When administered a single meal containing spores of Nosema michaelis and buquinolate, specimens of the blue crab, Callinectes sapidus, were less likely to acquire spore-ridden muscle tissue than individuals not allowed the drug. Those crabs presented the drug 48 hr preceding or following the introduction of spores also exhibited minimal incidence of infection. Even after 2 mo, spores were not observed in the musculature of most crabs

Overstreet, Robin M.

English

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnFaculty Publications from the Harold W. ManterLaboratory of Parasitology Parasitology, Harold W. Manter Laboratory of9-1975Buquinolate as a Preventive Drug to ControlMicrosporidosis in the Blue CrabRobin M. OverstreetGulf Coast Research Laboratory, robin.overstreet@usm.eduFollow this and additional works at: https://digitalcommons.unl.edu/parasitologyfacpubsPart of the Parasitology CommonsThis Article is brought to you for free and open access by the Parasitology, Harold W. Manter Laboratory of at DigitalCommons@University ofNebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Harold W. Manter Laboratory of Parasitology by an authorizedadministrator of DigitalCommons@University of Nebraska - Lincoln.Overstreet, Robin M., "Buquinolate as a Preventive Drug to Control Microsporidosis in the Blue Crab" (1975). Faculty Publicationsfrom the Harold W. Manter Laboratory of Parasitology. 875.https://digitalcommons.unl.edu/parasitologyfacpubs/875   Published in Journal of Invertebrate Pathology 26:2 (September 1975), pp. 213–216; doi: 10.1016/0022-2011(75)90051-8. Copyright © 1975 by Academic Press/Elsevier. Used by permission. Submitted October 8, 1974; published online December 15, 2004.   Buquinolate as a Preventive Drug to Control Microsporidosis in the Blue Crab1   Robin M. Overstreet  Gulf Coast Research Laboratory, Ocean Springs, Mississippi, USA  Abstract When administered a single meal containing spores of Nosema michaelis and buquinolate, specimens of the blue crab, Callinectes sapidus, were less likely to acquire spore-ridden muscle tissue than indi-viduals not allowed the drug. Those crabs presented the drug 48 hr preceding or following the in-troduction of spores also exhibited minimal incidence of infection. Even after 2 mo, spores were not observed in the musculature of most crabs.  Introduction  Buquinolate prevented microsporidosis caused by Nosema michaelis in most specimens of the blue crab, Callinectes sapidus, fed infective spores. The need to prevent infections in the blue crab is especially pronounced when considering the economic potential for rearing the crab for the soft-shelled crab industry, presently an underexploited fishery (Overstreet and Cook, 1972). Infection occurs when one crab feeds on spores from an infected individ-ual. After the spore is ingested, vegetative growth of the parasite ensues in hemocytes lo-cated in sinuses in the connective tissue surrounding the midgut, followed by sporogenesis in the sarcoplasm of the striated muscle cells (Weidner, 1970). Dissolution of both infected and adjacent uninfected muscle fibers is considerable and, under certain conditions, the host becomes immobilized (Overstreet and Weidner, 1974). Infected crabs die quickly when under severe stress.   O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  2 Methods and Materials  Crabs used in the study were collected from Mississippi Sound where N. michaelis infected only one individual out of a few thousand examined. Since death often accompanies molt-ing in rearing facilities, mature females, which rarely, if ever, molt, were utilized exclu-sively. Spores used to feed uninfected crabs came from infected C. sapidus in Lake Pontchartrain, Louisiana, where low rates of infection commonly existed. Because trials were conducted during widely separated periods, spores were collected and processed at different times. After being separated from host tissue using centrifugation and saline washes, the concentrated spores were aged in refrigerated distilled water for as long as 348 days before being fed to crabs in inoculated portions of fish approximately 3 mm3. Because the crab shreds its food while ingesting it, no accurate means could be established to meas-ure concentrations of the drug. Micronized buquinolate, ethyl 4-hydroxy-6, 7-diisobutoxy-3-quinoline-carboxylate lot no. CN 161 produced by the Norwich Pharmaceutical Com-pany, was, because it is nearly insoluble in water, administered with food that had been rolled in it. After the drug was introduced, the crab’s diet consisted of two or three feedings of untreated diced fish per week. Crabs always remained separated, two to a 25-by-35-cm divided plastic box. Five covered boxes and a force-flow-type filter incorporating charcoal and ground-up oyster shell constituted a column. Each test group was confined in one or two separate closed systems. For each system, except in the pilot test, two columns emptied water into a single collecting container from which a dual-purpose pump forced to the top boxes less than 350 liters/hr of water having a salinity of approximately 12 ppt. Water then flowed by gravity to the other boxes, with a 75-mm level continuously