### Materials

Bivoltine hybrid silkworm *Bombyx mori*, plastic rearing boxes, nylon net, iron stand, ant wells, ruler scale, weighing electrical balance, silver nitrate (AgNO_{3}), trisodium citrate (C_{6}H_{5}O_{7}Na_{3}), and double-distilled water were used throughout the experiment. The chemical used in this study was purchased from Hi media laboratory, New Delhi.

### Rearing of mulberry silkworm *Bombyx mori*

The third-instar larvae of Indian bivoltine hybrid silkworm *Bombyx mori* were collected from chawki worm rearing center, Kethanur, Tamil Nadu, India. The larvae were divided into 6 experimental groups including the control, each group consisting of 15 larvae. The larvae were reared in plastic boxes measuring 15 × 15 × 5 cm covered with nylon net and placed in an iron stand with ant wells. The control and treated MR2 mulberry leaves were fed to silkworms, four times per day (6 am, 10 am, 3 pm, and 10 pm). They were maintained up to the cocoon stage.

### Fabrication of silver nanoparticles

The silver nanoparticles were synthesized using chemical reduction method (Thangapandiyan & Prema, 2012). In this method, 150 ml of 1 × 10^{−3} M AgNO_{3} solution was heated to boiling. To this solution, 5 ml of 1% trisodium citrate was added drop by drop. During this process, the solution was mixed vigorously. Reaction mixture was heated until the color changes from colorless to pale yellow. Then, it was removed from the heating element and stirred until cooled to room temperature.

Mechanism of reaction could be expressed as follows:

4Ag^{+} + C_{6}H_{5}O_{7}Na_{3} + 2H_{2}O → 4Ag^{0} + C_{6}H_{5}O_{7}H_{3} + 3Na^{+} + H^{+} + O_{2} ↑

The solution was washed three times in double-distilled water and once in ethanol solution. The supernatant of the reaction solution was discarded, and particles were dried in hot air oven at 60 °C. The dried particles were used for further analysis.

### Preparation of Spirulina solution

Spirulina powder was purchased from Twenty First Century pharmaceuticals Pvt. Ltd., Chennai, and the experimental dose for 300 ppm concentration was prepared.

### Mulberry leaves treated with silver nanoparticles and Spirulina

Different concentrations of AgNPs (100 ppm, 300 ppm, 500 ppm), AgNPs with Spirulina, and Spirulina alone were prepared. Fresh MR2 mulberry leaves were separately soaked in these concentrations for 15 min and then were dried in air for 10 min. The treated mulberry leaves were used to feed the third-, fourth-, and fifth-instar larvae of the silkworm *Bombyx mori*. They were maintained up to the cocoon stage.

### Characterization of silver nanoparticles

The fabricated silver nanoparticles were confirmed by optical measurement using Shimadzu dual beam spectrophotometer (UV-2000) in the wavelengths ranging between 200 and 800 nm at the resolution of 1 nm. The morphological characterization of the prepared AgNPs was observed by scanning electron microscopy (JSM 35 CF JEOL) operated at a resolution of 60 A° at 15 kv magnification of 5.0 k. The scale was about 32 mm. Confirmation of elemental silver in the synthesized nanoparticles was carried out by EDS instrument (JSM 35 CF JEOL) in a resolution of 60 Å, operated at 15.0 kV with a magnification of about 5 k.

### Estimation of nutritional traits

The nutrigenetic traits estimation study was carried out in the fifth-instar larvae. Silkworm rearing was accompanied by a standard method under the acclaimed temperature and relative humidity until the fourth molt. On the first day of the fifth instar, from the entire 90 larvae, five healthy silkworms in each group were selected for estimation on nutritional traits analysis. Accurately weighed fresh mulberry leaves were fed five times a day to the experimental group and the control. Simultaneously, daily increases in larval weight were recorded separately. Silkworm rearing continued using appropriate plastic trays. The healthy larvae were counted daily in each group. Left over leaves and excreta were collected on each subsequent day, separated manually, and dried in a hot air oven daily at about 100 °C until they reached a constant weight using an air-tight electronic balance. When the larvae finished feeding, they were shifted to the mountage for spinning at normal ambient temperature of 25 ± 2 °C and RH 65 ± 5%. Cocoons were harvested 4–5 days later after completion of cocoon spinning. Harvested cocoons were retrieved for quantitative traits using the equations detailed below. The dry weight of leftover leaves, excreta, larvae, cocoon, and shell in each treatment was recorded.

