Effects of Antibiotic Treated Mulberry Leaves Feeding on Larval Growth, Cocoon Production, and Gut Bacteria of Silkworm

Areeba Mashaal, Mubashar Hussain*, Aleena Naeem, Kiran Aftab,

Ghulam Dastgeer, Shumaila Irum, Saira Bashir, and Suleman Hussain Shah

Department of Zoology, University of Gujrat, Gujrat 50700, Pakistan

ABSTRACT

The effect of different concentrations of Levofloxacin and Cefixime on bacterial profile, larval growth, and cocoon production of silkworms was evaluated by rearing under controlled conditions of temperature, relative humidity, and photoperiod (25 ± 1 °C, 75 ± 5 % and 16:08 h of light to darkness ratio, respectively). The larvae were fed mulberry leaves treated with two antibiotics i.e., Levofloxacin and Cefixime each with three concentrations (10, 20, and 30 ppm) during 3rd, 4th and 5th larval instars. The experiment was laid out in the Completely Randomized Design (CRD) and replicated thrice. The significant variations in larval length (cm) and larval body weight in 3rd, 4th and 5th instar larvae were recorded (p< 0.05). The antibiotic supplementation also affected cocoon weight and cocoon shell weight positively. Maximum larval body length and body weight were observed in both antibiotics at 20 ppm concentrations. Four surface bacterial strains (Bacillus cereus, Bacillus sp., Bacillus pumilus and Actinobacterium sp.) and three gut bacterial strains (Bacillus licheniformis, Actinobacterium sp., and Bacillus pumilus) associated with silkworm were isolated. These strains were identified based on 16S rRNA gene sequencing and were highly susceptible to Levofloxacin but showed resistance to Cefixime. The results demonstrated that both Levofloxacin and Cefixime enhanced silkworm growth, commercial traits and make them less susceptible to diseases when larvae were fed leaves treated with 20 ppm concentration.

Article Information

Received 01 December 2023

Revised 12 December 2024

Accepted 31 January 2024

Available online 03 May 2024

(early access)

Published 13 June 2025

Authors’ Contribution

AM and MH conceived the idea, designed, and organized project. MH supervised the work, analyzed data and revised the manuscript. AN, GD and SI helped in data analysis and write up. MH, KA, SB and SHS helped in data analysis and manuscript revisions.

Key words

Bacterial isolation, Levofloxacin, Susceptibility, Bombyx mori, Sericulture

DOI: https://dx.doi.org/10.17582/journal.pjz/20231201085224

* Corresponding author: [email protected]

0030-9923/2025/0004-1759 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The sericulture industry has benefitted mankind for centuries by providing natural animal silk (Holland et al., 2019; Karthik et al., 2015) of great quality like lightweight, natural sheen, an inherent affinity for dyes, drape, resilience, high absorbance, and vibrant color (Altman et al., 2003; Reddy, 2009; Karthik et al., 2015; Babu, 2018). This industry has great importance as an allied sector of agriculture (Neelaboina et al., 2018) and contributes to the rural economy in addition to its associated role in the ecosystem (Zhou et al., 2003; Neelaboina et al., 2018; Manjunath et al., 2020).

Continuous domestication of silkworm results in inbreeding depression that drastically lowers immunity and adds in the susceptibility to diseases (Adel et al., 2015). Silkworm disease prevalence results from several biological (nutritional, biological) or environmental factors (temperature and humidity) (Sakthivel et al., 2012). Commercial and biological features silkworm are prone to natural degradation (Adel et al., 2015). Continuous improvement in rearing technology and disease management is requisite for successful crop harvesting on sustainable basis. However, these two parameters have been remained an issued for making sericulture as an emerging industry despite its global importance for its silk production (Shah et al., 2007; Wurm, 2003; Hakimi et al., 2007).

During last decade, bacterial flacherie has become a major threat to silkworm larvae during rearing (Stockland, 2017; Gani et al., 2017; Chairman et al., 2012; Saad et al., 2019; Gore et al., 2014), contributing about 70% loss to sericulture annually (Manimegalai, 2003). Bacterial flacherie incur an estimated loss of 30-40% to cocoon crops (Anantha, 2011). In another study, the most prevalent silkworm disease was the flacherie (47.9 %) followed by grasserie (42.6 %), and then kenchu (9.5 %) (Manimegalai, 2003).