maintained by over-flow-tubes. It was exchanged periodically. A pilot test utilized one submersible pump for each column, but that type pump proved less reliable than the open-air type. It was con-ducted using one column per test group. The temperature of the water could not be regu-lated at a specific level, but, during the warmer months, an air conditioner maintained temperatures considerably less than the seasonal values. At the beginning of a trial imme-diately following initial ingestion of spores by the crabs, the temperature of the water was increased to about 30°C for 2 hr and then allowed to cool. Controls consisted of two groups: crabs presented spores without drugs and crabs presented neither spores nor drugs. Since some crabs died early in the experiment, and their deaths could not necessarily be at-tributed to the microsporidan, only crabs surviving after the first recognized infection were used in computations. All groups in the last two trials initially contained 40 individuals. Heavy mature infections appear conspicuous in live crabs because of the chalky opaque character of infected muscles seen through the integument at joints of the appendages. In this study, however, muscle tissue from near the base of the fifth walking leg and from the hindgut of crabs that died was examined using a compound microscope. Only after infec-tions with spores were determined to be well established in many crabs, were the remain-ing crabs sacrificed and examined.   O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  3 Results  The number of crabs infected and examined in each treatment group appears in Table l. In the first trial, running for 101 days, 71% of the control crabs were infected, compared to 13% of those administered buquinolate. Spores were first demonstrated at 25 days in the muscular tissue of the hindgut and at 36 days in the appendage muscle of dead crabs. Using more crabs in the second trial, lasting 29 days after the spores were presented, pro-duced similar results. However, in the last trial, which lasted 51 days, the outcome was the same, but all groups had fewer infected crabs than in the previous two trials. Spores used for the last trial had been aged nearly l yr compared with a much shorter time for the oth-ers. In the last two trials, the initial infection was noted 17 days after the spores were intro-duced.  Table 1. Percentage of Adult Callinectes sapidus Infected by Nosema michaelis With and Without Treatment with Buquinolate  I  II  III Trial Infected Exam-ined %  Infected Exam-ined %  Infected Exam-ined % No drug, no spores     0 35 0.0  0 35 0.0 No drug, spores 5 7 71.4  24 38 63.2  11 36 30.6 Drug and spores simultaneously 1 8 12.5  7 32 21.9  0 36 0.0 Drug 48 hr preceding spores         2 33 6.1 Drug 48 hr following spores         1 33 3.0  Because the number of infected crabs in each of the columns comprising a treatment group reflected similar rates of infection, all the crabs in one such group were combined. Spores usually infected muscles from both appendages and the intestine, but occasionally they were noted in either one or the other.  Discussion  Buquinolate unquestionably caused a reduction in the number of crabs developing spore-ridden muscle tissue, even if applied 2 days preceding or following presentation of spores. Perhaps, by administering more than a single dose, the disease would not become manifest in any crabs experimentally infected and held under conditions reported in this paper. The dose necessary to prevent the disease, the stage of the parasite affected, and the mechanism involved were not studied. In contrast to the finding of Weidner (1970), sporogenesis did occur in intestinal muscles of the infected crabs. My finding that in the trials continuing for about 2 and 3 mo fewer crabs given the drug developed spores in their muscles than those in the trial lasting 29 days does not support O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  4 the presence of a long-lasting latent infection in the hemocytes. If hemocytes contained trophozoites inhibited by the action of buquinolate, I would expect to find heavy infections in the muscle after a reasonable length of time. The drug, incorporated as the active ingredient in Bonaid, prevents coccidiosis in broiler chickens. As shown by the activity of buquinolate on the development of Eimeria tenella in embryonic chicken kidney cells, there is an inhibition or delay, depending on the concen-tration of the drug used, in the development of second-generation schizonts (McDougald and Galloway, 1973). In the chick, only the sporozoite when it first invades the host cell is susceptible to inhibition because the drug cannot penetrate deep enough into the gut wall to affect later stages (Ryley, 1967). Variation in the number of crabs with discernible microsporidosis in the last two trials could be a result of the age or other factors determining viability of the spores, the temper-ature of the water or other seasonal variables, the state of the crabs, or other factors. Studies delimiting some of these factors plus the effects of repeated introduction with spores or drug are planned. Charles A. Johnson of Duke University Marine Laboratory