During the silkworm nutritional study, data were collected on the biomass of larvae and cocoons for the 19 nutrigenetic traits on ingesta, digesta, excreta, approximate digestibility (AD), reference ratio (RR), consumption indices (CI), relative growth rate (RGR), respiration and metabolic rate (MR), and efficiency conversion of ingesta (ECI) and digesta (ECD) for larva, cocoon, and shell. Further, the ingesta and digesta required for producing 1 g of cocoon and shell (I/g and D/g) were collected and calculated as described by standard gravimetric methods.

The equations with brief description of the nutrigenetic traits evaluated are given below (Ramesha, Lakshmi, Kumari, Anuradha, & Kumar, 2012).

$$ \mathrm{Ingesta}\ \left(\mathrm{g}\right)=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{leaf}\ \mathrm{fed}-\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{left}\ \mathrm{over}\ \mathrm{leaf} $$

$$ \mathrm{Digesta}\ \left(\mathrm{g}\right)=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{leaf}\ \mathrm{ingested}-\mathrm{dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{litter} $$

### Approximate digestibility (%)

This directly indicates the assimilation efficiency of mulberry leaves and depends on the passage rate of food through gut in silkworm

$$ \mathrm{AD}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{Digesta}/\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{food}\ \mathrm{ingested}\ \mathrm{x}\ 100 $$

### Reference ratio

This is an indirect expression of absorption and assimilation of food and expresses the ingesta required per unit excreta produced

$$ \mathrm{RR}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{food}\ \mathrm{ingested}/\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{excreta} $$

### Consumption index

$$ \mathrm{CI}=\mathrm{Ingesta}/{5}^{\mathrm{th}}\ \mathrm{stage}\ \mathrm{mean}\ \mathrm{fresh}\ \mathrm{larval}\ \mathrm{weight}\ \left(\mathrm{g}\right)\ \mathrm{x}\ {5}^{\mathrm{th}}\ \mathrm{stage}\ \mathrm{larval}\ \mathrm{duration}\ \mathrm{in}\ \mathrm{days} $$

### Relative growth rate

Refers to larval gain biomass and indicates the efficiency of conversion of nutrition into larval biomass.

$$ \mathbf{RGR}=\frac{\mathbf{Weight}\ \mathbf{gain}\ \mathbf{of}\ \mathbf{the}\ \mathbf{larva}\ \mathbf{during}\ \mathbf{feeding}\ \mathbf{period}\ }{\mathbf{5}\mathbf{th}\ \mathbf{stage}\ \mathbf{mean}\ \mathbf{fresh}\ \mathbf{larva}\mathbf{l}\ \mathbf{weight}\ \left(\mathbf{g}\right)} \times {\mathbf{5}}^{\mathbf{th}}\ \mathbf{stage}\ \mathbf{larva}\mathbf{l}\ \mathbf{duration}\ \mathbf{in}\ \mathbf{days} $$

### Respiration

A catabolic reaction in which total oxidation of the digested or assimilated food for releasing energy required for the entire biological activities by break down of macromolecules into simpler molecules

$$ \mathrm{Respiration}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{food}\ \mathrm{digested}-\mathrm{Maximum}\ \mathrm{dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{larvae} $$

### Metabolic rate

Measure of total biochemical reactions involving both catabolic and anabolic reactions of an organism, associated with the degradation of macromolecules into smaller unit and vice versa

$$ \mathrm{MR}=\mathrm{Respiration}/{5}^{\mathrm{th}}\ \mathrm{stage}\ \mathrm{mean}\ \mathrm{fresh}\ \mathrm{larval}\ \mathrm{weight}\ \left(\mathrm{g}\right)\ \mathrm{x}\ {5}^{\mathrm{th}}\ \mathrm{stage}\ \mathrm{larval}\ \mathrm{duration}\ \mathrm{in}\ \mathrm{days} $$

### ECI/ECD to larva (%)

The expression of efficiency conversion of ingesta and digesta into larval biomass

$$ \mathrm{ECI}/\mathrm{ECD}\ \mathrm{to}\kern0.5em \mathrm{larva}=\frac{\mathrm{dry}\ \mathrm{weight}\ \mathrm{gained}\ \mathrm{by}\ \mathrm{larva}\mathrm{e}\ \mathrm{during}\ \mathrm{feeding}\ \mathrm{period}}{\mathrm{dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{ingesta}\ \mathrm{or}\ \mathrm{digesta}}\mathrm{x}\ 100 $$

Efficiency conversion of ingesta/efficiency conversion of digesta (ECI/ECD)

