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The spread of pebrine within a colony of the silkworm, Bombyx mori (Linnaeus)
JOURXAL
OF INVERTEBRATE
The
PATHOLOGY
Spread Silkworm, REN Sevicultzrral
7, 126-131
of
(1965)
Pebrine
within
a Colony
mori
(Linnaeus)
Bombyx ISHIHARA* Experiment
AND
TADASHI
Station,
Accepted
July
of
the
FUJIWARA
Sugivtami-Ku,
Tokyo,
Japan
6, i96J
The epizootic pattern of pebrine within a colony of the silkworm, Bonzbyn mori (Linnaeus), was investigated. The rate of infection was high in the first, the second, and the fifth instars, and low in the middle instars. This pattern corresponded to the change in the number of larvae excreting spores of Nosema bombycis Naegeli. MATERIALS
INTRODUCTION
Breeders of the silkworm, Bombyx mori (Linnaeus), in Japan, when they raise silkworms for cocoon production, do not use home-bred eggs of the silkworm. Commercially available eggs are used instead. Nowadays breeders suffer only minor lossesfrom pebrine (French pe’brine), owing to the rigid inspection of female moths that are reared for commercial breeding of eggs. Of the lots of moths subject to the inspection of pebrine, however, 1 to 2 percent of the lots are rejected by the inspection. How the moths in these lots acquire the disease is not yet clear. It is necessary to know the mode of dissemination of the disease in order to clarify the route of invasion and to establish effective control measures. Since Pasteur’s investigation (1870)) many works (Neriki and Ito, 1896; Ishiwata, 1897; Ichikawa, 1935; Ohshima, 1960) have been conducted concerning the epizootiology of pebrine? but its mode of dissemination, in an exact sense,is not completely clear. This paper deals with an analysis of one facet of this problem, using silkworms reared in the standard way of present-day Japan. 1 Present address: Insect Pathology Research stitute, Sault Ste. Marie, Ontario, Canada.
In-
126
Colony
AND
METHODS
of the Silkworm
Healthy silkworms, Bombyx mori, and pebrine carriers were mixed together one day after hatching in a ratio of 300:8, 300:32, or 900:96. The carriers were obtained from the eggs laid by the infected moths. Microscopical inspection of feces of these larvae was made to confirm the presenceof Sosema bonzbycis Saegeli spores before they were mixed with healthy larvae. Carriers or a part of healthy larvae were genetically marked with color patterns on the skin (Dominant chocolate; I-a, and Zebra; Ze: seeTanaka, 1953) for the purpose of identification. These marker geneswere introduce3 into silkworms of the same strain as those from which healthy larvae were obtained. Therefore the developmental pattern of genetically marked silkworms was almost the same as that of unmarked .4nalysis
of
the Spread
healthy
larvae.
oj the Disease
In order to determine the extent of spread of the diseaseduring each instar, somelarvae were taken out of the population and replaced with the samenumber of healthy larvae. The age of the larvae newly supplied was the sameas that of silkworms in the colony. This
EPIZOOTIC
PATTERN
sampling and replacement procedure was performed after the finish of every molting. The larvae taken out of the colony were reared individually in Petri dishes and daily observations of their feces were made until the insects died or matured. When they died or developed into moths, a pebrine inspection of the entire carcass was made. During the course of rearing, a decrease in the colony size was caused by death or loss in cleaning of rearing trays; the larvae lost were not replaced. The spread of the disease was analyzed utilizing the results of examination for pebrine infection in the larvae sampled. Details of the method of sampling, the length of period of contact with pebrine carriers, the size of colony, and the number of carrier larvae mixed will be described in the explanation of results. RESULTS
Variation in the Incidence Different Instars
of Pebrine
among
127
OF PEBRINE
1
I
/
I
TInI
/
Ip
I
P
instar
1. Variation of the incidence of pebrine among instars. Histograms in Part A show the rates of occurrence of pebrine (solid part) within the groups of silkworms reared with carriers during each instar, from the first instar to the fifth. Crosshatched lines denote the instar during which these groups of larvae were mixed with carrier larvae. Healthy insects and carriers were mixed in a ratio of 300:32 one day after hatching. Part B shows the frequency distributions of larvae excreting spores of Nosema bombycis in feces. Open and stippled histograms are obtained from the groups of larvae mixed with carrier insects during the first and the second instars, respectively. FIG.
