Impact of Adding some Algal Probiotics ( Spirulina Plantalis and Azolla Pinnata ) as Food Supplements to Silkworm, Bombyx mori L. for Strengthening the Sericulture Industry

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INTRODUCTION
Mulberry silkworm has an economic importance due to its ability to convert mulberry protein to silk protein.It has attention worldwide, especially countries that are interested in this industry.Lately, Egypt has given attention to this industry so, researchers have tried to improve the silk quality and quantity in different ways.
One of these ways is adding supplements such as proteins, amino acids, botanical extracts and probiotics as a food supplement.Silkworm is a monophagous insect, so the quality of mulberry leaves plays a vital role in the production of silk Stanley and Abraham (2016).Nutritious mulberry leaves are essential for the growth, development, and cocoon production of the larvae Masthan et al. (2011).Adding nutritional supplements like vitamins, amino acids, proteins, and probiotics to larval feed can enhance the nutritional efficiency and economic traits of the silkworm Amalarani et al. (2011) Probiotics are live microbial supplements that improve microbial balance and enhance cellular growth Shruti et al (2019).Several studies have shown that probiotics have a positive impact on insect health Dillon and Dillon (2004).Spirulina is a blue-green microalgae that contains a lot of amino acids, vitamins and B12, Ganeshprabu et al. (2011).Azolla pinnata is a floating water fern, rich in protein (25-30%) and contains the symbiont cyanobacterium Anabaena azollae, which fixes nitrogen from the atmosphere and also, has essential amino acids (7-10%), minerals (10-15%), vitamins (A, B12, and β-carotene), and growth promoter Shruti et al. (2019).After oral administration of eri silkworm with mulberry leaves supplemented with cyanobacteria, there was an increase in larval and shell weight, as well as commercial cocoon characteristics Kumar et al. (2009).The fifth instar silkworms' supplementation with azolla, showed positive impacts on cocoon features also, feeding B. mori mulberry leaf combined with spirulina improved the larval and cocoon characteristics Venkataramana et al. (2003).
The factors responsible for the growth and the production of silkworms are the food quality, physiological state of the larvae, enzyme activity and the type of additives Vitthalrao and Sucheta (2012).Silkworm hemolymph plasma almost contains protein particles Marwa (2023).Highly protein mulberry leaves have a positive impact on silk production and are responsible for the activity of the protease enzyme that converts mulberry protein to silk protein Kumar and Kalpana (2009).High quality of silk production depends on the silkworm protein content.The fifth instar hemolymph protein contributes to silk protein biosynthesis in the silk gland.High-quality protein-rich diets are essential for producing the finest silk Shivkumar and Subramanya (2015).Amylase is an important enzyme that concerns carbohydrate metabolism.It was identified with the analysis of digestive fluid or hemolymph Lokesh et al. (2012).Invertase enzyme belongs to glycoside hydrolyse family which has different functions in different organisms Cimini et al. (2016), Kulshrestha et al. (2013).It plays a fundamental role in converting sucrose which is a polysaccharide to glucose and fructose (monosaccharides) Guo et al (2018).
This investigation has provided new insights into improving the economic parameters of the sericulture industry by adding green probiotics to regulate the biochemical machinery that directly on the production.

MATERIALS AND METHODS
The experiment was carried out at the Sericultural Research Department, Plants Protection Institute, and Agricultural Research Centre in Giza.

A-Preparation of Stock Culture:
Silkworm seeds were imported from India (PM X CSR2) and then incubated at 27 ± 1°C and 80 ± 5% RH till hatching.Rearing was carried out under hygienic conditions according to Marwa (2020).Newly hatched silkworm reared in the summer season at 29-32 °C and relative humidity 80-90% RH.Silkworms fed on clean mulberry leaves from hatching till the end of the 3 rd larval instar.

B-Preparation of Probiotics Concentrations Solution Used for The Study:
Azolla was collected fresh from the Soil, Water and Environment Research Institute, Agriculture Research Centre, brought to the laboratory, washed thoroughly with clean tap water and subsequently with distilled water.Azolla was weighed 000 grams for extraction using 1000 ml distilled water.Initially, Azolla was grinded with 100 ml of distilled water (out of 1000 ml) and sieved, the residue was mixed with 100 ml of distilled water and grinded and sieved, and the procedure was repeated 4-5 times and finally, the filtrate volume was made up with the remaining distilled water and treated as stock solution.From this stock solution, different concentrations were prepared viz., one, two and three, percent and the same was used for feed supplementation as done by Vijaykumar et al (2016).
Spirulina powder was purchased from the Algal Department, National Research Centre, and 500 ml spirulina solution of different concentrations viz., one, two and three percent.

