Utilization of Garamycin for the control of bacterial disease: flacherrie in the larval instars of silkworm, Bombyx mori (L) (Race: Double Crossed)

The bacterial disease: flacherrie is the most significant parameter associated with the loss of silk yield. The loss of appetite; discharge watery feces and vomiting are the common symptoms of infection of bacteria to the larval instars of silkworm, Bombyx mori (L). The present attempt is dealing with utilization of antibiotic compound for the control of bacterial disease: flacherrie in the larval instars of silkworm, Bombyx mori (L) (Race: Bivoltine Double Hybrid). For the bacterial pathogens, the diseased black thorax septicemia infected larvae of silkworm, Bombyx mori (L) were crushed through the use of using mortar and pestle; the solution was filtered; the filtrate was centrifuged (at 4000-5000 rpm) for ten minutes; the precipitate (in the form of pellet) was used for bacterial inoculum. The bacterial sample (inoculum) was streaked in Luria Agar under aseptic conditions and processed for incubation (at 37oC overnight). After 24 hours, the growth of bacteria was noticed, and it was further processed for sub culture. A bacterial sample was taken through the use of loop; centrifuged for 15 minutes at 4000 rpm and the precipitate (in the form of pellet) was dissolved in distilled water. Soon after the second moult, larval instars were divided into four groups (Untreated control group; Water treated group; Bacterial inoculum treated (infected) group and the group treated with Garamycin antibiotics, each with hundred individuals. The larvae of bacterial inoculum treated (infected) group and the group of larvae for antibiotics treatment were infected (treated) with the aqueous solution of bacterial inoculum. This treatment was carried out through smearing the solution bacterial solution onto the surface of leaves of mulberry, Morus alba (L) (M.5 Variety) leaf surface. The treated leaves were allowed for draining. The treated leaves were fed four times to the third instar larvae on the first day (100 grams of leaves for the group of hundred larvae for each time). For the second day and third day, the larvae were fed with normal untreated mulberry leaves. The water treated group of larvae was fed with mulberry leaves smeared with distilled water. The larvae of untreated control and antibiotics treated group were fed with normal untreated leaves for the days: first, second and third. The antibiotics treatment was followed on the fourth day of the third instar. Hundred grams of mulberry leaves were immersed in four hundred milliliter aqueous solution of Garamycin (40 microgam/ml distal water) for half an hour. The leaves were drained completely. The Garamycin treated leaves were used for the feeding on the fourth day (four feedings at the rate of 100 grams of leaves for the group of hundred larvae for each time). Thereafter, the larvae were fed with untreated mulberry leaves to all the groups of larvae of third, fourth and fifth instars. The haemolymph from the larvae (ten larvae from each group) was collected on the fifth day of the fifth instar and processed for electrophoresis. The hundred percent effective rate of rearing (ERR) were reported for the Garamycin treated group. Single female cocoon weight: 1.564 (±0.429) units with the shell ratio: 24.744 units and single male cocoon weight: 1.193 (±0.055) with the shell ratio: 22.967 units were reported for the Garamycin treated International journal of Horticulture, Agriculture and Food science(IJHAF) Vol-4, Issue-4, Jul-Aug, 2020 https://dx.doi.org/10.22161/ijhaf.4.4.2 ISSN: 2456-8635 www.aipublications.com Page | 140 Open Access group. The variation was detected in the pattern of banding of the protein with significant polymorphism (88.3 percent) with two bands of monomorphic nature; twelve bands of polymorphic nature and three bands of “unique” nature. Keywords— Antibiotics, Bacterial Flacherrie, Bombyx mori, Garamycin.


