Techno-Mechanical and Structural Properties of Indian Mulberry Silkworm Fibers: An Insight into the Structure–Property Relationship

Abstract

Non-textile application of silk fiber is the major focus of the present scientific communities. Characteristics, i.e., structural, mechanical, are the key advantages of silk protein to make it promising candidates for its variable application. Keeping this in view, the present investigation has been conducted to understand the structural and mechanical variability of silk breeds, i.e., CSR₂ × CSR₄ (single hybrid), PM × CSR₂ (cross breed) and FC₁ × FC₂ (double hybrid) for their respective promising non-textile application. It is envisaged that FC₁ × FC₂ (double hybrid) has the highest tensile strength (431.47 ± 28.46 MPa), Young’s modulus (5.92 ± 0.45 GPa) and β-sheet content (46.62 ± 1.45%). The lowest nano-crystallite size (3.34 ± 0.22) and elongation % (10.85 ± 0.77) were also observed in the FC₁ × FC₂. Further, significant positive correlation was observed between β-sheet with crystalline % (p *** < 0.001; r = 0.95), crystalline % with tensile strength (p *** < 0.001; r = 0.91) and Young’s modulus with tensile strength (p * < 0.001; r = 0.80). This indicates that the higher the β-sheet content is, the higher the tensile strength and higher crystalline phase of the fiber will be. Crystallite size has a negative correlation with the β-sheet content, crystalline %, tensile strength and Young’s modulus, which shows that the lower the crystallite size, the more the compactness and strength will be.

1. Introduction

Bombyx mori silkworm silk is a high-performance natural fiber, with hundreds of thousands of tons produced annually for use in both the textile industry and emerging biomedical applications. Silk is a prized biopolymer, serving as a cornerstone of the textile industry due to its plethora of unique properties, including high tensile strength, extensibility, luster, and sheen. In addition, silk is highly valued in biomedical applications because of its biocompatibility and favorable processing characteristics [1].

Silkworms produce silk at the end of their larval stage to spin a protective cocoon within which they undergo pupation [2,3,4]. The cocoon functions as a highly specialized natural structure providing mechanical protection against injury and infection, as well as environmental buffering for temperature and humidity. The silk used to form this cocoon is pultruded as a continuous signal fiber up to hundreds of meters in length.

Raw silk produced by silkworms consists of two fibroin protein core filaments (brins), which provide the fiber’s load-bearing capacity [5]. These filaments are then enveloped by a sericin protein coating, which acts as a natural gumming agent, binding the filaments together and facilitating cocoon formation. To obtain usable silk fibers, the cocoons are typically subjected to a degumming process in which they are first softened by steaming or boiling, and then the sericin is removed with a boiling sodium carbonate solution. The filaments are then gently unwound from the cocoon, producing continuous silk threads ready for weaving or other uses.

The silk fibers extracted from these cocoons exhibit excellent mechanical properties, with an ultimate tensile strength of 300–700 MPa and a strain at failure of 4–26% [6,7]. In addition, the fibers’ relatively small diameter results in a material that is more comfortable than many synthetic fibers. These exceptional mechanical properties arise from the structural organization of silk, which consists of highly crystalline beta-sheet domains connected by relatively disordered amorphous regions [8]. However, several factors can impact the crystal-limited molecular orientation of the silk and, consequently, its mechanical properties. These factors include spinning conditions, climatic conditions (temperature and humidity), and the rearing conditions of the silkworm, such as feed quality and overall health [9,10,11,12].

One often underappreciated factor affecting the properties of produced silk is the genetic makeup of the silkworm, with the breed significantly influencing larval duration, cocoon yield, filament length, and the silk’s material properties. All these factors have a direct impact on the profitability and economics of sericulture. Therefore, it is fundamental to identify silkworm breeds that not only produce high silk yields but also exhibit superior mechanical and structural characteristics, balancing these traits according to the intended application of the silk. A breed/hybrid with favorable cocoon yield and long filament length may satisfy commercial textile requirements, but for advanced materials, the fiber’s micro-structure such as β-sheet content, crystallinity, crystallite size, and fiber diameter becomes increasingly critical. Therefore, in this study, we aim to compare three key Bombyx mori hybrids to develop an evidence-based understanding of their growth, cocoon-spinning, and the mechanical behavior of their spun silks. These hybrids viz., CSR₂ × CSR₄ (single hybrid), PM × CSR₂ (cross breed) and FC₁ × FC₂ (double hybrid) were taken, because these are popular at field level and the most mulberry cocoon production comes from these hybrids. This will also allow sericulture practitioners to make informed decisions on silkworm breed selection, while also providing insights into the mechanical and structural properties of these hybrids to support future silkworm breeding programs.

2. Materials and Methods

2.1. Rearing of Silkworm

The silkworm hybrids utilized in this study consisted of, bivoltine single hybrid (CSR₂ × CSR₄), multi x bivoltine hybrid (PM × CSR₂) and bivoltine double hybrid (FC₁ × FC₂). The general features and images of larvae, cocoon and silk fiber of these hybrids are summarized in

Table 1. Salient features of hybrids studied.