C-Experimental Design:
At the beginning of the 4 th larval instar, the larvae are divided into 6 groups each group consists of three replicates each one consists of 50 larvae.All groups fed on dipped mulberry leaves with aqueous solution prepared probiotics concentrations (1%, 2%, and 3%).keeping in shade for half an hour to remove excessive wetness, the leaves were fed to the larvae from the first day of the fourth instar.One feed of the probiotics-treated leaves and three feeds of the normal mulberry leaf were given to the silkworms on odd days till the spinning of cocoons.

F-Physiological Characters:
Hemolymph was collected from matured larvae of the 5 th larval instar by removal of the thoracic leg in 1.5 ml Eppendorf tubes with a small amount of phenyl thiourea crystal (PTU) as an anti-coagulant substance, Mahmoud (1988).The tubes were kept at -20°c, and the blood samples were centrifuged at 10000rpm for 10 minutes at 5°c. the supernatant was assayed to determine.a-Total protein, was determined by the method of Bradford (1976).b-Amylase, was determined by the method of the modifications of Amin (1998) to the method described by Ishaaya and Swirski(1976).c-Invertase, digestive enzymes were determined according to the modifications of Amin (1998) to the method described by Ishaaya and Swirski(1976).d-Protease, proteolytic enzyme was determined according to Tatchell et al. (1972) with some modification of measuring the increasing of amino acid according to Lee and Takabashi (1966).

G-Analysis of Proximate Compositions of The Primary Phytochemicals of Algae:
Powders of spirulina, and azolla were subjected to proximate composition analysis (crude protein, etheric extract, total ash, moisture and total carbohydrate) following the methodology of Castell and Tiews (1980) as given in AOAC (1995).

H-Preparation of Solvent Extracts of Algae:
The powdered samples of spirulina, and azolla (75g each) were taken and packed in Whatmann No. 1 filter paper and put into the soxhlet apparatus.The extracts were successively soaked with 300 ml (1:4 w/v) of a polar solvent, methanol (99.9% purity, Changshu Yangyuan Chemicals, China), and a non-polar solvent, petroleum ether (99.98% purity, SRL Pvt. Ltd.India) individually and separately for 6-9 h each (30 to 36 cycle) until a clear colorless solution was obtained.The extracts were filtered by using a doublelayered muslin cloth and concentrated at 40-50 °C using a rotary vacuum evaporator (ROTAVAP).The extracts obtained were vacuum-dried under 40°C and used for further investigation.The extracts obtained appeared as dark green, gummy solid.

A-Biological Parameters:
Feeding quality is an important factor affecting all biological parameters, larval weight is an indicator for healthy larvae and good cocoons produced.Table (1), showed weights of the larvae not affected by the additives during the 4 th instar, the maximum results recorded for Spirulina1%, Spirulina2%, Spirulina3% and Control on par (0.530 g., 0.519 g., 0.524 g. and 0.505 g.) respectively and the lowest weight recorded (0.441 g.) for Azolla1%.But the results differed during the 5 th larval instar where, the maximum weight recorded for the larvae that fed on Azolla3%, Azolla2% and Spirulina1% significantly, was 3.63g., 3.52 and 3.62 g. respectively and the control value was the minimum (2.64g.).
Regarding the overall growth rate, the results have the same direction of the larval weights, during 4 th instar.The highest values recorded for Spirulina1%, Spirulina2%, Spirulina3% and Control without any significance between each other were, 443.5, 431, 439 and 416 % respectively, and Azolla1% recorded the lowest value (354%).Significant results were obtained during the 5 th larval instar, the maximum results for overall growth weight were 674.5 and 647 % for Azolla2% and Azolla3%, respectively and the minimum value was (387.5 %) for the control.
In the larval duration or longevity, the data take another direction, whereas the control has significantly the highest result (768 hr.) representing 32 days but the larvae fed on Azolla with all concentrations recorded the lowest values (696 hr.) representing 29 days.
Fecundity is an indicator of the effect of protein on larvae, Table (1) showed that larvae fed on Azolla2% recorded maximum significant value (708 eggs), followed by other treatments that were 587, 581, 569, 553 and 549 for (Azolla3%, Spirulina1%, Spirulina3%, Spirulina2% and Azolla1%) respectively, without any significance between each other.But the lowest value was (464 eggs) for the control.