In the experiment, the results of which are shown in Fig. 1, the size of the colony was 332, of which 32 were pebrine carriers and 300 were healthy larvae at the beginning the second (stippled). The larvae began to of the experiment. Of healthy insects, 30 disseminate spores of the parasite from the carried genetical markings on the skin. These fourth instar, when the contact was made marked larvae were taken out of the colony in the first instar. The larvae infected with and replaced with 30 marked healthy larvae pebrine during the second instar excreted the after the finish of every molting. spores later. The decreaseof the number of Part A of Fig. 1 shows the incidence of larvae excreting the spores in a later period pebrine among larvae sampled from the of the fifth instar was caused by the death colony after the finish of every molting. The of larvae. level of infection was high in larvae which of Carriers Obtained from Eggs were in contact with carriers during the first, Longevity Laid by Moths Infected with Pebrine the second, and the fifth instars. The level of infection was low during the middle instars. Of carriers obtained from eggs laid by This pattern of incidence of pebrine was also moths infected with pebrine, 20 to 48 insects observed in the caseswhen the ratio of heal- were set aside before other carriers were thy insects to carriers was 300:8 and 900:96. mixed with healthy larvae. They were reared Part B shows frequency distributions of individually up to death, in order to estimate larvae excreting spores of the parasite. The the longevity of carrier larvae mixed in the larvae were in contact with pebrine carriers colony. Throughout five experiments carried during the first instar (open histogram) or out from 1962 to 1963, they died out within
128
ISHIHARA
AND
Sprmg 1963
IIITII
m
P
instar
30 Summer 1962
20 IO 0
IIlrn
m
n
lnstoi
Summer 1963
n
Fall
0
5
IO Time
15 I” days
Instar
1963
20
25
FIG. 2. Longevity of carriers obtained from eggs laid by moths infected with pebrine. Histograms showing the decrease in number of live carriers. Stipples denote the larvae excreting spores of Nosema bombycis in feces. The length of period of instar shown in the abscissa is not for carrier larvae, but for healthy insects in the colony.
15 days after hatching, except for the case of summer rearing in 1962 when one larvae survived for 27 days, arriving at the third instar, as is illustrated in Fig. 2. Spores of Nosema bombycis were found everyday in feces of most larvae. This result indicates that the number of carriers are also less in the colony in the middle instars. Effect of Length of Contact Period with Pebrine Carriers on the Intensity of Spread of Pebrine In the experiment of Fig. 1, the length of period of contact with pebrine carriers is limited to one instar. Under the natural rearing condition, however, the contact with carriers continues throughout the larval development. It is possible for larvae to begin to disseminate spores of Nosema bombycis ear-
FU JJWARA
lier when they are in contact with carriers continuously. If this is true, the pattern of incidence of pebrine under the natural condition, may be different from the pattern shown in Fig. 1. With the purpose of investigating this possibility, the experiment below was conducted. In three successive moltings, from the first to the third instar, 60 unmarked larvae were taken out of the colony in which healthy silkworms and pebrine carriers were mingled in a ratio of 900:96 one day after hatching. In this experiment, carriers were marked in turn and no larvae were supplied to replace insects lost by sampling. Of the sampled larvae, 30 were reared individually and the inspection of spores of Nosema bombycis in both carcasses and feces was made as was the case in the experiment of Fig. 2. The other 30 larvae were mixed with 30 healthy marked larvae. After the finish of the following molting, marked larvae were separated and replaced with 30 marked healthy insects. The procedures of separation and replacement were carried out after every succeeding molting. The results of inspection of carcasses and of feces are shown by histograms A1 to Aa and C, to CH, in Fig. 3, respectively. The rate of infection of pebrine among marked larvae that were mixed with insects sampled from the colony after three successive moltings, the first to the third, are illustrated by histograms Br, B?, and B:$. Histograms Ar to Aa show that the level of incidence of pebrine in larvae sampled from the colony is increas#ing as the length of rearing in the colony is longer, but the number of larvae excreting spores of the parasite in the fourth instar was not so different from each other, as is shown by histograms Cr, C?, and C:;. In addition, the incidence of pebrine within the group of silkworms which were mixed with larvae taken out of the colony after the third molting (histogram B:]-1) did not become so high, compared with that of groups of larvae which were mixed with insects sampled after the first or the