has infected zoea through adult stages, and his investigations should show the relationship among the rate of infection and the crab’s size and the water’s temperature and salinity. He (Johnson, pers. comm.) always obtained a frequency of infection less than 10% in young and adult stages, even though he achieved higher rates in zoea and megalops. The raised tempera-ture of circulating water for a short period following presentation of spores or the viability of spores may account for the higher frequencies I report. Several other drugs were tested in addition to buquinolate with less success, and these, as well as some of the problems encountered during the study, will be reported separately. Mention of two of the drugs, however, seems relevant because the two have been found effective against other microsporidans. Beekeepers consider bicyclohexylammonium fumagillin (2,4,6,8-decatetraenedioic acid 4-[l ,2epoxy-l, 5dimethyl-4-hexenyl]-5-methoxy-l-oxaspiro[2,5] oct-6-yl ester) the drug of choice to control Nosema apis in honeybees. Katznelson and Jamieson (1952) first demon-strated its use. The drug is also employed to control Perezia pyraustae in the European corn borer (Lynch and Lewis, 1971 ). A single dose of Fumidil B, the water-soluble form of the antibiotic produced by Abbott Laboratories, resulted in a slightly larger percentage of crabs becoming infected with N. michaelis than for the controls. One reason the drug may not be effective is that vegetative production of N. apis occurs in the intestinal cells, whereas in N. michaelis this stage exists in hemocytes. Jaronski ( 1972) demonstrated histochemically that Fumidil B inhibited RNA synthesis in the microsporidan Octosporea muscaedomesticae in the black blowfly. He also pointed out that in one fly the organisms in the apical, but not the basal, regions of the host cells were affected by the drug, suggesting fumagillin had difficulty entering the cell. Since vegetative growth of N. michaelis occurs in hemocytes, the inability of the drug to diffuse through the basal membrane of the gut epithelium, thereby not reaching the trophozoites, would explain the drug’s inefficacy. Benomyl, a systemic fungicide (methyl 1-[butylcarbamoyl]-2 benzimidazolecarbamate), was accidentally discovered by Hsiao and Hsiao (1973) to control Nosema sp. infecting the alfalfa weevil. In the concentration used for crabs, it was not nearly as effective as buquin-olate on the microsporidan infection, and it apparently killed more crabs. O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  5 Synergism between drugs allows a much lower concentration of drugs to be used. Chal-ley and Jeffers (1973) reported a drastic decrease in production of Eimeria acervulina Tyzzer oocysts in chicks given a combination of meticlorpindol and buquinolate than in those given either drug alone. This same drug, under the generic name clopidol (3,5-dichloro-2,6 dimethyl-4-pyridinol), did not enhance the effect of buquinolate on N. michaelis. In fact, in the only trial using the combination, of the fewer surviving crabs, more were infected than when buquinolate was used alone.  Acknowledgments – I appreciate Edward C. Whatley, Jr. for his assistance and struggles. The Nor-wich Pharmacal Company and Dow Chemical Company kindly provided the buquinolate and clopidol, respectively, and Milton Dudenhefer collected the naturally infected crabs.  Note  1. This study was conducted in cooperation with the U.S. Department of Commerce, NOAA, Na-tional Marine Fisheries Service, under PL 88-309, Project No. 2-174-R and NOAA, Office of Sea Grant, under Grant No. 04-3-158-53. The U.S. Government is authorized to produce and distrib-ute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.  References  Challey, J. R., and Jeffers, T. K. 1973. Synergism between 4-hydroxyquinoline and pyridone coccidi-ostats. J. Parasitol., 59, 502–504. Hsiao, T. H., and Hsiao, C. 1973. Benomyl: a novel drug for controlling a microsporidan disease of the alfalfa weevil. J. Jnvertebr. Pathol., 22, 303–304. Jaronski, S. T. 1972. Cytochemical evidence for RNA synthesis inhibition by fumagillin. J. Antibiot., 25, 327–331. Katznelson, H., and Jamieson, C. A. 1952. Control of nosema disease of honeybees with fumagillin. Science, 115, 70–71. Lynch, R. E., and Lewis, L. C. 1971. Reoccurrence of the microsporidan Perezia pyraustae in the Euro-pean corn borer, Ostrinia nubilalis, reared on diet containing Fumidil B. J. Jnvertebr. Pathol., 17, 243–246. McDougald, L. R., and Galloway, R. B. 1973. Eimeria tenella: anticoccidial drug activity in cell cultures. Exp. Parasitol., 34, 189–196. Overstreet, R. M., and Cook, D. W. 1972. An underexploited gulf coast fishery: soft shelled crabbing. Amer. Fish Farm. World Aquacul. News, 3, 12–17. Overstreet, R. M., and Weidner, E. 1974. Differentiation of microsporidian spore-tails in Inodosporus spraguei gen. et sp. n. Z. Parasitenkunde, 44, 169–186. Ryley, J. F. 1967. Studies on the mode of action of quinolone and pyridone coccidiostats. J. Parasitol., 53, 1151–1160. Weidner, E. 1970. Ultrastructural study of microsporidian development. I. Nosema sp. Sprague, 1965 in Callinectes sapidus Rathbun. Z. Zellforsch., 105, 33–54. 