INTRODUCTION
Silk is considered as the most superior fiber for human protection. Therefore, the silk deserve economic significance. The larval instars of silkworm, Bombyx mori (L) feed on the leaves of mulberry, Morus alba (L). The mature fifth instar larvae spin the silky cocoon around their body and enter into the pupal stage. The quality of silk depends on many factors like: race of silkworm; race of host plant (mulberry, Morus alba); health of individual instars in the life cycle; environmental conditions; … etc. There are several reports on attempts on the improvement of quality of health of larval instars of silkworm, Bombyx mori (L) for the qualitative yield of the silk. Most of the attempts on this line are concerned with the management of control of the diseases of silkworm and it's host plant mulberry. According to Taha (2002), the bacterial flacherrie is the most common disease of larval instars of mulberry silkworm. The bacterial infection in the larval instars of silkworm, Bombyx mori (L) deserves multiplicity (Choudhury et al., 2002). Babu, et al. (2005) identified and reported the principle of "Antivirosis" in the Spirulina platensis (L) against the bmnpv virus of polyhedrosis category in the silkworm, Bombyx mori (L). Therefore, the pathological knowledge on microbial diseases in silkworm is vast area and it should be fully understood.
The feeding with the infected or contaminated mulberry leaves of mulberry, Morus alba (L) is the reason for causing the disease of bacterial flacherie to the larval instars of the silkworm, Bombyx mori (L). The flacherie infected larval instars of silkworm, Bombyx mori (L) appear look weak. The flacherie infected larval instars of silkworm, Bombyx mori (L) can die from disease. The flacherie infected larval instars of silkworm, Bombyx mori (L) that are about to die become dark brown in color. Both, infectious and non-infectious flacherie use to cause death of the flacherie infected larval instars of silkworm, Bombyx mori (L). The pathogens of the flacherie are known for destruction of the gut tissues in the body of host. The Serratia marcescens (L) and species of Streptococcus and Staphylococcus are the bacterial pathogens contributing for the disease of bacterial flacherie in silkworm, Bombyx mori (L). The loss of appetite in larval instars; the sluggishness of larval instars; the growth of larval instars at the least rate; the shrinkage of larval instars; the swelling of thorax of larval instars; appearance of brown speeks on skin in the larval instars and straightened appearance of body of larval instars are the significant symptoms of bacterial flacherie in silkworm, Bombyx mori (L). According to Samson (1995), the other symptoms of bacterial flacherie in silkworm, Bombyx mori (L) include: the oral (and anal too) discharge; the liquefaction of visceral organs; the rupture of the larval skin and the brown fluid (with the foul smell).
According to Tanda and Kaya (1993), the loss in appetite; discharge of watery faecal matter (diarrhea) (discharge of watery feces) and vomiting are the significantly common symptoms of infection of bacteria to the larval instars of silkworm, Bombyx mori (L). Then, the larval body wall starts for softening and die emitting a foul odor. The only available practice at present is to discard large stocks of infected to avoid the spread of disease. There are no specific preventive methods known to the Indian sericulturist farmers. According to Acharya et al. (2002), sanitizations of the rearing room and rearing beds are going to help to avoid the spread of disease. Adoption of methods of the "Prophylactic and Curative" category should be aimed for the management of the control of the microbial diseases in the mulberry silkworm, Bombyx mori (L) is the prime concern for sericulturist farmers. The disease control methods should aimed taking into account the eco-friendly nature and cost effectiveness. According to Subramanian, et al. (2009), the sericulture should be with the use of the antibiotics as a component of bed disinfectants. The antibiotics are used as therapeutic applications against bacterial diseases. In the sericulture industries, the silk productivity and the silk quality mostly depends on the health of larval instars of silkworm; quality of mulberry leaves used for feeding; the growth of the silkworm larvae and the favorable conditions of the environment. The physiological process within the body of larval instars of silkworm, Bombyx mori (L) are contributing for growth and development. Improvement in the methods of rearing; quality of the mulberry leaves Open Access (nutrition) and upkeep of health of larval instars of silkworm is going to orchestrate the progression of sericulture practice towards the improvement of silk quality. The silkworm larvae are highly susceptible for the infection through significant group of microbial pathogens. Attempt on use of antibiotics for the control of bacterial diseases in silkworm, Bombyx mori (L) is not new. The use of antibiotics (examples: penicillin, streptomycin, tetracycline and chloramphenicol) for the control of bacterial diseases in silkworm, Bombyx mori (L) was already reported. According to Venkatesh and Srivastava (2010) According to Philips, et al. (2004), the use of antibiotics in silkworm rearing is allowed for four reasons. These reasons (purposes of use of antibiotics for the rearing of larval instars of silkworm) include: treating the diseased larvae with antibiotics stops the heavy loss through the bacterial diseases in silkworms; antibiotic treatment prevent the larvae from diseases; antibiotic treatment control diseases in silkworms; antibiotic treatment help for health maintenance and promotion of growth of silkworms. For the innate immunity in silkworms, the haemolymph deserves a key role. According to Hou, et al. (2010), the body of larvae (through the innate immunity) in silkworm get triggered for responding for the entry of microbial pathogens. Tanaka and Yamakawa (2011) opined the effective innate immunity system in the body of insects against foreign microbial pathogens. The humoral reactions and cellular reactions are the two significant types in the body of insects for the innate immunity response. Humoral reactions involve soluble proteins in the hemolymph such as The production of soluble proteins like enzyme (ex. Phenoloxidase); anti-microbial protein (AMP); the lysozymes and the lectins in the haemolymph are the examples for humoral reactions in insects like, silkworm, Bombyx mori (L). The processes like phagocytosis, encapsulation and nodule formation are contributing as the cellular reactions in insects like, silkworm, Bombyx mori (L). According to Jannatun, et al.