EPIZOOTIC
PATTERN
129
OF PERRINE
rearing with carriers in the colony was short or long. Time of Death of Larvae Infected with brine by the Contact with Carriers
Pe-
Table 1 illustrates that larvae infected with pebrine in the first or the second instar by contact with carriers, succumbed to the diseasebefore pupation. In this experiment, the infection did not occur in the middle instar, but the observation of infected larvae obtained from the experiment of Fig. 2 showed that larvae developed to moths when the infection occurred in the third or later instars. In Table 1, the same pattern of incidence of pebrine as that of Fig. 1 is also observable. DISCUSSION
Fm. 3. Effect of 1eng;h of period of contact with pebrine carriers on the intensity of spread of pebrine. Histograms A,, A,, and A, show the incidence of pebrine among larvae which were in contact with pebrine carriers during the first instar, the first to the second, and the first to the third instar, respective!y. Histograms B,, B,, and B, show the occnrrence of pebrine caused by rearing with larvae which were sampled from the colony after the contact with carriers for the same periods as those of !arvae in histograms A,, A,, and A,, respectively. Histograms c c,. C, show the frequency distributions of la:bae excreting spores of Nosema bombycis in feces. These larvae were those used in histograms A,, A,, and A,. The colony consisted of 900 healthy insects and 96 carrier larvae at the beginning of the experiment.
second molting (histogram Br-3 and BZ-2). Histograms Br-4, Bz-3, and BR-2 show that the transmission of the parasite mainly occurred during the fifth instar, whether the
The results of the present investigations show that the epizootic pattern of pebrine is a monophasic wave, which is high in the first, the second,and the fifth instars, and low in the middle instars, when experiments are initiated by the mixing of healthy larvae with carriers one day after hatching. The pattern did not change even though the size of colony and the ratio of healthy larvae to carriers extended from 332 to 996 and 300:8 to 900:96, respectively. Therefore, this pattern may be a basic feature of spread of pebrine within a mass colony of the silkworm. The carriers obtained from eggs laid by moths infected with pebrine succumbed to the diseasebefore healthy larvae develop to the fourth molting, as is shown in Fig. 2. On the other hand, larvae infected with pebrine
by the contact
with
carriers
in earlier
instars began to excrete sporesof the parasite from the fourth instar (Fig. 1). In addition. the time of excretion of the spores did not start earlier, even though the length of period of contact continued from the first instar to the third (Fig. 3). Therefore, the carrier is
possibly smaller in number in the middle instars within
compared with other instars a mass colony of the silkworm.
even
Thus the change of the number of carrier larvae
130
ISHIHARA
AND
FU JIWARA
TABLE CLASSIFICATION
OF SILKWORMS
WHICH
WERE
1 WITH
MIXED
PEBRINE
Period I Time Larva,
of death
II Infected
before spinning after spinning
10/11a
O/j
CARRIERS
of mixing
BY
III insects
9/18
among
THE
TIME
OF
DEATH
(instar) IV
v
dead silkworms
O/3
o/4
O/3 o/3
O/l O/l
O/5
2/5
Pupa
O/7
O/l
Moth
O/4
O/6
O/5 O/16
O/5 o/19
16/l?
10/27
9/28
o/30
o/30
18/27
37 3
32 2
0 0
0 0
67 3
Total Rate of infection Disannearanceb
(%)
a Infected/total dead insects. b Of 30 marked larvae mixed with pebrine carriers, these insects were not recovered from the colony. The colony consisted of 8 carriers, 30 marked, and 270 unmarked susceptible silkworms at the beginning of the experiment (one day after hatching). Marked larvae were sampled and replaced with other marked insects after the finish of every molting.