Buquinolate as a Preventive Drug to Control Microsporidosis in the Blue Crab

UNL | Libraries

University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Faculty Publications from the Harold W. Manter
Laboratory of Parasitology Parasitology, Harold W. Manter Laboratory of
9-1975
Buquinolate as a Preventive Drug to Control
Microsporidosis in the Blue Crab
Robin M. Overstreet
Gulf Coast Research Laboratory, robin.overstreet@usm.edu
Follow this and additional works at: https://digitalcommons.unl.edu/parasitologyfacpubs
Part of the Parasitology Commons
This Article is brought to you for free and open access by the Parasitology, Harold W. Manter Laboratory of at DigitalCommons@University of
Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Harold W. Manter Laboratory of Parasitology by an authorized
administrator of DigitalCommons@University of Nebraska - Lincoln.
Overstreet, Robin M., "Buquinolate as a Preventive Drug to Control Microsporidosis in the Blue Crab" (1975). Faculty Publications
from the Harold W. Manter Laboratory of Parasitology. 875.
https://digitalcommons.unl.edu/parasitologyfacpubs/875
 
 
 
Published in Journal of Invertebrate Pathology 26:2 (September 1975), pp. 213–216; doi: 10.1016/0022-
2011(75)90051-8. 
Copyright © 1975 by Academic Press/Elsevier. Used by permission. 
Submitted October 8, 1974; published online December 15, 2004. 
 
 
Buquinolate as a Preventive Drug to Control 
Microsporidosis in the Blue Crab1 
 
 
Robin M. Overstreet 
 
Gulf Coast Research Laboratory, Ocean Springs, Mississippi, USA 
 
Abstract 
When administered a single meal containing spores of Nosema michaelis and buquinolate, specimens 
of the blue crab, Callinectes sapidus, were less likely to acquire spore-ridden muscle tissue than indi-
viduals not allowed the drug. Those crabs presented the drug 48 hr preceding or following the in-
troduction of spores also exhibited minimal incidence of infection. Even after 2 mo, spores were not 
observed in the musculature of most crabs. 
 