Hybrid	Fecundity	Egg Color	Newly Hatched Larvae Color	Larval Pattern	Cocoon Shape	Cocoon Color	Feature
CSR2 × CSR4	550–600	Yellow	Black	Plain bluish white	Hybrid shape	White	Productive single hybrid
PM × CSR2	450–500	Light Yellow	Black	Plain	Oblong shape	Light greenish yellow	Cross breed with robustness
FC1 × FC2	550–600	Yellow	Black	Marked brownish white	Hybrid shape	Bright white	Productive double hybrid

and Figure 1. The selected silkworm hybrids were reared employing the standard rearing technique as advocated by Dandin et al. 2014 [13]. The effective rate of rearing was estimated utilizing Equation (1).

2.2. Recording of Cocoon and Reeling Parameters

Key parameters related to the mechanical properties of the hybrid cocoons were measured. All experiments for the three hybrid types were carried out in triplicate unless otherwise stated. Total Larval Duration (h) was calculated as the total hours taken from the date of brushing to the mounting of ripe worms for each hybrid group. Fifth Instar Larval Duration (h) was calculated as the total hours taken by larvae from the first day of 5th instar up to mounting of the ripe larvae for each hybrid group. The average weight of mature larvae (g) was determined by randomly selecting 10 mature larvae from each hybrid group. The average cocoon weight (g) was determined by weighing 10 cocoons from each hybrid group. Single cocoon shell weight (g) was determined by carefully extracting a single shell from each cocoon in for each hybrid group and weighing it individually. The shell ratio percentage and filament length was calculated as per Equations (2) and (3) respectively.

$$\mathrm{E}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{R}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{g} (\mathrm{E}\mathrm{R}\mathrm{R}) \% = {\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{o}\mathrm{n}\mathrm{s} \mathrm{h}\mathrm{a}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}}{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{a}\mathrm{r}\mathrm{v}\mathrm{a}\mathrm{e} \mathrm{b}\mathrm{r}\mathrm{u}\mathrm{s}\mathrm{h}\mathrm{e}\mathrm{d}}}\times 100$$

(1)

$$\mathrm{S}\mathrm{h}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o} \% = {\displaystyle \frac{\mathrm{S}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{l}\mathrm{e} \mathrm{S}\mathrm{h}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{g}\right)}{\mathrm{S}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{l}\mathrm{e} \mathrm{C}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{o}\mathrm{n} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} (\mathrm{g})}}\times 100$$

(2)

$$\mathrm{L} = \mathrm{R}\times 1.125 \mathrm{m}$$

(3)

where L-Length of the filament (m); R-Number of revolutions; 1.125 m-circumference of the eprouvette reel.

The NBFL, denier, renditta, raw silk percentage and BOR were estimated using Equations (4)–(8) respectively.

$$\mathrm{N}\mathrm{B}\mathrm{F}\mathrm{L}={\displaystyle \frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{F}\mathrm{i}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}}{1 + \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{k}\mathrm{s}}}$$

(4)

$$\mathrm{D}\mathrm{e}\mathrm{n}\mathrm{i}\mathrm{e}\mathrm{r}={\displaystyle \frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{f}\mathrm{i}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{f}\mathrm{i}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}}\times 9000$$

(5)

$$\mathrm{R}\mathrm{e}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{t}\mathrm{t}\mathrm{a}={\displaystyle \frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{e}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{d}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{r}\mathrm{a}\mathrm{w} \mathrm{s}\mathrm{i}\mathrm{l}\mathrm{k} \mathrm{o}\mathrm{b}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}}}$$

(6)

$$\mathrm{R}\mathrm{a}\mathrm{w} \mathrm{S}\mathrm{i}\mathrm{l}\mathrm{k} \%={\displaystyle \frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{r}\mathrm{a}\mathrm{w} \mathrm{s}\mathrm{i}\mathrm{l}\mathrm{k} \mathrm{o}\mathrm{b}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{e}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{d}}}\times 100$$

(7)

$$\mathrm{B}\mathrm{O}\mathrm{R} \%={\displaystyle \frac{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} - \mathrm{D}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}}\times 100$$

(8)

2.3. Sample Preparation

Cocoons were boiled in distilled water for 10 min to soften the shell. The softened cocoons were then wet-reeled to obtain continuous fibers, taking care to avoid breaks and entanglements during the reeling process. The reeling was done with laboratory eprouvette.

To Degum the silks reeled fibers were washed with distilled water. A solution of 0.2 wt. % of Na₂CO₃ (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 wt. % of Marseille Soap in distilled water was made. The solution was heated until boiling and the fibers were placed in the solution for 45 min. After 45 min, the fibers were removed and thoroughly washed three times with distilled water to remove all soap. They were then soaked in fresh distilled water for 5 min, after which the distilled water was replaced, and the fibers were left for an additional 5 min before being dried at ambient temperature.

To obtain scanning electron microscopy (SEM) images and diameter, the silk fibers were gold-coated with a thickness of 2 nm using S-3400N, model: Noran system 7 (Japan). The minimum of 10 fibers were analyzed for the same.

Mechanical properties such as tensile strength, Young’s modulus and elongation of the silk fiber of the silkworm hybrids were assessed by using a mechanical testing apparatus (Instron Micro-Tester; EXCEED Model E43 MTS USA). The test was conducted under the following specifications: Gauge length, 30 mm; Strain rate, 50 mm/min and a pretension of 0.5 N (load cell) at ambient temperature (27.2 °C) and humidity (65%).