Table (1):
Effect of Azolla pinnata and Spirulina plantalis as a probiotic on the biological characteristics of the silkworm larvae Bombyx mori L.
Means with the same letter is not significant

B-Biochemical Analysis:
Pooled analysis of silkworm heamolymph showed fluctuations in all biochemical analyses of the larvae fed on enriched mulberry leaves with different concentrations of algal probiotics under study.

Total Protein:
The larvae fed on fortified mulberry leaves with Azolla2% recorded the maximum results, which was a significance of 73. 8 mg/ml.Followed by Azolla 1%, Azolla 3% and Spirulina 3% on par were 60.26, 60.30, and 62.26 mg/ml..The lowest value was 38.86 mg/ml, for the control.

Amylase:
The results of the amylase enzyme have the same direction as the total protein data, whereas the maximum significant data for larvae fed on Azolla2% (90.96 u glucose/min/ml) followed by Azolla3% (33u glucose/min/ml) and the lowest result were 11.3, 11.8 and 12.06 for Spirulina2%, Spirulina3%, and the control without any variance between each other.

Protease:
The data of protease enzyme have the maximum significant value (188.66 ug alanin/min/ml) for Spirulina3%.Followed by the control which was 142.33 ug alanin/ min/ ml and the lowest data were 66.70, 74.43, and 72 ug alanin/min/ml significantly for Azolla1%, Azolla2%, and Azolla3% respectively, Table 3: Effect of the Impact of Azolla pinnata and Spirulina platensis on some biochemical analysis of the silkworm larvae Bombyx mori L.
Means with the same letter is not significant

DISCUSSION
Friendly microorganisms (probiotics) are a very important factor in maintaining a healthy intestine and raising immunity.Although silkworm gut contains different types of microorganisms adding algal probiotics supplements positively on all characters.

A-Biological Parameters:
The previous results indicate that adding probiotics during the 4 th larval instar affect negatively on larval weight and 4 th overall growth rate of silkworms.These findings are in line with Saad et al. (2014) who concluded that full-grown larvae were affected significantly by adding the supplements.Marwa (2023) stated that Echinacae botanical extract affects negatively the larvae during the 4 th instar.However, the results changed during the 5 th larval instar and the additives affect positively on 5 th larva weight, 5 th overall growth rate, longevity and fecundity.These results are in agreement with Mala et al. (2017) who supplemented mulberry leaves with Alo vera that gave a positive impact on silkworm growth.Shruti et al. (2019) reported that adding Azolla and Spirulina to mulberry leaves increased body weight.Shinde et al. (2014) silk industry was affected positively by mulberry variety and nutritive additives.Also, Masthan et al. (2017) reported the positive impact of Spirulina on silkworm larval weight.The overall growth rate increased by adding the honey bee to the mulberry leaves Sharma et al (2023).
Longevity or larval duration is an important economic factor in sericulture, the reduction of larval duration saved the consumed mulberry leaves.In this investigation adding Azolla pinnata or Spirulina reduced the larval duration.Mulberry leaves enriched with soya bean reduced the larval duration Horie and Watanabe (1983).Larval duration is reduced by adding Azolla to mulberry leaves during the spring season.Furthermore, increasing larval duration depends on the additives and the type of probiotics Shruti et al. (2019).Silkworms fed on mulberry leaves smeared with Spirulina fusiforma during the 5 th instar consumed 6 days otherwise untreated group took 9 days Mathavan et al. (1984).
As shown previously, moths that emerged from the larvae fed on mulberry leaves sprayed with 2% azolla pinnata and laid the maximum number of eggs significantly.This result was confirmed with the female pupal weight that recorded the highest weight in this investigation.These results are in line with Singh and Prasad (1987)who referred to the positive correlation between pupal weight and fecundity.There was a linear correlation between spirulina concentrations and the number of laid eggs Kumar and Bakiya (2023).The number of eggs laid increased as a result of adding soya bean to the feeding of mulberry silkworm Sawsan (2020).