Introduction 
 
Buquinolate prevented microsporidosis caused by Nosema michaelis in most specimens of 
the blue crab, Callinectes sapidus, fed infective spores. The need to prevent infections in the 
blue crab is especially pronounced when considering the economic potential for rearing 
the crab for the soft-shelled crab industry, presently an underexploited fishery (Overstreet 
and Cook, 1972). Infection occurs when one crab feeds on spores from an infected individ-
ual. After the spore is ingested, vegetative growth of the parasite ensues in hemocytes lo-
cated in sinuses in the connective tissue surrounding the midgut, followed by sporogenesis 
in the sarcoplasm of the striated muscle cells (Weidner, 1970). Dissolution of both infected 
and adjacent uninfected muscle fibers is considerable and, under certain conditions, the 
host becomes immobilized (Overstreet and Weidner, 1974). Infected crabs die quickly 
when under severe stress. 
  
O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  
2 
Methods and Materials 
 
Crabs used in the study were collected from Mississippi Sound where N. michaelis infected 
only one individual out of a few thousand examined. Since death often accompanies molt-
ing in rearing facilities, mature females, which rarely, if ever, molt, were utilized exclu-
sively. Spores used to feed uninfected crabs came from infected C. sapidus in Lake 
Pontchartrain, Louisiana, where low rates of infection commonly existed. Because trials 
were conducted during widely separated periods, spores were collected and processed at 
different times. After being separated from host tissue using centrifugation and saline 
washes, the concentrated spores were aged in refrigerated distilled water for as long as 348 
days before being fed to crabs in inoculated portions of fish approximately 3 mm3. Because 
the crab shreds its food while ingesting it, no accurate means could be established to meas-
ure concentrations of the drug. Micronized buquinolate, ethyl 4-hydroxy-6, 7-diisobutoxy-
3-quinoline-carboxylate lot no. CN 161 produced by the Norwich Pharmaceutical Com-
pany, was, because it is nearly insoluble in water, administered with food that had been 
rolled in it. After the drug was introduced, the crab’s diet consisted of two or three feedings 
of untreated diced fish per week. Crabs always remained separated, two to a 25-by-35-cm 
divided plastic box. Five covered boxes and a force-flow-type filter incorporating charcoal 
and ground-up oyster shell constituted a column. Each test group was confined in one or 
two separate closed systems. For each system, except in the pilot test, two columns emptied 
water into a single collecting container from which a dual-purpose pump forced to the top 
boxes less than 350 liters/hr of water having a salinity of approximately 12 ppt. Water then 
flowed by gravity to the other boxes, with a 75-mm level continuously maintained by over-
flow-tubes. It was exchanged periodically. A pilot test utilized one submersible pump for 
each column, but that type pump proved less reliable than the open-air type. It was con-
ducted using one column per test group. The temperature of the water could not be regu-
lated at a specific level, but, during the warmer months, an air conditioner maintained 
temperatures considerably less than the seasonal values. At the beginning of a trial imme-
diately following initial ingestion of spores by the crabs, the temperature of the water was 
increased to about 30°C for 2 hr and then allowed to cool. Controls consisted of two groups: 
crabs presented spores without drugs and crabs presented neither spores nor drugs. Since 
some crabs died early in the experiment, and their deaths could not necessarily be at-
tributed to the microsporidan, only crabs surviving after the first recognized infection were 
used in computations. All groups in the last two trials initially contained 40 individuals. 
Heavy mature infections appear conspicuous in live crabs because of the chalky opaque 
character of infected muscles seen through the integument at joints of the appendages. In 
this study, however, muscle tissue from near the base of the fifth walking leg and from the 
hindgut of crabs that died was examined using a compound microscope. Only after infec-
tions with spores were determined to be well established in many crabs, were the remain-
ing crabs sacrificed and examined. 
  
O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  
3 
Results 
 
The number of crabs infected and examined in each treatment group appears in Table l. In 
the first trial, running for 101 days, 71% of the control crabs were infected, compared to 
13% of those administered buquinolate. Spores were first demonstrated at 25 days in the 
muscular tissue of the hindgut and at 36 days in the appendage muscle of dead crabs. 
Using more crabs in the second trial, lasting 29 days after the spores were presented, pro-
duced similar results. However, in the last trial, which lasted 51 days, the outcome was the 
same, but all groups had fewer infected crabs than in the previous two trials. Spores used 
for the last trial had been aged nearly l yr compared with a much shorter time for the oth-
ers. In the last two trials, the initial infection was noted 17 days after the spores were intro-
duced. 
 