For assessment of structural properties, Fourier Transform Infrared Spectroscopy (FTIR) spectra were collected on a PerkinElmer Spectrum Version (Ethernet, L1600401; SL No.-94012 Made in Llantrisant, UK) spectrometer ranging from 600 to 4000 cm⁻¹ with 32 scans followed by deconvolution of FTIR spectra to measure β-sheet content as suggested by Belton et al. 2018 [14]. Deconvolutional analysis of the amide I band (1700–1600 cm⁻¹) spectra was first subjected to baseline correction using a straight line between 1600 and 1700 cm⁻¹ followed by multipeak Gaussian peak deconvolution. The detection of peaks was carried out by analyzing the characteristic points for β-sheet, random coil, α-helices and β-turns as described in the literature. The peak half-width was fixed in the range from 10 to 30 cm⁻¹. All analysis was undertaken using Origin-Pro 2024 software.

Measurement of crystallinity: An X-ray diffractometer (Righaku, smartlab 3 Kv, Japan) with Cu Ka radiation operated at 40 kV and 30 mA was used to evaluate the crystalline features of silk fibers. Samples were scanned from 3 to 60 at a step of 0.02, by utilizing a curved position sensitive detector relating to the transmission mode. The crystalline phase present in fibroin of selected silkworm breeds and hybrids were identified by X-ray diffraction technique (XRD) as per the protocol suggested by Wang et al. 2021 [15]. The crystalline percentage and crystallite size were calculated as per Equations (9) and (10):

$$\mathrm{C}\mathrm{r}\mathrm{y}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{e} \%={\displaystyle \frac{\mathrm{X}}{\mathrm{Y}}}\times 100$$

(9)

where X is the net area of the diffracted peaks and Y is the net area of the diffracted peaks + background area.

$$\mathrm{C}\mathrm{r}\mathrm{y}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{e} \mathrm{s}\mathrm{i}\mathrm{z}\mathrm{e} \left(\mathrm{\mu }\mathrm{m}\right)={\displaystyle \frac{\mathrm{ĸ}\mathsf{\lambda }}{\mathsf{\beta }\mathrm{C}\mathrm{o}\mathrm{s}\mathsf{\theta }}}$$

(10)

where K is a dimensionless shape factor, λ is the wave length of X-ray beam, β is the full width at half-maximum (FWHM) of the peak and θ is the Braggs angle. Here, K is taken as a value of 0.9 according to a previous study.

2.4. Statistical Analysis

The experiments were conducted in a completely randomized design (CRD). A One-way analysis of variance (ANOVA)/Kruskal–Wallis test was used to determine the significant difference among the attributes. Subsequently, multiple comparisons were performed using Tukey’s HSD (Honestly Significant Difference) or Dunn’s tests. All statistical tests were undertaken using R software 4.4 (R Core Team 2024) [16].

3. Results

3.1. Rearing and Cocoon Parameters

The bivoltine double hybrid (FC₁ × FC₂) recorded significantly maximum larval weight (44.20 ± 0.28 g/10 larvae) followed by PM × CSR₂ (39.78 ± 0.74 g/10 larvae). Statistically, the weight of ten mature larvae (χ² = 5.45; p = 0.065) did not vary significantly among the hybrids studied. The Dunn’s test showed significant differences between single hybrid and double hybrid (p = 0.04) and double hybrid and cross breed (p = 0.04). The total larval duration (TLD) ranges from 552.00 ± 0.00 h (PM × CSR₂) to 576.00 ± 0.00 h (FC₁ × FC₂). The TLD (χ² = 8.00; p = 0.018) varied significantly among the hybrids and the Dunn’s test also showed significant differences between single hybrid and cross breed (p = 0.014) and between double hybrid and cross breed (p = 0.014). The lowest fifth stage larval duration was observed in PM × CSR₂ which was 126.00 ± 0.00 h followed. The fifth stage larval duration (χ² = 8.00; p = 0.018) varied significantly across the hybrids. The FC₁ × FC₂ double hybrid observed highest effective rate of rearing (86.67 ± 0.57%). The effective rate of rearing (%) varied significantly among hybrids (F = 10.46; p = 0.011). Tukey’s HSD Test multiple comparisons test found that the ERR% significantly differed between cross breed and single hybrid (p = 0.04) and also between double hybrid and single hybrid (p = 0.01). The Yield/100 Dfl is main character of any breed/hybrid while taken into the study. In this study the maximum Yield/100 Dfl was found in the double hybrid FC₁ × FC₂ (76.18 ± 1.88 kg) followed by the cross breed, CSR₂ × CSR₄ (70.12 ± 0.16 kg) (

Table 2. Rearing and cocoon performance of silkworm hybrids (Mean ± SD).

Hybrid	Mature Larva (g/10)	Total Larval Duration (h)	Fifth Instar Larval Duration (h)	Effective Rate of Rearing (%)	Yield/100 Dfl	Cocoon Weight (gm)	Shell Weight (gm)	Shell Ratio %
CSR2 × CSR4	39.74 ± 0.55 b	576.00 ± 0.00 a	130.00 ± 0.00 a	84.03 ± 0.95 b	70.12 ± 0.16 b	1.70 ± 0.02 ab	0.35 ± 0.01 a	20.35 ± 0.23 a
PM × CSR2	39.78 ± 0.74 bc	552.00 ± 0.00 b	126.00 ± 0.00 b	85.89 ± 0.58 a	68.00 ± 2.61 b	1.59 ± 0.05 b	0.30 ± 0.01 b	18.66 ± 0.56 b
FC1 × FC2	44.20 ± 0.28 a	576.00 ± 0.00 a	130.00 ± 0.00 c	86.67 ± 0.57 a	76.18 ± 1.88 a	1.82 ± 0.08 a	0.41 ± 0.02 c	22.75 ± 0.52 c
F/χ2 value	5.45	8.00	8.00	10.46	15.76	13.19	77.08	59.00
p value	0.65	0.018	0.018	0.011	0.004	0.006	0.000	0.000
Shapiro–Wilk W	0.79	0.61	0.61	0.93	0.90	0.95	0.91	0.91
p	0.15	0.00	0.00	0.54	0.31	0.75	0.38	0.38

Superscript letters indicate statistical differences. Number with same letters indicate no significant differences between hybrids.