Sericulturists suggest management of bacterial flacherie is key to successful silk crop production (Yuan et al., 2006; Gani et al., 2017). Bacterial disease management in silkworms has been achieved by exposing larvae to different antibiotics with encouraging results (Hamamoto et al., 2004a; Javaid et al., 2021; Rafiq et al., 2021a). Antibiotics are used in the sericulture industry as an element of bed sterilizers and therapeutic impact in bacterial diseases management (Venkatesh and Srivastava, 2010; Srivastava and Kumar, 2009; Mahmoud and Taha, 2012; Subramanian et al., 2010). Such antibiotics include penicillin, ampicillin, tetracycline, erythromycin, and chloramphenicol that showed promising results in bacterial disease management of silkworm larvae Subramanian et al., 2010; Venkatesh and Srivastava, 2010; Ashok and Ramakrishna, 2014; Prakash and Puttaraju, 2006; Kaito et al., 2011; Kaito and Sekimizu, 2007; Meng et al., 2017). Silkworm being domesticated has specific bacterial microbiota associated with their gut which has not been evaluated for the impact of antibiotics in Pakistan. This study highlighted the effect of antibiotic-treated mulberry leaves on bacteria associated with silkworm larval gut. Additionally, the larval growth, and cocoon production was also assessed.

MATERIALS AND METHODS

Stock culture preparation

The disease-free silkworm eggs were obtained from the Sericulture Research Laboratory, Lahore. All equipment, rearing room and food were cleaned before shifting the larvae, the rearing room was disinfected with a 4% formalin solution and was kept airtight for 24 h (Ito, 1969; Krishnaswami, 1978). The eggs were incubated in hatching plates at 25 ± 1 °C temperature and 75 ± 5% relative humidity (Hussain et al., 2011). Temperature and humidity were monitored by thermo- hygrometer (HM 16) to monitor.

Larval rearing

Larval rearing was carried out at temperature of 25 ± 1 °C, 75 ± 5% relative humidity (RH), and photoperiod of 12 h of light:12 h of dark (Rahmathulla, 2012). Three different concentrations (10, 20, 30 ppm) of two analytical grade antibiotics were sprayed on fresh mulberry leaves which were then chopped and fed to the larvae. The feeding schedule for larval rearing comprised of 3 times a day during 1st two instars and 04 times a day during 3rd, 4th and 5th instar (Hussain et al., 2011). Antibiotics used in this experiment were Cefixime (C16H15N5O7S2) and Levofloxacin (C18H20FN3O4).

Experimental design

In the present work, the effect of three different concentrations (10, 20, 30 ppm) of Levafloxacian and Cefixime treated mulberry leaves were studied on larval growth, cocoon production, and gut bacteria associated with silkworms. The research work was carried out in Complete Randomized Design (CRD) and replicated thrice. At the beginning of the 3rd instar, the larvae were divided into 21 groups, each replicate containing 30 larvae. For bacterial isolation, 4th and 5th instar silkworm larvae were randomly sampled from each replicate and were stored in disinfected airtight sealed vials at -18oC in the refrigerator.

Data on larval growth and cocoons

At the end of 3rd, 4th and 5th instar, larval weight (g) and larval length (cm) were measured by using electronic balance, and measuring tape, respectively. Larval weight (g) was measured by taking mean values of randomly selected five larvae from each replication of all treatment groups. Larval length (cm) was taken as mean of randomly selected five larvae (Zulfiqar et al., 2022).

Cocoon weight (g) was recorded on 7th day of spinning and shell weight (g) was calculated by removing pupae from the same cocoons which were used to calculate cocoon weight and shell weight by following formulae:

Isolation of bacteria

Bacteria associated with silkworm surface and gut were isolated as follows:

To obtain bacterial samples from the surface of silkworm larvae, the refrigerated larvae were sterilized by dipping in 2% sodium hypochlorite (5% of reagent grade) for three to five minutes, then washed with autoclaved distilled water (Ayoade et al., 2014; Adel et al., 2015). The suspension was then serially diluted up to six times (Adel et al., 2015). Then 0.1 mL suspension was inoculated with the help of a micropipette onto a nutrient agar plate and incubated for 24 h at 37°C (Ayoade et al., 2014). Distinct colonies (based on color and shape) were then isolated and streaked for further analysis.