among instars corresponded to that of the rate of occurrence of pebrine. This fact provides an explanation for the pattern of the epizootic of pebrine which was shown in the present experiment: The incidence of pebrine in the colony is determined by the number of carrier larvae in the colony. The spread of the disease in early instars is caused by the carriers obtained from eggs, laid by infected moths and the spread in the fifth instar is determined by the larvae infected with pebrine in early instars. It seems to be quite reasonable that the change of the epizootic pattern corresponded to the change in number of larvae excreting spores of LVosem8a bombycis. However, other factors are possibly concerned in the epizootic pattern of pebrine. Silkworm larvae become resistant to the inoculation of spores of N. bornbycis as they grow. Therefore, the incidence of pebrine may become lower as their age progresses, even if the number of carriers in the colony is fixed. On the other hand, the chance of eating Nosenza spores increases as they grow, since the amount of feeding becomes greater with development, i.e. 88 percent of the total amount of food is injested during the fifth instar, only 0.5 percent during the first to the second, and less than 2
percent during the third, respectively (Matsumma and Takeuchi, 1950). Besides, the length of an instar is longer in later instars, as is shown in Fig. 1. These factors may influence the rate of occurrence of pebrine among grown larvae, although the number of carriers may be primarily of importance for determining the pattern of spread of pebrine. Judging from the results of Table 1, the pressure of pebrine on the harvest of silkworm cocoons depends on the level of infection during the first and the second instars, since the larvae infected with pebrine in early instars fail to spin their cocoons. This fact also implies, as was already mentioned by Pasteur (1870), that the parasites are transmitted to the next generation by the insects infected with Nosenza bombycis during advanced stages, including the third instar. ACKNOWLEDGMENTS
The authors Dr. K. Aizawa tion, Tokyo, manuscript.
are grateful to Dr. Z. Kuwana and of the Sericultural Experiment Stafor their critical readings of the REFERENCES
T. 1935. Studies on the spread of pebrine within a population of the silkworm, Bombyx mori Linnaeus. Sanshikaiho, 44, 244-250. (In Japanese.)
ICHIKAWA,
EPIZOOTIC
PATTERN
ISHIWATA, T. 1897. Effect of transovum transmission of X’osema bombycis Naegeli on the silkworm rearing. Tokyo Sangyo Aoshujo Hokoku, 13, 142-195. (In Japanese.) MATSUMURA, S. AND TAKEUCHI, K. 1950. Studies on the nutrition of the silkworm, Bombyx nzori Linnaeus. I. Nutrition of silkworms of F, hybrids recently approved. J. Serici&. Sci., 19, 192-200. (In Japanese.) NERIKI, Y. AND ITO, T. 1896. Studies on p&brine disease. Tokyo Sangyo Koshujo Hukoku, 12, 236-273. (In Japanese.)
OF PEBRINE
131
OHSHIMA, K. 1960. Investigation on the distribution of the moth of silkworm (Bombyx mori L.) suffering from pebrine in a lot and its sampling method. Japan J. Appl. Bntomol. Zool., 4, 212-225. (In Japanese with English summary.) PASTEUR, L. 1870. “La Pebrine.” In “titudes sur la maladie des vers a soie,” pp. 49-206. Gauthier-Villars, Paris. TANAKA, Y. 1953. Genetics of the silkworm, Bombyx moui. Advan. Genetics, 5, 240-317.
OF INVERTEBRATE
The
PATHOLOGY
Spread Silkworm, REN Sevicultzrral
7, 126-131
of
(1965)
Pebrine
within
a Colony
mori
(Linnaeus)
Bombyx ISHIHARA* Experiment
AND
TADASHI
Station,
Accepted
July
of
the
FUJIWARA
Sugivtami-Ku,
Tokyo,
Japan
6, i96J
The epizootic pattern of pebrine within a colony of the silkworm, Bonzbyn mori (Linnaeus), was investigated. The rate of infection was high in the first, the second, and the fifth instars, and low in the middle instars. This pattern corresponded to the change in the number of larvae excreting spores of Nosema bombycis Naegeli. MATERIALS
INTRODUCTION
Breeders of the silkworm, Bombyx mori (Linnaeus), in Japan, when they raise silkworms for cocoon production, do not use home-bred eggs of the silkworm. Commercially available eggs are used instead. Nowadays breeders suffer only minor lossesfrom pebrine (French pe’brine), owing to the rigid inspection of female moths that are reared for commercial breeding of eggs. Of the lots of moths subject to the inspection of pebrine, however, 1 to 2 percent of the lots are rejected by the inspection. How the moths in these lots acquire the disease is not yet clear. It is necessary to know the mode of dissemination of the disease in order to clarify the route of invasion and to establish effective control measures. Since Pasteur’s investigation (1870)) many works (Neriki and Ito, 1896; Ishiwata, 1897; Ichikawa, 1935; Ohshima, 1960) have been conducted concerning the epizootiology of pebrine? but its mode of dissemination, in an exact sense,is not completely clear. This paper deals with an analysis of one facet of this problem, using silkworms reared in the standard way of present-day Japan. 1 Present address: Insect Pathology Research stitute, Sault Ste. Marie, Ontario, Canada.