). The Yield/100 Dfl (F = 15.76; p = 0.006) varied significantly across the hybrids and Tukey’s HSD Test multiple comparisons test also found that the yield significantly differed between double hybrid and single hybrid (p = 0.01) and also between double hybrid and cross breed (p = 0.00).

Among the different hybrids studied, the double hybrid (FC₁ × FC₂) recorded a highest cocoon weight of 1.82 ± 0.08 g and the lowest was recorded in PM × CSR₂ (1.59 ± 0.05 g). The performance of different hybrids varied significantly (F = 13.19; p = 0.006) and Tukey’s HSD Test multiple comparisons test also found that the single cocoon weight significantly differed between double hybrid and cross breed (p = 0.00). Shell weight is an important trait for high productivity, which can be mostly achieved by the heterosis effect [17]. The double hybrid performed well, recording the highest shell weight of 0.41 ± 0.02 g, which was found to be statistically superior over all other hybrids The single shell weight (F = 77.08; p = 0.0000) varied highly significant across the hybrids and Tukey’s HSD Test multiple comparisons test also confirms that the single shell weight significantly differed between cross breed and single hybrid (p = 0.00), double hybrid and single hybrid (p = 0.00) and double and cross breed (p = 0.00). Shell ratio is a very important trait, which is considered one of the main breeding targets for silkworm breeders. The highest shell ratio of 22.75 ± 0.52 percent was recorded in FC₁ × FC₂ followed by bivoltine single hybrid (20.35 ± 0.23%) (Table 2). The shell ratio percentage (F = 59.00; p = 0.0000) varied significantly across the hybrids and Tukey’s HSD Test multiple comparisons test also confirms that the shell ratio significantly differed between double hybrid and single hybrid (p = 0.000), cross breed and single hybrid (p = 0.01) and double and cross breed (p = 0.000) (Figure 2).

3.2. Reeling Parameters

There was great variation in the length of silk filament among the different hybrids studied (

Table 3. Reeling performance of silkworm hybrids (Mean ± SD).

Hybrid	Average Filament Length (m)	Average Non-Breakable Filament Length (m)	Denier	Reelability (%)	Renditta (kg)	Raw Silk (%)	Raw Silk Recovery (%)	Neatness (%)	Boil-Off Loss of Ratio (%)
CSR2 × CSR4	939 ± 14.01 a	885 ± 5.51 a	2.74 ± 0.05 a	84 ± 0.84 a	7.26 ± 0.10 a	14.65 ± 0.41 a	72.21 ± 0.17 a	87 ± 1.53 a	25.85 ± 0.78 a
PM × CSR2	705 ± 48.34 b	660 ± 44.44 b	2.93 ± 0.06 b	82 ± 0.35 b	7.59 ± 0.18 b	13.82 ± 0.10 a	65.26 ± 3.01 b	88 ± 2.08 a	25.95 ± 0.98 a
FC1 × FC2	1072 ± 9.29 c	1033 ± 11.06 b	2.80 ± 0.08 ab	88 ± 0.48 c	6.50 ± 0.10 c	16.22 ± 0.42 b	76.11 ± 1.17 a	94 ± 1.53 b	25.59 ± 0.97 a
F/χ2 value	118.20	148.00	7.322	118.00	55.00	37.87	25.99	12.70	0.12
p value	0.000	0.000	0.024	0.000	0.000	0.001	0.006	0.006	0.889
Shapiro–Wilk W	0.86	0.87	0.94	0.86	0.90	0.89	0.86	0.89	0.86
p	0.11	0.14	0.59	0.11	0.27	0.22	0.11	0.20	0.11

Superscript letters indicate statistical differences. Number with same letters indicate no significant differences between hybrids.

). Among the various hybrids, the double hybrid (FC₁ × FC₂) recorded the highest silk filament length of 1072 ± 9.29 m, which was found to be statistically superior (F = 118.20; p = 0.0000) over all other hybrids. The significantly lowest silk filament length of 705 ± 48.34 m was recorded in PM × CSR₂, which is mostly practiced by the farmers in tropical regions. Tukey’s HSD comparisons test confirms that the filament length significantly differed between cross breed and single hybrid (p = 0.000), double hybrid and single hybrid (p = 0.00) and double and cross breed (p = 0.000). Non-breakable filament length (NBFL) is a key quality characteristic of reeling cocoons of any hybrid or breed which envisages achievable production speed. Among the hybrids investigated the double hybrid shows positive results in terms of NBFL of fiber (1033 ± 11.06). Further, the NBFL (F = 148.00; p = 0.000) varied highly significantly among the hybrids and also Tukey’s HSD comparisons test confirms that the NBFL significantly differed between cross breed and single hybrid (p = 0.000), double hybrid and single hybrid (p = 0.001) and double hybrid and cross breed (p = 0.000).