(2020), in the body of larval instars of silkworm, Bombyx mori (L), there are six different groups of anti-microbial proteins (AMPs) and they include: the cecropin; the attacin; the lebocin; the moricin; the gloverin and the defensin. One lysozyme is reported in the silkworm, Bombyx mori (L). The three lysozyme-like proteins are reported in the silkworm, Bombyx mori (L). One of the lysozyme like protein is reported for involvement in elimination of invading microbial pathogens. There are no reports on use of Garamycin antibiotics for the management of bacterial diseases in of silkworm, Bombyx mori (L). Therefore, present attempt was planned with the aim of enhancement of resistance in the body of silkworm, Bombyx mori (L) against the infections of bacterial pathogens through the use of Garamycin antibiotics.

II. MATERIAL AND METHODS
The attempt on the utilization of antibiotic compound,

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rearing of silkworm instars was carried out. The DFLs (disease free layings) of double hybrid bivoltine race (CSR6 x CSR26) x CSR2 x CSR27) of silkworm, Bombyx mori (L) were procured through the "Dr. APIS" Laboratory and processed for black boxing, rearing of early instars, rearing of late age instars, regular feeding with leaves of mulberry, provision of mountage for spinning the cocoon and cocoon harvesting through the standard methods.
(B). Isolation of Bacteria Causing the Disease: Flacherrie in the Larval instars of silkworm, Bombyx mori (L): The method explained by Aneja (2003) was utilized for the isolation of the bacteria causing the flacherrie disease in the larval instars of silkworm, Bombyx mori (L). According to Tanda and Kaya (1993), the loss in appetite; discharge of watery fecal matter (diarrhea) (discharge of watery faeces) and vomiting are the significantly common symptoms of infection of bacteria to the larval instars of silkworm, Bombyx mori (L). Then, the larval body wall starts for softening and die emitting a foul odor. The flacherrie diseased larval stages of silkworm, Bombyx mori (L) exhibit the black thorax. This black thorax condition of the larval instars of silkworm, Bombyx mori (L) is recognized as "Septicemia" in sericulture practice. The larval instars of silkworm, Bombyx mori (L) exhibiting "Septicemia" were collected from the fifth instar bed of Malegaon Sheti Farm of Agricultural Development Trust, Baramati (India). The diseased larval instars of silkworm, Bombyx mori (L) were crushed through the use of little amount of distilled water; mortar and pestle. The aqueous solution was processed for filtration. The filtrate was then processed for centrifugation (at 4000-5000 rpm) for ten minutes. According to Aneja (2003), the supernatant should be discarded and the precipitate in the form of pellet should used for further processing. Accordingly, the supernatant was discarded and the precipitate (in the form of pellet) was used for bacterial culture after re-suspending in distilled water.