Table 1. Percentage of Adult Callinectes sapidus Infected by Nosema michaelis With and Without 
Treatment with Buquinolate 
 I  II  III 
Trial Infected 
Exam-
ined % 
 
Infected 
Exam-
ined % 
 
Infected 
Exam-
ined % 
No drug, no spores     0 35 0.0  0 35 0.0 
No drug, spores 5 7 71.4  24 38 63.2  11 36 30.6 
Drug and spores 
simultaneously 1 8 12.5 
 
7 32 21.9 
 
0 36 0.0 
Drug 48 hr 
preceding spores 
        
2 33 6.1 
Drug 48 hr 
following spores 
        
1 33 3.0 
 
Because the number of infected crabs in each of the columns comprising a treatment 
group reflected similar rates of infection, all the crabs in one such group were combined. 
Spores usually infected muscles from both appendages and the intestine, but occasionally 
they were noted in either one or the other. 
 
Discussion 
 
Buquinolate unquestionably caused a reduction in the number of crabs developing spore-
ridden muscle tissue, even if applied 2 days preceding or following presentation of spores. 
Perhaps, by administering more than a single dose, the disease would not become manifest 
in any crabs experimentally infected and held under conditions reported in this paper. The 
dose necessary to prevent the disease, the stage of the parasite affected, and the mechanism 
involved were not studied. 
In contrast to the finding of Weidner (1970), sporogenesis did occur in intestinal muscles 
of the infected crabs. 
My finding that in the trials continuing for about 2 and 3 mo fewer crabs given the drug 
developed spores in their muscles than those in the trial lasting 29 days does not support 
O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  
4 
the presence of a long-lasting latent infection in the hemocytes. If hemocytes contained 
trophozoites inhibited by the action of buquinolate, I would expect to find heavy infections 
in the muscle after a reasonable length of time. 
The drug, incorporated as the active ingredient in Bonaid, prevents coccidiosis in broiler 
chickens. As shown by the activity of buquinolate on the development of Eimeria tenella in 
embryonic chicken kidney cells, there is an inhibition or delay, depending on the concen-
tration of the drug used, in the development of second-generation schizonts (McDougald 
and Galloway, 1973). In the chick, only the sporozoite when it first invades the host cell is 
susceptible to inhibition because the drug cannot penetrate deep enough into the gut wall 
to affect later stages (Ryley, 1967). 
Variation in the number of crabs with discernible microsporidosis in the last two trials 
could be a result of the age or other factors determining viability of the spores, the temper-
ature of the water or other seasonal variables, the state of the crabs, or other factors. Studies 
delimiting some of these factors plus the effects of repeated introduction with spores or 
drug are planned. Charles A. Johnson of Duke University Marine Laboratory has infected 
zoea through adult stages, and his investigations should show the relationship among the 
rate of infection and the crab’s size and the water’s temperature and salinity. He (Johnson, 
pers. comm.) always obtained a frequency of infection less than 10% in young and adult 
stages, even though he achieved higher rates in zoea and megalops. The raised tempera-
ture of circulating water for a short period following presentation of spores or the viability 
of spores may account for the higher frequencies I report. 
Several other drugs were tested in addition to buquinolate with less success, and these, 
as well as some of the problems encountered during the study, will be reported separately. 
Mention of two of the drugs, however, seems relevant because the two have been found 
effective against other microsporidans. 
Beekeepers consider bicyclohexylammonium fumagillin (2,4,6,8-decatetraenedioic acid 
4-[l ,2epoxy-l, 5dimethyl-4-hexenyl]-5-methoxy-l-oxaspiro[2,5] oct-6-yl ester) the drug of 
choice to control Nosema apis in honeybees. Katznelson and Jamieson (1952) first demon-
strated its use. The drug is also employed to control Perezia pyraustae in the European corn 
borer (Lynch and Lewis, 1971 ). A single dose of Fumidil B, the water-soluble form of the 
antibiotic produced by Abbott Laboratories, resulted in a slightly larger percentage of 
crabs becoming infected with N. michaelis than for the controls. One reason the drug may 
not be effective is that vegetative production of N. apis occurs in the intestinal cells, whereas 
in N. michaelis this stage exists in hemocytes. Jaronski ( 1972) demonstrated histochemically 
that Fumidil B inhibited RNA synthesis in the microsporidan Octosporea muscaedomesticae 
in the black blowfly. He also pointed out that in one fly the organisms in the apical, but 
not the basal, regions of the host cells were affected by the drug, suggesting fumagillin had 
difficulty entering the cell. Since vegetative growth of N. michaelis occurs in hemocytes, the 
inability of the drug to diffuse through the basal membrane of the gut epithelium, thereby 
not reaching the trophozoites, would explain the drug’s inefficacy. 
Benomyl, a systemic fungicide (methyl 1-[butylcarbamoyl]-2 benzimidazolecarbamate), 
was accidentally discovered by Hsiao and Hsiao (1973) to control Nosema sp. infecting the 
alfalfa weevil. In the concentration used for crabs, it was not nearly as effective as buquin-
olate on the microsporidan infection, and it apparently killed more crabs. 
O V E R S T R E E T ,  J O U R N A L  O F  I N V E R T E B R A T E  P A T H O L O G Y  2 6  (1 9 7 5 )  
5 
Synergism between drugs allows a much lower concentration of drugs to be used. Chal-
ley and Jeffers (1973) reported a drastic decrease in production of Eimeria acervulina Tyzzer 
oocysts in chicks given a combination of meticlorpindol and buquinolate than in those 
given either drug alone. This same drug, under the generic name clopidol (3,5-dichloro-2,6 
dimethyl-4-pyridinol), did not enhance the effect of buquinolate on N. michaelis. In fact, in 
the only trial using the combination, of the fewer surviving crabs, more were infected than 
when buquinolate was used alone. 
 