Denier is one of the main characteristics of raw silk, as uniform filament size results in better weaving and reeling efficiencies by a reduction in breakages. The thickness of silk filament was significantly varied in different hybrids. Among the different hybrids tested, the single hybrid (CSR₂ × CSR₄) recorded the lowest denier of 2.74 ± 0.05. The denier (F = 7.32; p = 0.024) varied significantly among the hybrids and Tukey’s HSD comparisons test confirms that the denier significantly differed between PM × CSR₂ and CSR₂ × CSR₄ (p = 0.02), FC₁ × FC₂ and PM × CSR₂ (p = 0.02). The reelability percentage was recorded highest in FC₁ × FC₂, (88 ± 0.48%) followed by CSR₂ × CSR₄ (84 ± 0.84) and the lowest reelibilty was recorded in PM × CSR₂ which was 82 ± 0.35. The reelability percentage (F = 118.00; p = 0.0000) varied significantly among the hybrids and Tukey’s HSD comparisons test approves that the reelability significantly differed between PM × CSR₂ and CSR₂ × CSR₄ (p = 0.000), FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.003) and FC₁ × FC₂ and PM × CSR₂ (p = 0.000). The results showed that the lowest renditta (6.50 ± 0.10 kg) found in FC₁ × FC₂ and the highest renditta (7.59 ± 0.18 kg) was found in cross breed, PM × CSR₂. The renditta (χ² = 55.00; p = 0.000) varied significantly among the hybrids and comparisons test also confirms that the renditta significantly differed between PM × CSR₂ and CSR₂ ×CSR₄ (p = 0.04), FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.00) and FC₁ × FC₂ and PM × CSR₂ (p = 0.00). The raw silk percentage was recorded highest in FC₁ × FC₂ (16.22 ± 0.42) and the lowest was recorded in cross breed, PM × CSR₂ (13.82 ± 0.10). The raw silk percentage (F = 37.87; p = 0.000) varied significantly among the hybrids and comparisons test also confirms that the raw silk % significantly differed between FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.00) and FC₁ × FC₂ and PM × CSR₂ (p = 0.00) (Figure 3).

Raw silk recovery percentage (RSR) denotes to the quantity of raw silk actually reeled relative to the total silk fiber present in the cocoons which are determined by various factors like shell ratio, filament length and uniformity, reeling conditions and machinery and also degumming methods. The maximum RSR was found in FC₁ × FC₂ (76.11 ± 1.17%) and the lowest in PM × CSR₂ (65.26 ± 3.01%). The RSR % varied significantly among the hybrids (F = 25.99; p = 0.006) and comparisons test (Tukey’s HSD) also confirms that the RSR % significantly differed between PM × CSR₂ and CSR₂ × CSR₄ (p = 0.00) and FC₁ × FC₂ and PM × CSR₂ (p = 0.00). The neatness of the cocoon filament is important to produce the high-quality fabrics and the selection of this trait is very important from the breeding point of view. This parameter, shows high heritability and the breed with high neatness is used as breeding resource material and the selection is usually carried out only in early generations. In this study, the neatness was recorded highest in FC₁ × FC₂ (94 ± 1.53%) and this parameter (F = 12.70; p = 0.006) varied significantly among the hybrids and comparisons test (Tukey’s HSD) also confirms that the neatness % significantly differed between FC₁ × FC₂ and CSR₂ ×CSR₄ (p = 0.00) and FC₁ × FC₂ and PM × CSR₂ (p = 0.01). The boil-off loss ratio is one of the most important qualitative traits in silkworm breeding. The BOR% does not varied significantly (F = 0.12; p = 0.889) among the hybrids (Figure 3).

3.3. Diameter of Silk Threads/Scanning Electron Microscope Observations

Each silk sample bears morphologically similar characteristics irrespective of varietal difference (Figure 4). An absence of sericin on the silk surface confirms the effectivity of the degumming method employed in the present investigation. The lowest diameter was recorded in the cross breed PM × CSR₂ which was 10.39 µm and the highest was recorded in FC₁ × FC₂ (11.74 µm). The diameter does not vary significantly across the hybrids studied (χ² = 2.827; p = 0.098) (

Table 4. Diameter (µm) of tested silkworm hybrids.

Hybrid	Mean	Min	Max	SD	SE	Variance %	F Value	p Value
CSR2 × CSR4	10.42	9.77	11.60	0.83	0.37	8.005	2.827	0.098
PM × CSR2	10.39	8.57	12.70	1.49	0.67	14.310
FC1 × FC2	11.74	9.99	13.60	1.43	0.64	12.203

and Figure 5).

3.4. Mechanical Properties

The tensile Force- displacement curves of the three types of silkworm silk fibers is shown in Figure 6 and the calculated mechanical parameters are summarized in

Table 5. Mechanical properties of silkworm hybrids (Mean ± SD).

Hybrid	Tensile Strength (MPa)	Young’s Modulus (GPa)	Elongation (%)
CSR2 × CSR4	320.30 ± 28.58 a	4.35 ± 0.51 a	13.41 ± 4.32 a
PM × CSR2	386.97 ± 6.18 b	4.88 ± 0.31 ab	11.75 ± 2.96 a
FC1 × FC2	431.47 ± 28.46 b	5.92 ± 0.45 b	10.85 ± 0.77 a
F/χ2 value	16.92	10.34	0.425
p value	0.003	0.011	0.800
Shapiro–Wilk W	0.95	0.96	0.80
p	0.69	0.82	0.02

Superscript letters indicate statistical differences. Number with same letters indicate no significant differences between hybrids.