For the isolation of silkworm gut bacteria, dissection of previously surface-sterilized silkworm larvae was carried out. Surface sterilized larvae were placed on a dissection tray which were sterilized with 95% ethanol (Ayoade et al., 2014). The larvae were fixed with fine pins in a dorsoventral position on a dissection board. An incision was made carefully with a pair of sterilized blades and scissors through the dorsal midline from posterior to anterior end and the digestive tract was extracted. The extracted gut was then ground and deposited in 200 mL saline solution in a sterilized Eppendorf tube (Ayoade et al., 2014). The suspension was then serially diluted up to 6 times. Then 0.1 mL suspension was inoculated with the help of a micropipette onto a nutrient agar plate and incubated for 24 h at 37°C (Ayoade et al., 2014). Distinct colonies (based on color and shape) were then isolated and streaked for further analysis.

Antimicrobial susceptibility

Antibiotic sensitivity was measured by using analytical grade antibiotic susceptibility discs. Nutrient agar plates were prepared and 50μl of nutrient broth containing bacterial isolates was spread on them separately. Two different antibiotic disks levofloxacin and cefixime were placed with the help of sterilized forceps on the surface of inoculated plates. Plates were then incubated for 24 h at 37°C (Bauer et al., 1966). The appearance of the transparent area around the antibiotic disk was taken as an indicator of sensitivity while the growth of colonies around the discs showed the resistance of isolates. Diameter of zone of bacterial growth inhibition was measured in millimeters (mm) with the help of a scale (Kabir et al., 2013; Aditi et al., 2017).

PCR amplification of 16S rRNA sequence

The genomic DNA of bacterial strains was extracted using a Dream TaqTM Green PCR Master Mix which was stored at -18°C until used. Universal primers were employed for the molecular characterization of bacterial strains. The forward primer was 16F (5’-GAGTTTGATCCTGGCTCAG-3’) and the reverse primer was 1510R (5’ –GGCTACCTTGTTACGA- 3’) (Satokari et al., 2001; Leitch et al., 2007; Carroll et al., 1999). PCR amplification was performed in a total volume of 25μl containing 12.5μl of Master Mix, 1μl of Reverse Primer, 1μl of forwarding Primer, 5.5μl of dd H2O, and 5μl of DNA template. PCR was performed in a thermo-cycler with successfully optimized PCR conditions. The thermal cycle comprised of incubation at 95 oC for 5 min followed by 35 cycles, each of denaturation at 95 oC for 45 sec, annealing at 50 oC for 30 sec and extension at 72 oC for 1 min. Final extension was done at 72 oC for 5 min.

The amplified products (~1,500 bp) were subjected to 2% agarose gel electrophoresis and sequenced directly in the Center of Excellence In Molecular Biology (CEMB), Lahore. The similarity of the sequences was compared using the NCBI BLAST database (http://www.ncbi.nlm.nih.gov/Blast.cgi). CHROMAS 2 was used for the editing of FASTA files while CLUSTALW was used for sequence alignment. Mega X software (Hall, 2005) was used to prepare phylogenetic trees for each strain. Phylogenetic trees were constructed using the Neighbor-joining tree method based on the Tamura-Nei model (Tamura and Nei, 1993). Furthermore, antibiotic sensitivity was also measured by using analytical-grade antibiotic susceptibility discs.

Data collection and analysis

To determine significance, the data were subjected to ANOVA. While the treatment means were compared by Tukey’s post hoc test.

RESULTS

Larval length (cm)

ANOVA results of larval length during the rearing of larvae from 3rd to 5th instar at the end of each instar showed significant differences (p < 0.05). Maximum larval length was recorded in T(Lev-20ppm) (3.37cm) during 3rd instar whereas lowest larval length was observed in T(control) (2.53cm) (Fig. 1A). The larval lengths recorded at the end of the 4th instar showed significant differences between means obtained from larvae reared under different treatment groups (p < 0.05). Mean maximum larval length was found T5(Lev-20ppm) (3.9cm) whereas minimum length in T0(control) (3.47cm) (Fig. 1B). Body length in 5th instar larvae demonstrated significant differences in means between treatment groups. Mean larval length was observed maximum in T5(Lev-20ppm) (7.08cm) and lowest in T0(control) (6.37cm) (Fig. 1C).

Body length (cm) Body weight (g)

 

Larval weight (g)

Mean larval weight recorded at the end of 3rd, 4th, and 5th instar larvae showed significant differences between treatment groups (p < 0.05). The larval weight recorded at the end of 3rd instar showed maximum larval weight (0.239g) in T(Ce-30ppm) whereas minimum larval weight (0.194g) was observed in T(control) (Fig. 1A).