In-
126
Colony
AND
METHODS
of the Silkworm
Healthy silkworms, Bombyx mori, and pebrine carriers were mixed together one day after hatching in a ratio of 300:8, 300:32, or 900:96. The carriers were obtained from the eggs laid by the infected moths. Microscopical inspection of feces of these larvae was made to confirm the presenceof Sosema bonzbycis Saegeli spores before they were mixed with healthy larvae. Carriers or a part of healthy larvae were genetically marked with color patterns on the skin (Dominant chocolate; I-a, and Zebra; Ze: seeTanaka, 1953) for the purpose of identification. These marker geneswere introduce3 into silkworms of the same strain as those from which healthy larvae were obtained. Therefore the developmental pattern of genetically marked silkworms was almost the same as that of unmarked .4nalysis
of
the Spread
healthy
larvae.
oj the Disease
In order to determine the extent of spread of the diseaseduring each instar, somelarvae were taken out of the population and replaced with the samenumber of healthy larvae. The age of the larvae newly supplied was the sameas that of silkworms in the colony. This
EPIZOOTIC
PATTERN
sampling and replacement procedure was performed after the finish of every molting. The larvae taken out of the colony were reared individually in Petri dishes and daily observations of their feces were made until the insects died or matured. When they died or developed into moths, a pebrine inspection of the entire carcass was made. During the course of rearing, a decrease in the colony size was caused by death or loss in cleaning of rearing trays; the larvae lost were not replaced. The spread of the disease was analyzed utilizing the results of examination for pebrine infection in the larvae sampled. Details of the method of sampling, the length of period of contact with pebrine carriers, the size of colony, and the number of carrier larvae mixed will be described in the explanation of results. RESULTS
Variation in the Incidence Different Instars
of Pebrine
among
127
OF PEBRINE
1
I
/
I
TInI
/
Ip
I
P
instar
1. Variation of the incidence of pebrine among instars. Histograms in Part A show the rates of occurrence of pebrine (solid part) within the groups of silkworms reared with carriers during each instar, from the first instar to the fifth. Crosshatched lines denote the instar during which these groups of larvae were mixed with carrier larvae. Healthy insects and carriers were mixed in a ratio of 300:32 one day after hatching. Part B shows the frequency distributions of larvae excreting spores of Nosema bombycis in feces. Open and stippled histograms are obtained from the groups of larvae mixed with carrier insects during the first and the second instars, respectively. FIG.
In the experiment, the results of which are shown in Fig. 1, the size of the colony was 332, of which 32 were pebrine carriers and 300 were healthy larvae at the beginning the second (stippled). The larvae began to of the experiment. Of healthy insects, 30 disseminate spores of the parasite from the carried genetical markings on the skin. These fourth instar, when the contact was made marked larvae were taken out of the colony in the first instar. The larvae infected with and replaced with 30 marked healthy larvae pebrine during the second instar excreted the after the finish of every molting. spores later. The decreaseof the number of Part A of Fig. 1 shows the incidence of larvae excreting the spores in a later period pebrine among larvae sampled from the of the fifth instar was caused by the death colony after the finish of every molting. The of larvae. level of infection was high in larvae which of Carriers Obtained from Eggs were in contact with carriers during the first, Longevity Laid by Moths Infected with Pebrine the second, and the fifth instars. The level of infection was low during the middle instars. Of carriers obtained from eggs laid by This pattern of incidence of pebrine was also moths infected with pebrine, 20 to 48 insects observed in the caseswhen the ratio of heal- were set aside before other carriers were thy insects to carriers was 300:8 and 900:96. mixed with healthy larvae. They were reared Part B shows frequency distributions of individually up to death, in order to estimate larvae excreting spores of the parasite. The the longevity of carrier larvae mixed in the larvae were in contact with pebrine carriers colony. Throughout five experiments carried during the first instar (open histogram) or out from 1962 to 1963, they died out within
128
ISHIHARA
AND
Sprmg 1963
IIITII
m
P
instar
30 Summer 1962
20 IO 0
IIlrn
m
n
lnstoi
Summer 1963
n
Fall
0
5
IO Time
15 I” days
Instar
1963
20
25
FIG. 2. Longevity of carriers obtained from eggs laid by moths infected with pebrine. Histograms showing the decrease in number of live carriers. Stipples denote the larvae excreting spores of Nosema bombycis in feces. The length of period of instar shown in the abscissa is not for carrier larvae, but for healthy insects in the colony.