. The tensile strength varied significantly among the hybrids studied (F = 16.92; p = 0.003). The highest tensile strength is recorded in FC₁ × FC₂ (431.47 ± 28.46 MPa) followed by bivoltine single hybrid, CSR₂ × CSR₄ (320.30 ± 28.58 MPa). Tukey’s HSD comparisons test confirms that the tensile strength significantly differed between PM × CSR₂ and CSR₂ × CSR₄ (p = 0.03) and FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.00). The lowest Young’s modulus was recorded in bivoltine single hybrid, CSR₂ × CSR₄ (4.35 ± 0.51 GPa) and the highest was recorded in FC₁ × FC₂ (5.92 ± 0.45 GPa). Young’s modulus (F = 10.34; p = 0.011) varied significantly among the hybrids. Tukey’s HSD comparisons test also confirms that the Young’s modulus significantly differed between and FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.01) and FC₁ × FC₂ and PM × CSR₂ (p = 0.05). The highest elongation % was recorded in CSR₂ × CSR₄ (13.41 ± 4.32%) and the lowest percentage was observed in FC₁ × FC₂ (17.85 ± 0.77) which confirms that single hybrid has highest extensibility as compared to double hybrid. These results are in line with our tensile strength parameters which shows that higher the elongation at break lower will be the tensile strength of that breed. Further, the elongation % (χ² = 0.425; p = 0.80) does not varied significantly among the hybrids (Figure 7)

3.5. Secondary Structure

3.5.1. FTIR Spectral Characteristics

The raw and deconvoluted FTIR spectra are presented in Figure 8 and Figure 9 respectively. Four characteristic peaks were observed at 1616–1637, 1638–1655, 1656–1162 and 1663–1696 cm⁻¹. The first peak at 1616–1637 cm⁻¹ was ascribed to the β-sheet structure owing to the N–H deformation, the second peak was assigned to random coil due to C=O stretching along the silk back bone, the third peak from 1656 to 1662 cm⁻¹ to α-helices and the fourth, and last, from 1663 to 1696 cm⁻¹ were assigned to β-turn structures proposed by Belton et al. 2018 [14].

The present results showed that the highest β-sheet content was found in the double hybrid FC₁ × FC₂ (46.62 ± 1.45%) followed by PM × CSR₂ (40.85 ± 1.21%) (

Table 6. Secondary structural properties of tested silkworm hybrids (Mean ± SD).

Hybrid	β-Sheet %	Random Coil %	α-Helices %	β-Turns %	Crystalline %	Crystallite Size (µm)
CSR2 × CSR4	39.65 ± 0.61 a	29.78 ± 0.39 a	18.10 ± 0.35 ab	12.49 ± 0.56 a	36.35 ± 2.07 a	4.62 ± 0.13 a
PM × CSR2	40.85 ± 1.21 a	30.02 ± 0.97 a	17.68 ± 0.17 a	11.50 ± 0.45 ab	39.08 ± 1.67 a	4.46 ± 0.15 ab
FC1 × FC2	46.62 ± 1.45 b	23.29 ± 0.75 a	19.73 ± 1.49 b	10.37 ± 0.83 b	43.79 ± 1.58 b	3.34 ± 0.22 b
F/χ2 value	31.83	5.6	6.48	8.426	13.33	6.25
p value	0.000	0.060	0.038	0.018	0.006	0.040
Shapiro–Wilk W	0.85	0.78	0.76	0.97	0.94	0.83
p	0.07	0.01	0.00	0.94	0.62	0.04

Superscript letters indicate statistical differences. Number with same letters indicate no significant differences between hybrids.

). The β-sheet content (F = 31.83; p = 0.000) varied highly significant across the hybrids that were studied. Tukey’s HSD comparisons test also confirms that the β-sheet significantly differed between and FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.00) and FC₁ × FC₂ and PM × CSR₂ (p = 0.002). The random coil content was recorded and was not found significantly (χ² = 5.6; p = 0.060) among the hybrids. The FC₁ × FC₂ recorded lowest content (23.10 ± 1.25%) and the highest was observed in PM × CSR₂ which was 30.02 ± 0.97%. The α-helices content was recorded lowest in CSR₂ × CSR₄ (18.10 ± 0.35%) followed by PM × CSR₂ in which it was 17.68 ± 0.17% (Table 6). The α-helices content (F= 6.48; p = 0.038) varied significantly among the hybrids and Tukey’s HSD comparisons test also confirms that the α-helices significantly differed between and FC₁ × FC₂ and PM × CSR₂ (p = 0.002). The β-turns content was recorded lowest in FC₁ × FC₂ (10.37 ± 0.83%) followed by PM × CSR₂ in which it was 11.50 ± 0.45%. The β-turns content (F = 13.33; p = 0.006) varied significantly among the hybrids and Tukey’s HSD comparisons test confirms that the β-turns significantly differed between FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.014).