The larval weight recorded at the end of 4th instar showed significant differences in mean larval weight between treatments (p < 0.05). Maximum larval weight (0.865g) was observed in T5(Ce-20ppm), and minimum larval weight (0.715g) was recorded in T0(control) (Fig. 1B). The larval weight (g) recorded at the end of 5th instar showed significant differences in means (p < 0.05). Maximum larval wight (3.90g) was recorded in T5(Lev-20ppm) followed by T2(Ce-20ppm) (3.710g). While minimum larval weight (3.47g) was recorded in T0(control) (Fig. 1C).

Cocoon weight (g)

Cocoon weight (g) showed significant difference in mean values among different treatment groups (p < 0.05). Maximum weight was observed in T5(Ce-20ppm) followed by T2(Lev-20ppm) (Fig. 2A).

Shell weight (g)

Shell weight (g) recorded at the end of spinning after 5th instar demonstrated significant variations in means in different treatment groups (p < 0.05) (Fig. 2B).

 

Isolation of bacteria

A total of seven bacterial strains were isolated from silkworm out of which four were from the body surface, and three were isolated from the gut of 4th and 5th instar silkworm larvae. These isolates were classified based on color, colony shape, and morphological properties (Table I).

 

Table I. Morphological characterization of bacterial isolates from B. mori (in the column two under the title strains, the codes represent: The first letter shows that whether the strain was isolated from the gut (G) or surface (S), while the next two characters symbolize the isolate’s color and shape).

S. No.	Strains	Colony, morphology and color
1.	SYR	Surface yellow round
2.	SGA	Surface green amorphous
3.	SYCR	Surface yellowish creamy round
4.	SWAE	Surface white amorphous elevated
5.	GRW	Gut round white
6.	GPW	Gut pure white
7.	GWSP	Gut white spreading pure

 

Table II. Isolation of bacterial strains from silkworm in different antibiotic concentrations of Levofloxacin and cefixime. (note: “+” indicate presence of bacterial strains and “-”.

Bacterial strains	Cefixime (Ce)			Levofloxacin (Lev)			Control
	10 ppm	20 ppm	30 ppm	10 ppm	20 ppm	30 ppm
SYR	+	−	+	-	-	-	+
SGA	-	-	-	+	-	-	+
SYCR	-	-	-	+	+	-	+
SWAE	+	-	-	-	-	-	+
GRW	-	-	-	-	+	-	+
GPW	-	-	+	-	-	-	+
GWSP	-	+	-	-	-	-	+

 

Moreover, each antibiotic concentration has a different effect on bacterial strains (Table II). For this reason, the presence of each bacterial strain in different antibiotic concentrations was also recorded to measure the effect of each concentration on bacteria (Table II).

Antibiotic susceptibility measurement

Antibiotic sensitivity was measured by using analytical-grade antibiotic susceptibility discs. The diameter of the zone of bacterial growth inhibition was measured in millimeters (mm) with the help of a scale. Antibiotic susceptibility measurement demonstrated that bacterial strains were resistant to cefixime but sensitive to levofloxacin. The multiple antibiotic resistance (MAR) index value was greater than 0.2 that indicated a high-risk source of contamination (Table III).

 

Table III. Antibiotics zone diameter (mm) and MAR index.

S. No.	Strains	Cefixime Ce (5µg)	Levofloxacin Lev (5µg)	MAR index
1	SYR3	R	16.50	0.5
2	SGA11	R	13.75	0.5
3	SYCR12	R	15.00	0.5
4	SWAE15	R	15.25	0.5
5	GRW	R	16.00	0.5
6	GPW	R	17.50	0.5
7	GWSP	R	15.00	0.5

 

 

Phylogenetic analysis

The phylogenetic tree and 16S rRNA gene sequence analysis showed that GPW, GRW, GWSP, SYR, SWCR, SGA, and SYCR had sequence homology with Bacillus licheniformis (24%), Actinobacterium sp. (87%), Bacillus pumilus (81%), Bacillus cereus (100%), Bacillus sp. (99%), Bacillus pumilus (46%) and Actinobacterium sp. (76%), respectively (Figs. 3 and 4).