### ECI/ECD to cocoon (%)

This is the most economically important trait used by the sericulture industry. It was the expression of efficiency conversion of ingesta and digesta into the cocoon, also referred to as the leaf-cocoon conversion rate. This nutrigenetic trait was kept as the ultimate index for assessing the superiority of group for nutritional efficiency in this investigation

$$ \mathrm{ECI}/\mathrm{ECD}\ \mathrm{to}\kern0.5em \mathrm{cocoon}=\frac{\mathrm{dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{cocoon}}{\mathrm{dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{food}\ \mathrm{ingested}\ \mathrm{or}\ \mathrm{digested}}\mathrm{x}\ 100 $$

Efficiency conversion of ingesta/efficiency conversion of digesta (ECI/ECD)

### Ingesta required producing 1 g of cocoon/cocoon shell

This was another important trait of profitable significance to assess silkworm breed performance in nutrigenetic analysis. It was the expression of total ingesta required for the production of 1 g of cocoon

$$ \mathrm{Ingesta}/\mathrm{g}\ \mathrm{cocoon}/\mathrm{shell}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{Ingesta}/\mathrm{Dry}\ \mathrm{cocoon}\ \mathrm{or}\ \mathrm{cocoon}\ \mathrm{shell}\ \mathrm{weight} $$

### Digesta required producing 1 g of cocoon / cocoon shell

The total digesta requisite for the production of 1 gram of cocoon and shell

$$ \mathrm{Digesta}/\mathrm{g}\ \mathrm{cocoon}/\mathrm{shell}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{Digesta}/\mathrm{Dry}\ \mathrm{cocoon}\ \mathrm{or}\ \mathrm{cocoon}\ \mathrm{shell}\ \mathrm{weight} $$

### Analysis of economic traits

Five days after spinning, the cocoons were harvested. About five cocoons from each group were taken for analysis of their economic traits.

### Length and weight of silkworm

Five larvae were randomly selected in each group and the larval weight was measured using electronic balance, and it was expressed in grams (g). The lengths of the third-, fourth-, and fifth-instar larvae were measured using a ruler scale, and it was expressed in centimeter (cm).

### Weight of silk gland

At the end of the fifth instar, when the larvae stopped eating and emptied their gut, five larvae were randomly selected from each group and anesthetized with water. The silk glands were removed and washed thoroughly with water. The weight of the silk glands was measured in each group separately.

### Weight of cocoon

Five days after spinning, the cocoons were harvested and weighed using electronic balance.

### Weight of pupae

After taking the cocoon weight, the cocoon was cut open and the pupal weight was measured.

### Cocoon shell ratio

It is the ratio between the weight of the shell, and the whole weight of the cocoon expressed as percentage. It is calculated by using this formula (Rajitha & Savithri, 2015)

$$ \mathrm{cocoon}\ \mathrm{shell}\ \mathrm{ratio}\ \left(\%\right)=\frac{\mathrm{cocoon}\ \mathrm{shell}\ \mathrm{weight}}{\mathrm{cocoon}\ \mathrm{weight}}\times 100 $$

Cocoon shell ratio helps to estimate the raw silk yield as well as the price of the cocoons. The ratio is altered based on the cocoon age.

### Length of silk filament

About four cocoons from each group was taken and immersed in hot water (65–75°C) for 10 min. The tightly spun thread became loose. The silk filament was reeled out using a handy reeling machine “Epprouvette”, and the total length for each cocoon was measured being expressed in meters (m), and also, the filament is weighed. It can be estimated by using the following formula (Rajitha & Savithri, 2015)

$$ \mathrm{Silk}\ \mathrm{filament}\ \mathrm{length}\ \left(\mathrm{m}\right)=\mathrm{Revolutions}\ \mathrm{of}\ \mathrm{epprouvette}\ \mathrm{X}\ \mathrm{Wheel}\ \mathrm{circumference}\ \left(\mathrm{m}\right) $$

### Estimation of sericin and fibroin content

After the completion of the spinning, the cocoons were collected and opened to remove the pupae. The shells were dried at 80 °C and weighed. The cocoon shell were treated with 0.5% KOH for 6 h and thoroughly washed in hot water. The following formulae were used to estimate the sericin and fibroin content (Mondal, Kanika, & Nirmal, 2007)

$$ \mathrm{Sericin}\ \mathrm{content}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{cocoon}-\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{cocoon}\ \mathrm{after}\ \mathrm{alkali}\ \mathrm{treatment} $$

$$ \mathrm{Fibroin}\ \mathrm{content}=\mathrm{Dry}\ \mathrm{weight}\ \mathrm{of}\ \mathrm{cocoon}-\mathrm{Sericin}\ \mathrm{content} $$

### Statistical analysis

SPSS 20 version were used for the determination of Duncan’s multiple range test (DMRT), correlation, and mean ± standard deviation