3.5.2. X-Ray Diffraction Characteristics

The crystalline structure and the respective resultant patterns of silk are depicted in Figure 10. Minimal marked differences were observed among curves, irrespective of silkworm varieties (Figure 10). Six diffraction peaks of all curves were exhibited at around 20.9, 25.5, 26.3, 28.6, 30.9 and 44.34 (Figure 10). These peaks were assigned to the characteristic peaks of the β-sheet crystalline structure [18]. The highest crystalline percentage was observed in FC₁ × FC₂ 43.79 ± 1.58% which have the highest β-sheet percentage also. The lowest crystalline percentage was found in CSR₂ × CSR₄ which confirms the results of β-sheet content (Table 6). The crystalline percentage shows significant variation among the hybrids studied (F = 13.33; p = 0.006) and Tukey’s HSD comparisons test confirms that the crystalline percentage significantly differed between FC₁ × FC₂ and CSR₂ × CSR₄ (p = 0.005) and also between FC₁ × FC₂ and PM × CSR₂ (p = 0.04). The lowest crystallite size was recorded in FC₁ × FC₂ 3.34 ± 0.22 µm followed by PM × CSR₂ 4.46 ± 0.15 µm. The crystallite size (F = 6.25; p = 0.040) varied significantly among the hybrids Tukey’s HSD comparisons test confirms that the crystalline size significantly differed between CSR₂ × CSR₄ and FC₁ × FC₂ (p = 0.04) only (Figure 11).

3.6. Structure–Property Relationship

The structure–property relationship is considered highly significant for physical properties of silk especially mechanical [19]. In this study, we have correlated various parameters by the method of Pearsons correlation coefficient (r). the bench mark for significance, non-significant and highly significant are mentioned in Figure 12 and are represent as ns: p > 0.05; p * < 0.05; p ** < 0.01 and p ***< 0.001. the weight of mature larvae is found significant and have positive correlation with shell ratio % (r = 0.86), single shell weight (r = 0.88) and single cocoon weight (r = 0.80). However, single cocoon weight is found highly significant with a p *** < 0.001 and have positive correlation with single shell weight (r = 0.95), shell ratio % (r = 0.84) and filament length (r = 0.89). The filament length was also found significant (p *** < 0.001) and is positively correlated with the shell ratio % (r = 0.91) and single shell weight (r = 0.94). The β-sheet content is found highly significant (p *** < 0.001) and positively correlated with crystalline percentage (r = 0.95). This parameter was also found significant with a significance level of p ** < 0.01 and was positively correlated with tensile strength (r = 0.82) and Young’s modulus (r = 0.81). The crystalline % was found highly significant (p *** < 0.001) and positively correlated with tensile strength (r = 0.91) and Young’s modulus (r = 0.83). The crystallite size was negatively correlated with the tensile strength (r = −0.82), Young’s modulus (r = −0.80), β-sheet content (r = −0.95) and crystalline percentage (r = −0.91). This indicates that lower the crystallite size higher will be the mechanical and secondary structure properties. The tensile strength has positive correlation (r = 0.80) with Young’s modulus with a significance level of p * < 0.05 (Figure 12).

4. Discussion

Silk derived from Bombyx mori silkworms has long been prized as a valuable textile commodity. However, in recent years, it has become clear that silk has many additional uses far beyond textiles. Therefore, it is imperative to understand the behavioral characteristics and silk properties of different silkworm breeds/hybrid to maximize the economic value provided to the sericulture industry. This study aimed to support sericulturists by evaluating key properties required for rearing of three key hybrids of silkworms, as well as the characteristics of the silk they produce.

In this study, the hybrid PM × CSR₂ exhibited the shortest larval duration of 552 h, compared to FC₁ × FC₂, which demonstrated the longest duration of 576 h. A short larval duration is advantageous as not only does it reduce the risk of disease incidence but also allows for a higher turnover of silkworm generations, leading to quicker cocoon availability. Similarly, the shortest fifth instar duration was also observed in PM × CSR₂. However, although PM × CSR₂ demonstrated the fastest turnover rate with the shortest larval duration, the effective rate of rearing a key indicator of seed-to-cocoon conversion was actually highest in FC₁ × FC₂, with 86.67% of larvae successfully reaching cocoon formation. Naseema Begam et al., [20] also reported superior rearing efficiency in bivoltine hybrids under diverse climatic conditions. Cocoon weight, which reflects the total yield of cocoon spun was the highest in FC₁ × FC₂, while PM × CSR₂ produced the smallest cocoons. The ability of a breed to efficiently convert mulberry leaf input into cocoon output is a critical determinant of commercial profitability, and the superior cocoon weight of FC₁ × FC₂ confirms its strong potential for large-scale silk production. Similar trends of enhanced cocoon weight in bivoltine double hybrids have been documented by several researchers [21]. Furthermore, all hybrids in the present study exhibited acceptable shell weights, exceeding the commercial benchmark of ≥0.45 g [22]. The FC₁ × FC₂ hybrid showed particularly strong performance in characteristics related to quantity of silk production, consistent with earlier reports [17,23,24], all of whom noted superior shell weights and ratios in double hybrids compared to single or multivoltine hybrids. Suresh Kumar et al., 2011 [25] also observed a significantly higher shell ratio (22.38%) in double hybrids, supporting the present findings. The varied shell percentage (18.86–21.29%) among the selected bivoltine breeds were also recorded by Murali et al. 2018 [26].