B-Cocoon Indices:
Cocoon weight is an important economic indicator in sericulture that refers to the approximate quantity of raw silk output.As shown previously feeding enhanced with probiotics (Azolla pinnata, and Spirulina plantalis) had a positive impact on cocoon parameters.Our results conform with Venkataramana et al. (2003) who reported the positive impact of adding Spirulina to the feeding of silkworm larvae during 5 th instar on cocoon characters.Feeding silkworm larvae on mulberry leaves smeared with Spirulina and thyroxine during the 5 th instar affected positively on the cocoon parameters ( cocoon weight, shell weight, and shell ratio) Maqbool et al. (2023).Vijaykumar et al. (2016) referred to the increase in cocoon weight, and shell weight resulting when added protein source to silkworm larvae feeding.Cocoon weight and cocoon shell increased by adding Azolla pinata 50% over control Shruti et al. (2019).Also, our results are comparable with Sujatha et al. (2015) who reported that certain concentrations of algal extract improved the biological and economic characteristics of silkworm larvae.Adding 3% of probiotics increased the cocoon parameters of silkworm larvae Pachiappan et al. (2021).Sekar et al. (2016) reported that larvae fed on mulberry leaves coated with Lactobacillus Casei improved cocoon parameters.

C-Biochemical Analysis:
Previous findings of the present investigation imply that adding algal probiotics as a food supplementary has a good impact on enzymatic activity that increases total protein, amylase, invertase and protease enzymes in the treated larvae compared to untreated larvae.Protein is an aid in the development, growth and silk production our investigation recorded the highest activity for larvae fed on 2% of Azolla pinnata.This results in agreement with Wani et al. (2021) hemolymph protein content varied according to the rate of protein metabolism.Larvae fed on mulberry leaves coated with different concentrations of chlorella pyrinoidosa increased heamolymph protein Dyanaselin (2021).
Hemolymph protein increased at 2% of probiotics normagut in silkworm larvae Anisha et al. (2022).Although Spirulina plantalis had a high protein content of about 61% from its dry weight of protein its effect was less than Azolla in all biological economic and biochemical analyses.These results conform with Marwa (2024) who referred to the negative effect of high doses of egg albumen on silkworm production.Amylase enzyme plays a vital role in complex carbohydrate digestion so increasing the activity does not mean an increase in the production directly but supports the larvae with energy to complete its life cycle.An increase in hemolymph amylase activity results from increasing metabolism that raises enzyme secretion Lokes et al. (2012).Pachiappan et al. (2021) concluded that amylase and invertase activity increased by increasing the concentration of Bifidobacterium longum in silkworm feeding.
About the protease enzyme, the increase in the activity in the larva fed on mulberry leaves enriched with spirulina resulted from its acting as an inducer for releasing and activating enzyme.Our findings are consistent with Bhuvaneswari et al. (2023) who reported the importance of spirulina for the activation of protease enzyme.Enzyme activity varied due to the food chemical constituted Kumari et al. (1997).Spirulina is an important factor in protein digestion by raising protease activity Selin et al. (2018).In this investigation although larvae fed on mulberry leaves smeared with 3% of Spirulina showed high activity of protease enzyme but recorded lower protein content and biological parameters than Azolla pinnata.These results are in line with Ruth et al. (2019) who referred to the larvae fed on Johart mulberry leaves recorded a high increase of protease enzyme but larvae fed on TR10 mulberry leaves recorded high protein content that resulted from the ability of feeding to facilitate the reaction of enzyme on its substrate.

CONCLUSION:
Adding algal probiotics (Azolla or Spirulina) to the feeding of silkworms had a positive impact on growth and enzyme activity.Although the most effective concentrations in all criteria under study were Azolla (2% and 3%), and Spirulina (1% and 3%) the most recommended one was Azolla2%.It affects significantly the fecundity in the females and the shell ratio in the males.Also, reduced larval duration than Spirulina.Declarations: Ethical Approval: Ethical Approval is not applicable.Competing interests: The authors declare no conflict of interest.Funding: No funding was received.

Table 2 :
Effect of the Impact of Azolla pinnata and Spirulina platensis as a probiotic on the cocoon indices of the silkworm larvae Bombyx mori L.
C.w: Cocoon weight C.S.W: Cocoon shell weight P.W: Pupa weight C.S.R%: Cocoon shell ratio Means with the same letter is not significant