Acknowledgments – I appreciate Edward C. Whatley, Jr. for his assistance and struggles. The Nor-
wich Pharmacal Company and Dow Chemical Company kindly provided the buquinolate and 
clopidol, respectively, and Milton Dudenhefer collected the naturally infected crabs. 
 
Note 
 
1. This study was conducted in cooperation with the U.S. Department of Commerce, NOAA, Na-
tional Marine Fisheries Service, under PL 88-309, Project No. 2-174-R and NOAA, Office of Sea 
Grant, under Grant No. 04-3-158-53. The U.S. Government is authorized to produce and distrib-
ute reprints for governmental purposes notwithstanding any copyright notation that may appear 
hereon. 
 
References 
 
Challey, J. R., and Jeffers, T. K. 1973. Synergism between 4-hydroxyquinoline and pyridone coccidi-
ostats. J. Parasitol., 59, 502–504. 
Hsiao, T. H., and Hsiao, C. 1973. Benomyl: a novel drug for controlling a microsporidan disease of 
the alfalfa weevil. J. Jnvertebr. Pathol., 22, 303–304. 
Jaronski, S. T. 1972. Cytochemical evidence for RNA synthesis inhibition by fumagillin. J. Antibiot., 
25, 327–331. 
Katznelson, H., and Jamieson, C. A. 1952. Control of nosema disease of honeybees with fumagillin. 
Science, 115, 70–71. 
Lynch, R. E., and Lewis, L. C. 1971. Reoccurrence of the microsporidan Perezia pyraustae in the Euro-
pean corn borer, Ostrinia nubilalis, reared on diet containing Fumidil B. J. Jnvertebr. Pathol., 17, 
243–246. 
McDougald, L. R., and Galloway, R. B. 1973. Eimeria tenella: anticoccidial drug activity in cell cultures. 
Exp. Parasitol., 34, 189–196. 
Overstreet, R. M., and Cook, D. W. 1972. An underexploited gulf coast fishery: soft shelled crabbing. 
Amer. Fish Farm. World Aquacul. News, 3, 12–17. 
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Buquinolate as a Preventive Drug to Control Microsporidosis in the Blue Crab

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