From a textile industry perspective, FC₁ × FC₂ again showed the most favorable performance, producing the longest silk fibers and unbroken filaments, as well as exhibiting the highest neatness among the three hybrids evaluated. These attributes are highly valued in silk manufacturing because longer and cleaner filaments minimize waste during reeling, enhance spinning efficiency, and result in smoother, more lustrous fabrics with fewer imperfections. The capacity to reel long, continuous filaments also improves the tensile strength and uniformity of woven silk, both of which are essential for high-end textile applications. In terms of denier, FC₁ × FC₂ ranked second lowest, surpassed by CSR₂ × CSR₄. Nevertheless, all three hybrids exceeded the performance of many synthetic and other natural fibers in this metric [27,28]. The raw silk percentage was recorded highest in FC₁ × FC₂, double hybrid followed by CSR₂ × CSR₄. The lowest raw silk percentage was recorded in cross breed, PM × CSR₂. The variation in raw silk percentage shows the potential of any breed or hybrid in terms of silk generation. Majority of economic traits of silk were regulated by polygene, for example, neatness is regulated by a combination of a semi-dominant major gene and few minor genes [21]. However, the environment also affects significantly the expression level of these genes to regulate the economic traits [29]. The boil-off loss ratio (BOR%) serves as a key quality parameter in silk reeling, representing the percentage of material lost during degumming. BOR% values were comparable among the hybrids studied, ranging from 25.59 ± 0.97% in FC₁ × FC₂ to 25.95 ± 0.98% in PM × CSR₂. These findings align with those of Kumar et al. [21], who reported BOR values between 22% and 24% in CSR hybrids. The enhanced BOR might be due to genetic variabilities, i.e., variable gene frequencies at different loci of voltinism cadre which express variability under changing environmental conditions [30,31]. Thus, it is ascertain that the performance of silkworm race/breed/hybrid is the combination effect of hereditary potential and its expressibility under the given environment.

The continuing superiority of FC₁ × FC₂ silk quality is further supported by its mechanical performance, with this hybrid exhibiting the highest ultimate tensile strength and Young’s modulus among the tested groups. The tensile strength of 431.47 ± 28.46 MPa places FC₁ × FC₂ silk at the in the mid of the range reported for Bombyx mori fibers, typically between 300 and 700 MPa, depending on reeling rate, humidity, and post-processing conditions [6,7]. This high strength is consistent with the hybrid’s elevated degree of molecular order, as reflected in its β-sheet content of 46.62 ± 1.45%, substantially higher than that of CSR₂ × CSR₄ (39.65 ± 0.61%) and PM × CSR₂ (40.85 ± 1.21%). The corresponding crystallinity value as determined by XRD of 43.79 ± 1.58% for FC₁ × FC₂ was again the highest among the hybrids. It is suggested that fibers produced by this hybrid have a more densely packed and have a well-aligned crystalline network. This enhanced β-sheet formation provides the structural reinforcement and load-bearing capacity that underpin the observed improvements in tensile strength and stiffness. This structural organization may also help to explain why FC₁ × FC₂ silk produced the longest continuous filaments during reeling as the fiber’s crystalline domains and β-sheet alignment allow it to withstand higher tensile forces without fracture. However, this increased crystallinity and β-sheet dominance appear to come at the cost of extensibility. FC₁ × FC₂ showed the lowest elongation at break among the hybrids, consistent with the reduced proportion of random coil structures (23.29 ± 0.75%) relative to the other groups. This trade-off mirrors the established relationship in silk mechanics, where greater β-sheet content enhances stiffness and tensile strength but limits molecular mobility and extensibility [5,32,33]. Recently, Gull and Kumar [34] conducted a study of three silkworm breeds (CSR₄, CSR₆ and CSR₂₇) in which CSR₂₇ shows better results in terms of both economical and mechanical properties which also corroborates with this study as CSR₂₇ is one of the parents of double hybrid (FC₁ × FC₂). Although the decrease in elongation at break was not statistically significant, it indicates a trend toward a more rigid, crystalline network. Further, the structure–property relationship was drawn to various characters in which the β-sheet content was found highly linked with the crystalline % of the fiber. This parameter also show positive correlation with tensile strength and Young’s modulus of fiber. The crystallite size was found negatively correlated with β-sheet content, tensile strength, Young’s modulus as well as crystalline percentage of the silk fiber. This indicates that the lower the crystallite size, the higher the strength of the fiber will be. Our results are also corroborated by various previous studies which confirms that high β-sheet content and more crystalline phase gives the maximum strength and toughness of silk fiber [35,36,37].

5. Conclusions

Silk fibers of three silkworm hybrids (CSR₂ × CSR₄, PM × CSR₂, and FC₁ × FC₂) were investigated using a combination of characterization SEM, X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). The biological and economic parameters of these hybrids were also examined. Based on the findings of the present investigation, it can be envisaged that remarkable variability exists among the hybrids studied with respect to biological and economic characteristics of silkworm hybrids and silk fibers characteristics with a few exceptions. While the cross breed PM × CSR₂ completes its life cycle faster, this occurs at the cost of smaller larvae and lower silk yield. In contrast, FC₁ × FC₂ larvae exhibit slower growth but reach larger sizes and produce significantly higher silk quantities, offering greater commercial value despite the extended rearing duration. In addition, the quality of silk produced by FC₁ × FC₂ is superior, showing significantly greater filament length and strength. Furthermore, the information obtained regarding the mechanical and structural properties of these hybrids can be utilized in breeding programs to develop new silkworm breeds capable of producing higher-quality silk fibers. Such advances may also support the development of silk products for diverse applications, including in the biomedical and cosmetic fields. Therefore, it is necessary to focus on the utilization of different silkworm strains and to conduct further studies on this topic.