15 days after hatching, except for the case of summer rearing in 1962 when one larvae survived for 27 days, arriving at the third instar, as is illustrated in Fig. 2. Spores of Nosema bombycis were found everyday in feces of most larvae. This result indicates that the number of carriers are also less in the colony in the middle instars. Effect of Length of Contact Period with Pebrine Carriers on the Intensity of Spread of Pebrine In the experiment of Fig. 1, the length of period of contact with pebrine carriers is limited to one instar. Under the natural rearing condition, however, the contact with carriers continues throughout the larval development. It is possible for larvae to begin to disseminate spores of Nosema bombycis ear-
FU JJWARA
lier when they are in contact with carriers continuously. If this is true, the pattern of incidence of pebrine under the natural condition, may be different from the pattern shown in Fig. 1. With the purpose of investigating this possibility, the experiment below was conducted. In three successive moltings, from the first to the third instar, 60 unmarked larvae were taken out of the colony in which healthy silkworms and pebrine carriers were mingled in a ratio of 900:96 one day after hatching. In this experiment, carriers were marked in turn and no larvae were supplied to replace insects lost by sampling. Of the sampled larvae, 30 were reared individually and the inspection of spores of Nosema bombycis in both carcasses and feces was made as was the case in the experiment of Fig. 2. The other 30 larvae were mixed with 30 healthy marked larvae. After the finish of the following molting, marked larvae were separated and replaced with 30 marked healthy insects. The procedures of separation and replacement were carried out after every succeeding molting. The results of inspection of carcasses and of feces are shown by histograms A1 to Aa and C, to CH, in Fig. 3, respectively. The rate of infection of pebrine among marked larvae that were mixed with insects sampled from the colony after three successive moltings, the first to the third, are illustrated by histograms Br, B?, and B:$. Histograms Ar to Aa show that the level of incidence of pebrine in larvae sampled from the colony is increas#ing as the length of rearing in the colony is longer, but the number of larvae excreting spores of the parasite in the fourth instar was not so different from each other, as is shown by histograms Cr, C?, and C:;. In addition, the incidence of pebrine within the group of silkworms which were mixed with larvae taken out of the colony after the third molting (histogram B:]-1) did not become so high, compared with that of groups of larvae which were mixed with insects sampled after the first or the
EPIZOOTIC
PATTERN
129
OF PERRINE
rearing with carriers in the colony was short or long. Time of Death of Larvae Infected with brine by the Contact with Carriers
Pe-
Table 1 illustrates that larvae infected with pebrine in the first or the second instar by contact with carriers, succumbed to the diseasebefore pupation. In this experiment, the infection did not occur in the middle instar, but the observation of infected larvae obtained from the experiment of Fig. 2 showed that larvae developed to moths when the infection occurred in the third or later instars. In Table 1, the same pattern of incidence of pebrine as that of Fig. 1 is also observable. DISCUSSION
Fm. 3. Effect of 1eng;h of period of contact with pebrine carriers on the intensity of spread of pebrine. Histograms A,, A,, and A, show the incidence of pebrine among larvae which were in contact with pebrine carriers during the first instar, the first to the second, and the first to the third instar, respective!y. Histograms B,, B,, and B, show the occnrrence of pebrine caused by rearing with larvae which were sampled from the colony after the contact with carriers for the same periods as those of !arvae in histograms A,, A,, and A,, respectively. Histograms c c,. C, show the frequency distributions of la:bae excreting spores of Nosema bombycis in feces. These larvae were those used in histograms A,, A,, and A,. The colony consisted of 900 healthy insects and 96 carrier larvae at the beginning of the experiment.