(C). Luria Agar (LA) Medium Preparation and the Preparation of Bacterial Culture: The sample of bacterial was streaked in LA under aseptic conditions in a laminar air flow chamber with the help of streaking loop. It was then incubated at 37ºC overnight. After twenty four hours, the growth of bacteria was noticed. The system was then processed for further sub culture. The bacterial sample was taken away with the help of a loop. It was allowed for centrifugation (at 4000 rpm) for 15 minutes. The larvae of untreated control group were fed with normal untreated leaves for the days: first, second, third and fourth (four feedings per day at the rate of hundred grams leaves for the group of hundred larvae for each feeding) ( Table-1: Schedule of feeding the larvae of silkworm, Bombyx mori (L) (Race: Double Hybrid).
The larvae of water untreated control group were fed with water treated leaves for the first day (four feedings at the rate of hundred grams leaves for the group of hundred larvae for each feeding). For the second, third and fourth days, this group (water treated group of the larvae) was fed with normal untreated leaves (four feedings per day at the rate of hundred grams leaves for the group of hundred larvae for each feeding) ( Table-1: Schedule of feeding the larvae of silkworm, Bombyx mori (L) (Race: Double Hybrid).
The larvae of bacterial culture treated (infected) group was fed with the bacterial culture treated leaves of mulberry for the first day (four feedings at the rate of hundred grams leaves for the group of hundred larvae for each feeding). For the second, third and fourth days, this group (bacterial culture treated group of the larvae) was fed with normal untreated leaves (four feedings per day at the rate of hundred Open Access grams leaves for the group of hundred larvae for each feeding). The treatment was carried out through smearing the solution of bacterial culture onto the surface of leaves of mulberry, Morus alba (L) (M.5 Variety) leaf surface. The treated leaves were allowed for draining and then used for feeding to the respective group of the larvae ( Table-1: Schedule of feeding the larvae of silkworm, Bombyx mori (L) (Race: Double Hybrid).
The group of larvae of antibiotic treatment was fed with the bacterial culture treated leaves of mulberry for the first day (four feedings at the rate of hundred grams leaves for the group of hundred larvae for each feeding). For the second and third days, this group (antibiotics treatment group of the larvae) was fed with normal untreated leaves (four feedings per day at the rate of hundred grams leaves for the group of hundred larvae for each feeding).
The antibiotics treatment was followed on the fourth day of the third instar. Hundred grams of mulberry leaves were immersed in four hundred milliliter aqueous solution of Garamycin (40 microgams/ml distal water) for half an hour. The leaves were drained completely. The Garamycin treated leaves were used for the feeding on the fourth day of the third instared larvae of the group of antibiotic treatment (four feedings at the rate of hundred grams of leaves for the group of hundred larvae for each time) ( Table-1

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Thereafter, the larvae of all the groups (in third instar, fourth instar and fifth instar) were fed with normal untreated mulberry leaves of mulberry.
(E). Preparation of Haemolymph Sample for the Protein: Preparation of the haemolymph sample for the protein analysis was carried on the fifth day of the fifth instar. Ten larvae from each group were selected randomly. The hemolymph was collected from larvae (on the fifth day of the fifth instar). The abdominal legs were cut through the use of sterilized scizor. The haemolymph from each group was collected separately in an eppendrof tube. Equal volume of of buffer solution (Tris buffer with pH 6.8) was taken. The buffer solution was mixed in haemolymph sample. Thus, the dilution of the haemolymph was carried out. From each group of the larvae in the attempt, fifteen microliters diluted (with Tris buffer solution pH 6.8) haemolymph sample was taken in another eppendrof tube. The haemolymph sample in the eppendrof tube was mixed thoroughly. This mixing was carried out through the use of the vertex. The sample solution was heated in boiling water for 2-3 min to ensure complete interaction between proteins and SDS, and a pinch of Bromophenol blue is used as a tracking dye.
(F).The Qualitative Analysis of the Haemolymph Proteins through the Electrophoresis: Qualitative pattern of proteins in haemolymph of the fifth instared larvae of silkworm, Bombyx mori (L) (Race: Double Hybrid) were analysed by sodium dodecylsulfate (SDS) polyacrylamide slab gels through the use of method explained by Laemmli (1970). The method of Laemmli (1970) is dealing with a discontinuous system of sodium dodecylsulfate (SDS) system. This method is the most widely used in electrophoretic system in recent times. The treated peptides are concentrated in a stacking gel before gets entered into the separating gel. Therefore, the method of Laemmli gel deserve excellent quality of resolution.
