Kingston, Ontario Accepted iVovenlber 5, 1965
in
Grasshoppers
bilituratus and 10 percent of M. bivittatus adults were infected and sex did not significantly influence the rate of infection. Schizogregarine infection was not the principal cause of mortality in the population. Most deaths were caused by infection with a white strain of the bacterium, Serratia marcescens which has been reported as a pathogen of Canadian grasshoppers (G. E. Bucher. J. Insect Pathol., 1, 391-405, 1959). The schizogregarines developed in the fat body, which became filled with a mass of spherical cysts each of which contained several spores or developing sporeblasts (Figs. l-4). Cysts commonly contained from four to eight spores, but a few were found with nine or ten spores. It appears that the development of the zygote in this specieswould produce a maximum of 16 spores/cyst and that a lessernumber would result from failure of some zygotic divisions. Spores were about 8 p in length, fusiform with sharp ends, and had smooth walls resistant to stain when mature. The taxonomic position of this schizogregarine cannot be fixed with certainty as: early stagesin schizogony were not observed. The number and structure of the spores and the infection of the fat body indicate a rela-
FIGS. 1-4. Smears of infected fat body of grasshoppers showing schizogregarine cysts containing various numbers of spores. Mature spores are 8 u long and all figures are at the same magnification. Fig. 1. Unstained wet mount. Figs. 2-4. Smears fixed and stained with Giemsa; the small dots and rods in the background are bacteria (including Serratia mavcescens) that are slightly below the focal plane of the picture and thus are not in sharp focus; the large number of bacteria shows the heavy bacterial infection that was common when the grasshoppers died. Fig. 2. Four cysts each containing five or six spores; the group of black cells (indicated by the arrow) may be gamont stages of the schizogregarine. Fig. 3. A cyst containing 11 developing sporeblasts that take the stain. Fig. 4. A cyst containing the common number of eight mature spores that resist staining. 128
129
NOTES
tionship to the genus Lipotropha Keilin Weiser, J. Protozool., 2, 6-12, 1955).
(J.
At death the typically infected grasshopper resembled one that had been starved ; the gut lacked food particles and the fat body contained few fat globules, so that the nuclei of the fat cells were visible scattered through the tissue, much of which was filled with schizogregarine cysts. Bacteria rapidly invaded the hemocoel of dead insects and decomposed them. Invasion by the pathogen S. marcescens killed most of the schizogregarine-infected insects prematurely, and in such cases the dead insects had typical symptoms of bacterial disease, which included large numbers of bacteria in the body cavity (Figs. 3, 4) and extensive destruction of the tissues. An attempt was made to transmit the pathogen so that early stages of its development could be studied. Purified spores were obtained by centrifuging them from a suspension of infected insects macerated in water. Spores on lettuce leaves were fed to a healthy population of M. bivittatus bred in the laboratory. Several infected insects were ground up with alfalfa meal and fed to the same population in the dry food. The experimental insects, consisting of 65 nymphs in instars three to five and of 40 young adults,
The
Potential Combination
Role
of with
Virus
were examined at intervals for 30 days, but none developed schizogregarine infection. The failure to transmit the disease to halfgrown nymphs and to adults may indicate that only very young nymphs are susceptible to invasion. If this be so, then the disease in any given individual must progress slowly, as diagnosable infections occurred only in adults or mature nymphs. Moreover. some infected adults in the field collection did not die until 20 days after their arrival at the laboratory. Schizogregarine infection appears to be estremely rare in grasshoppers, for it was not encountered a second time, though many field collections of grasshoppers from western Canada have been examined. E. A. Steinhaus and G. Marsh (Hilgardia, 33, 349-490: 1962) recorded schizogregarine infection in adult grasshoppers from Montana, Melanoplus bilituratus (M. mexicanus), and an unidentified species. This appears to be the only previous report of schizogregarine infection in the family Acrididae. GORDON E. BUCHER Research Institute, Canada Department of Agriculture, Belleville, Ontario, Canada Accepted December
a Feeding the of
Nuclear
Stimulant
8, 1965
Used
in
Polyhedrosis
Heliothisl
Larvae of the corn earworm, Heliothis zea, are highly responsive to a water-soluble feeding stimulant or arrestant extracted from corn silks and kernals by Starks et al. (Ann. Entomol. Sot. Am., 58, 74-76). In this study laboratory tests were conducted: (a) to determine if the corn extract would elicit an increased feeding response by
H. zea when applied to cotton foliage and (b) to determine if a mixture of stimulant and inclusion bodies of the nuclear polyhedrosis virus (PIB’s) would increase the insecticidal efficiency of the virus. A lyophilized preparation of the feeding stimulant was prepared by Starks et al. (op. cit.).’ Six parts of corn extract were mixed with one part
1 Publication Number 1341, Mississippi Agricultural Experiment Station in cooperation with Entomology Research Division, U.S. Department of Agriculture.
2 Corn extract preparations of the feeding stimulant were provided by K. J. Starks, Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, Tifton, Georgia.