A Study on Assessments of the Variants of Mulberry Silkworm (Bombyx Mori L.)Breeds ‘Nistari’ using Biochemical & Molecular Markers_Open Access Journal of Biogeneric Science and Research
Abstract
Nistari, is an indigenous poly voltine breed of mulberry silkworm (B.mori L.). Though Nistari is not a highly productive, but gene(s) imparting to thermo-tolerance and resistant many pathogens are conserved in this poly voltine silkworm breed. Among poly voltine silkworm breeds, Nistari is the oldest breed that is being reared in India. Sericulture in eastern India is highly dependent on ‘Nistari’ as the breed can survive extreme climatic variations and disease-resistant properties. In fact, in the high temperature and humidity conditions of West Bengal, the Nistari breed is used as one of the components of hybrids (Multi x Multi and Multi x Bi) commercially reared during five seasons February, April, June, September, and November. On the basis of acclimatization in different regions of eastern India, different variants (=strains) of Nistari are available in West Bengal. Depending upon the locality/area of availability, such variants are known as Nistari-Batepur, Nistari-Chalsa, Nistari-Shivnivas, and Nistari-Debra. Moreover, some other strains like Nistari-Plain, Nistari-Marked, Nistari-White and Nistari-Sex-limited variants are quite frequent in different areas of West Bengal. These variant names are on the basis of larval and cocoon characteristics. All these mentioned Nistari strains exhibit phenotypic variations like cocoon weight, shell weight and markings on larval surface. With this background following study is proposed to study variation among strains of Nistari at biochemical (using enzymatic and non-enzymatic cellular components, molecular level (using microsatellite markers)) and repairing of DNA damage ability and along with the differences in yield traits of Nistari variants available in Institute’s repository.
Keywords: Sericulture; Nistari; DNA; Molecular markers; Coccon
Abbreviations: ROS - Reactive Oxygen Species; CAT - Catalase Activity; SOD - Superoxide Dismutase Specific Activity; TSS - Total Soluble Sugar Content; LPO - Lipid Peroxidation as a Function of Malonaldehyde Oxidation; APOX - Ascorbate Peroxidase Activity; H2O2 - Hydrogen Peroxide Titre
Introduction
Sericulture of eastern region is highly dependent on Nistari as it is the hardy polyvoltine breed which can survive extreme climatic conditions of this region. Nistari is used for producing cocoon on commercial scale in West Bengal and other states of eastern India. On the basis of acclimatization in different region of eastern India it has been called by area names such as Nistari-Batepur, Nistari-Chalsa, Nistari-Shivnivas and Nistari-Debra. Moreover there are other strains too named as Nistari-Plain, Nistari-Marked, Nistari-White and Nistari-Sex-limited [1]. All strains of Nistari reportedly conserved genes imparting thermo tolerance and disease resistant though the breed is not productive 2. Some strains of Nistari show better fecundity, larval and cocoon characters 3.Sericulture in Eastern India demands region and season specific breeds due to its fluctuating climatic conditions 4.Nistari plain reportedly having better survival and cocoon yield; while Nistari marked has high shell weight and shell ratio.
Chalsa and Debra have better performance over plain and marked strains of Nistari. Nistari Debra+P which is derived from the crossing of Nistari Plain and marked, has enhanced fecundity and yield parameters 3. Studies suggest that phenotypic variability among Nistari strains is due to environmental variability rather than genetic variability. But inadequate information is available on the reason of variation among Nistari strains found in Eastern India. To study the genetic variability among various available strains of Nistari molecular markers can be used. Reactive oxygen species (ROS) are produced by cellular metabolism and by exogenous agents in the cells. ROS include a number of chemical reactive molecules derived from oxygen, such as super-oxide anions, singlet oxygen, hydrogen-peroxide and hydroxyl radicals. Over production of ROS can cause oxidative damage of various bio-molecules, especially lipids and proteins. The lipid per oxidation induced cellular leakage due to free radicals is one of the major reasons of cell death .The DNA is also the major target of direct and/or indirect action of generated ROSs. The harmful effect of the free radicals can however, be blocked by antioxidant substances. Like many insects, during biotic and a biotic challenges imbalance between oxidant generation and antioxidant defese occurs in silkworm. Reactive oxygen species (ROS) and associated anti-oxidative enzyme system plays an important role in biotic /abiotic stress resistance in various insects. Various stress inducted ROS usually reacts with proteins, lipids and DNA, causing oxidative damage and impairing the normal functions of cells 4.Many researchers correlated some of the reactive oxygen species (ROS) associated enzymes like superoxide dismutase (ROS), ascorbate per oxidase (APOX), catalyse (CAT) and non-enzymatic component like H2O2 generations and lipid per oxidation. Besides, stress induced DNA damage is also considered as a major event during a biotic stresses. Though some strains of Nistari showed varying degree of biotic and stress tolerances, but information is lacking on the comparative analysis of these major anti oxidative groups among the available strains of Nistari. The relationship with radical scavenging potentials and oxidative stress induced DNA damage repair abilities of the available Nistari resources are also not known.