The SDS-PAGE using 4% stacking gel and 10% separating gel was performed under denaturing condition. The electrophoresis of the assay sample was carried out in the four percent stacking gel and ten percent separating gel. The gel was allowed for run at 150V for about ten to twenty minutes until the tracking dye moved till the end of the gel.
After the completion of electrophoretic run, gel was removed and it was stained with the Coomassie Brilliant Blue for 45 min-1hr. Standard molecular weight markers were used for estimating the molecular weight.
(G). Analysis of commercial parameters (characters of cocoons): The cocoons were harvested (separated from the mountage) on sixth day after the provision of mountage for spinning. Fifty cocoons from each group were selected randomly. Weight of each individual cocoon was recorded. Each individual cocoon was deflossed. The weight of individual deflossed cocoon was recorded. Each cocoon in particular group was cut vertically using the blade. The weight of individual silk shell from individual cocoon was recorded.
For knowing the weight of pupa within individual cocoon, the reading of the weight of silk shell of respective individual cocoon was subtracted from weight of respective individual deflossed cocoon. Weight of entire deflossed cocoon; weight of silk shell of individual cocoon and weight of pupa from individual cocoon were noted. The shell ratio of the cocoon is commercial or economic parameter. The shell ratio is the percentage of content of silk within the individual entire cocoon. The silk shell percentage (correctly called as shell ratio) was calculated through the use of readings of weight of whole deflossed cocoon and weight of silk shell in cocoon. The reading of silk shell weight was divided by reading of weight of whole cocoon without floss. The quotient thus obtained was processed for multiplication with hundred. Shell ratio or shell percentage is the outcome of this attempt. In sericulture, this silk shell percentage is called as shell ratio. Sericulture farmers get the price for the cocoon yield on the basis of "Shell Ratio".
(H). Statistical Analysis of the data: The statistical analysis involves collection and scrutinization of the data sample. Statistical analysis can be divided into five different discrete steps. These steps include: description of the nature of the data to be used for analysis; exploration the relation of the data to the underlying group; establishment of the model to summarize understanding of how the data relates to the underlying group; establishment of proof for the validity of the model and to employ the "Predictive Analytics" to proceed the scenarios that will help to guide future actions. The final goal of analysis through statistical methods is to identify trends (Norman and Baily, 1955). The present attempts in experimentation were repeated for three times. The purpose of repetition of attempts in experimentation is to get the consistent results. The data, in the form of mean, standard deviation and percent change was collected. This data was subjected for

III. RESULTS AND DISCUSSION
The The weight (gm) of entire deflossed female cocoon; weight (gm) of female silk shell (gm) and the weight (gm) of female pupa (gm) of the group of the bacterial cultured treated followed by the treatment with antibiotics (Garamycin) group were measured 1.564 (±0.429); 0.387 (±0.049) and 1.177 respectively. The ratio of female silk shell to the entire cocoon in the group of "bacterial cultured treated followed by the treatment with antibiotics (Garamycin)" was recorded 24.744 (table-2).
The weight (gm) of entire deflossed male cocoon; weight (gm) of male silk shell (gm) and the weight (gm) of male pupa (gm) of the untreated control group were measured 1.158 (±0.053); 0.238 (±0.019) and 0.920 respectively. The ratio of male silk shell to the entire cocoon in the group of "untreated control" was recorded 20.552 (table-2). The weight (gm) of entire deflossed male cocoon; weight (gm) of male silk shell (gm) and the weight (gm) of male pupa (gm) of the water treated control group were measured 1.158 (±0.069); 0.238 (±0.098) and 0.920 respectively. The ratio of male silk shell to the entire cocoon in the group of "water treated control" was recorded 20.552 (table-2).