second molting (histogram Br-3 and BZ-2). Histograms Br-4, Bz-3, and BR-2 show that the transmission of the parasite mainly occurred during the fifth instar, whether the
The results of the present investigations show that the epizootic pattern of pebrine is a monophasic wave, which is high in the first, the second,and the fifth instars, and low in the middle instars, when experiments are initiated by the mixing of healthy larvae with carriers one day after hatching. The pattern did not change even though the size of colony and the ratio of healthy larvae to carriers extended from 332 to 996 and 300:8 to 900:96, respectively. Therefore, this pattern may be a basic feature of spread of pebrine within a mass colony of the silkworm. The carriers obtained from eggs laid by moths infected with pebrine succumbed to the diseasebefore healthy larvae develop to the fourth molting, as is shown in Fig. 2. On the other hand, larvae infected with pebrine
by the contact
with
carriers
in earlier
instars began to excrete sporesof the parasite from the fourth instar (Fig. 1). In addition. the time of excretion of the spores did not start earlier, even though the length of period of contact continued from the first instar to the third (Fig. 3). Therefore, the carrier is
possibly smaller in number in the middle instars within
compared with other instars a mass colony of the silkworm.
even
Thus the change of the number of carrier larvae
130
ISHIHARA
AND
FU JIWARA
TABLE CLASSIFICATION
OF SILKWORMS
WHICH
WERE
1 WITH
MIXED
PEBRINE
Period I Time Larva,
of death
II Infected
before spinning after spinning
10/11a
O/j
CARRIERS
of mixing
BY
III insects
9/18
among
THE
TIME
OF
DEATH
(instar) IV
v
dead silkworms
O/3
o/4
O/3 o/3
O/l O/l
O/5
2/5
Pupa
O/7
O/l
Moth
O/4
O/6
O/5 O/16
O/5 o/19
16/l?
10/27
9/28
o/30
o/30
18/27
37 3
32 2
0 0
0 0
67 3
Total Rate of infection Disannearanceb
(%)
a Infected/total dead insects. b Of 30 marked larvae mixed with pebrine carriers, these insects were not recovered from the colony. The colony consisted of 8 carriers, 30 marked, and 270 unmarked susceptible silkworms at the beginning of the experiment (one day after hatching). Marked larvae were sampled and replaced with other marked insects after the finish of every molting.
among instars corresponded to that of the rate of occurrence of pebrine. This fact provides an explanation for the pattern of the epizootic of pebrine which was shown in the present experiment: The incidence of pebrine in the colony is determined by the number of carrier larvae in the colony. The spread of the disease in early instars is caused by the carriers obtained from eggs, laid by infected moths and the spread in the fifth instar is determined by the larvae infected with pebrine in early instars. It seems to be quite reasonable that the change of the epizootic pattern corresponded to the change in number of larvae excreting spores of LVosem8a bombycis. However, other factors are possibly concerned in the epizootic pattern of pebrine. Silkworm larvae become resistant to the inoculation of spores of N. bornbycis as they grow. Therefore, the incidence of pebrine may become lower as their age progresses, even if the number of carriers in the colony is fixed. On the other hand, the chance of eating Nosenza spores increases as they grow, since the amount of feeding becomes greater with development, i.e. 88 percent of the total amount of food is injested during the fifth instar, only 0.5 percent during the first to the second, and less than 2
percent during the third, respectively (Matsumma and Takeuchi, 1950). Besides, the length of an instar is longer in later instars, as is shown in Fig. 1. These factors may influence the rate of occurrence of pebrine among grown larvae, although the number of carriers may be primarily of importance for determining the pattern of spread of pebrine. Judging from the results of Table 1, the pressure of pebrine on the harvest of silkworm cocoons depends on the level of infection during the first and the second instars, since the larvae infected with pebrine in early instars fail to spin their cocoons. This fact also implies, as was already mentioned by Pasteur (1870), that the parasites are transmitted to the next generation by the insects infected with Nosenza bombycis during advanced stages, including the third instar. ACKNOWLEDGMENTS
The authors Dr. K. Aizawa tion, Tokyo, manuscript.
are grateful to Dr. Z. Kuwana and of the Sericultural Experiment Stafor their critical readings of the REFERENCES
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