 

DISCUSSION

Impact of different antibiotics on the development and cocoon production of silkworm has been evaluated and established in some studies (Javaid et al., 2021; Rafiq et al., 2021b). Antibiotic administration significantly enhanced the biological and commercial traits of silkworm (Rahmathulla et al., 2003; Hamamoto et al., 2004b; Adel et al., 2015; Shyamala et al., 2013; Savithri, 2007; Rahmathulla and Nayak, 2017; Kushelaf et al., 2014; Mahdi et al., 2017b; Mahmoud and Taha, 2012). In our study, three concentrations of each levofloxacin and cefixime showed significant effects on larval growth, cocoon production, and bacteria associated with silkworms. Both antibiotics are of broad spectrum and are proven to work against a wide range of Gram-positive and Gram-negative bacteria (Anderson and Perry, 2008; Hurst et al., 2002; Brogden and Campoli-Richards, 1989). Antibiotics improved growth of silkworm with increased concentrations (Rafiq et al., 2021b). Comparatively higher mean values of larval length and larval weight in the antibiotic treatment is indicative of improved cocoon production (Rafiq et al., 2021b).

Similarly, improved cocoon weight and cocoon shell was recovered from antibiotic treated groups. While concentration within antibiotic treatment resulted in statistically significant differences. Such results were reported in earlier studies where antibiotic concentrations showed variations in the response of larval performance for cocoon production (Rafiq et al., 2022). Higher larval body length and body weight improvement may be attributed to higher efficiency of food ingestion, approximate digestibility and resistance to diseases (Kaito et al., 2002; Kushelaf et al., 2014; Savithri, 2007; Nayak, 2006; Shyamala et al., 2013; Mahdi et al., 2017; Mahmoud and Taha, 2012; Venkatesh and Srivastava, 2010; Kumar and Srivastava, 2020; Li et al., 2010). In both antibiotics, the concentration of varying degree of impact on larval growth and cocoon production. While the concentration of 20 ppm in both antibiotics has more pronounced impact than other two concentrations (10 ppm and 30 ppm) more effective and significantly enhanced larval length, larval weight, cocoon and shell weight, and shell percentage as compared to control. Such findings were reported in some studies highlighting the effect of antibiotics and their varying concentrations (Rasool et al., 2018; Saranya et al., 2019; Mahdi et al., 2017; Rahmathulla and Nayak, 2017).

Through phylogenetic analysis, isolated gut bacterial strains were identified as Bacillus licheniformis (GPW), Actinobacterium sp. (GRW) and Bacillus pumilus (GWSP) (Fig. 3). These results were supported by other studies reporting these species associated with silkworm (Adel et al., 2015; Chen et al., 2018, 2020; Dong et al., 2018; Liu et al., 2018; Yeruva et al., 2019). However, surface bacterial strains were identified as Bacillus cereus (SYR), Bacillus sp. (SWCR), Bacillus pumilus (SGA) and Actinobacterium sp. (SYCR) (Fig. 4). The results were supported in earlier studies in which these species were isolated and identified (Sakthivel et al., 2012; Dong et al., 2017, 2018; Liu et al., 2018; Chen et al., 2018, 2020; Adel et al., 2015; Li et al., 2015, 2019; Yeruva et al., 2019).

From all the isolated strains, Bacillus cereus is known as the highly threatening hemolytic strain (Li et al., 2015, 2019; Pandiarajan et al., 2011; Dong et al., 2017). While Bacillus pumilus, Actinobacterium sp., and Bacillus licheniformis are silkworm gut associated strains. They play a vital role in silkworm health as they enhance the development and growth of larvae and reduced larval mortality (Mala and Vijila, 2018; Yeruva et al., 2019; Bhuyan et al., 2018). Bacillus pumilus has antiviral activity against silkworm nucleopolyhedrovirus in vitro (Liu et al., 2018). Bacillus licheniformis produces protease inhibitors that provides resistance against entomopathogenic fungi (Beauveria bassiana) (Li et al., 2012). While Actinobacterium sp. provides resistance against the toxic effects of insecticides (Chen et al., 2020).

CONCLUSION

The impact of different levels (10, 20, and 30 ppm) of levofloxacin and Cefixime enhanced larval growth performance and cocoon production. The use of Levofloxacin and Cefixime (20 ppm) can be used to in sericulture to enhance larval growth and cocoon production of silkworm.

Acknowledgement

Authors are grateful to Mr. Rizwan Khursheed (Sericulture Research Laboratory, Lahore) for the provision disease-free silkworm eggs and technical assistance.

Funding

The study received no external funding.

Statement of conflict of interest

The authors have declared no conflict of interest.

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