The weight (gm) of entire deflossed male cocoon; weight (gm) of male silk shell (gm) and the weight (gm) of male pupa (gm) of the bacterial cultured treated group were measured 1.074 (±0.055); 0.221 (±0.018) and 0.853 respectively. The ratio of male silk shell to the entire cocoon in the group of "bacterial cultured treated" was recorded 20.577 (table-2) The weight (gm) of entire deflossed male cocoon; weight (gm) of male silk shell (gm) and the weight (gm) of male pupa (gm) of the group of the bacterial cultured treated followed by the treatment with antibiotics (Garamycin) group were measured 1.193 (±0.059); 0.274 (±0.021) and 0.919 respectively. The ratio of male silk shell to the entire cocoon in the group of "bacterial cultured treated followed by the treatment with antibiotics (Garamycin)" was recorded 22.967 (table-2 In the body of poikilotherm animals the situation is exactly opposite to that of homeotherm animals (animal which maintains thermal homeostasis). Silkworm, Bombyx mori (L) belongs to the "Poikilotherm" group. The larval instars of the silkworm, Bombyx mori (L) use to respond very quickly for the changes in the environment. The temperature and the relative humidity are the environmental factors that use to affect the quality of life of the larval instars of the silkworm, Bombyx mori (L). The environmental condition of higher or lower temperature and humidity, ventilation and quality of food material exert adverse influence on the physiological functions of the silkworm, Bombyx mori (L). The larval instars of the silkworm, Bombyx mori (L) become highly susceptible to diseases.

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The electrophoretic pattern of the haemolymph samples of the larvae in the attempt are presented in figure -1 The electrophoretic pattern of haemolymph samples of the "Healthy Larvae" of untreated control group, the "Healthy Larvae" of infected (bacterial culture treated) group and "Healthy Larvae" of antibiotics treated group exhibited total "Fourteen" protein bands with molecular weight as listed here: 68. 30    , this 49.08 KDa protein deserve the significant role in the immunity system in silkworms. According to Kaito et al., (2002), the antibiotic compounds are used clinically for the human health and also they have therapeutic effects against silkworms injected with Staphylococcus aureus and Pseudomonas aeruginosa (L). The attempts of Hossain et al., (2006) were concerned with the estimation of the bacterial exotoxins with the capability of killing the silkworms. The fifty percent lethal dosage (LD50) of the staphylococcal alpha-toxin is 12μg/g. The fifty percent lethal dosage (LD50) of the staphylococcal betatoxin is 9μg/g. The fifty percent lethal dosage (LD50) of the Pseudomonas exotoxin A is 0.14μg/g. The fifty percent lethal dosage (LD50) of the diphtheria toxin is 1.1μg/g. Most of the fifty percent lethal dosage (LD50) values obtained in silkworm, Bombyx mori (L) were similar to that reported fifty percent lethal dosage (LD50) values in mice. This is suggesting that, silkworms can be utilized as a model animal to study the general effects of the bacterial exotoxins on multicellular organisms, including human beings. If we agree the opinion of Yamakawa and Tanaka (1999), we are free for a broad range of expectation from the studies on immunity system of silkworm for the contribution for the field of medicine, in the field of production of antibiotic compounds based on the bacterial proteins. The field of agriculture is also waiting for the establishment of the transgenic crops (at least in sericulture) with antimicrobial gene proteins through such attempts.

IV. CONCLUSION
The hundred percent effective rate of rearing (ERR) were reported for the Garamycin treated group. Double Hybrid -(CSR6 x CSR26) x CSR2 x CSR27)] in the diseased larvae in the present attempt may be related to the proteins of the immune system. Feeding the silkworm with mulberry leaves treated with bacterial culture (the bacterial infection) may be used for the surveying the genome of the host (silkworm larvae) as significant reactions (or response). The most significant reactions (or the response) by the larval instars of the silkworm, Bombyx mori (L) for feeding them with mulberry leaves treated with bacterial culture (the bacterial infection) is the "Innate Immune Response" to the microbial pathogen at the level of transcription. The provision of "Another Detailed Comprehension of the Interaction between the Pathogen and the Host" is supposed to be one more expected reaction (or the response) by the larval instars of the silkworm, Bombyx mori (L) for feeding them with mulberry leaves treated with bacterial culture (the bacterial infection). A lot of "Basal Metabolic Pathways" were modulated significantly. According to Huang, et al. (2009), the genes with reference to the poisoning are also regulated. Further, the genes with reference to the poisoning might be with a key role to control (naturally) the bacterial septicemia disease in the silkworm, Bombyx mori (L).