Objectives of Study
a. Assessment of variability of available ‘Nistari’ variants using ROS stabilizing enzymatic and non-enzymatic components of mulberry silkworm
b. Evaluation of stress induced DNA damage repairing ability of ‘Nistari’ variants
c. Assessment of genetic variability of ‘Nistari’ variants using Microsatellite markers.
Materials and Methods
Six strains (=variants) of poly voltine silkworm (Bombyxmori L.) breed Nistari were selected for the study. These six [Nistari strains- plain, marked, chalsa, Debra, white and Sex-limited Y (YSL)] strains were reared under recommended temperature and humidity. While, for different biochemical assessments, haemolymph of fifth instar (4th day) larvae was collected by pricking the 1st abdominal proleg of each larva using 1.5mL of sterile microfuge tube contacting 5-10mg of phenylthiourea. Subsequently, haemocytes were separated from haemolymph by centrifugation at 5000rpm for 10min at 4 °C and haemolymph plasma was stored at -35 °C for further use.
Results and Discussion
1.1. Assessment of Variability of Available ‘Nistari’ Variants Using ROS Associated Non-Enzymatic and Enzymatic and Components
Six strains of Nistari namely Plain, Marked, Chalsa, Debra+P, White and Y-Sex-limited are maintained in institute’s repository which were reared twice during January-February 2018 and April-May 2018 following standardized protocol. Haemolymph was collected from 5th instar larvae and store at -35 °C in 0.5mL aliquots for estimation / assessment of different parameters.
Nistari variants (=strain) originated from B.mori L. varied significantly for the haemolymph contents of H2O2, lipid peroxidation (LPO) and total soluble protein (TSS) as well as the activities of ascorbate peroxidase (APOX), catalase (CAT) and superoxide dismutase (SOD; Table 1). Variations of antioxidative enzyme activities in the tested strains of Nistari were generally much higher than the anti-oxidative non-enzymatic components and TSS. Among the enzymes, variation of APOX activity was maximum (5.4 fold), followed by CAT (3.5 fold); while SOD was the least variable (1.6 fold).Among the tested non-enzymatic components, variation of LPO was maximum (3.3fold).
Nistari, is an indigenous poly voltine breed of mulberry silkworm (B.mori L.). Though Nistari is not a highly productive, but gene(s) imparting to thermo-tolerance and resistant many pathogens are conserved in this poly voltine silkworm breed. Among poly voltine silkworm breeds, Nistari is the oldest breed that is being reared in India. Sericulture in eastern India is highly dependent on ‘Nistari’ as the breed can survive extreme climatic variations and disease-resistant properties. In fact, in the high temperature and humidity conditions of West Bengal, the Nistari breed is used as one of the components of hybrids (Multi x Multi and Multi x Bi) commercially reared during five seasons February, April, June, September, and November. On the basis of acclimatization in different regions of eastern India, different variants (=strains) of Nistari are available in West Bengal. Depending upon the locality/area of availability, such variants are known as Nistari-Batepur, Nistari-Chalsa, Nistari-Shivnivas, and Nistari-Debra. Moreover, some other strains like Nistari-Plain, Nistari-Marked, Nistari-White and Nistari-Sex-limited variants are quite frequent in different areas of West Bengal. These variant names are on the basis of larval and cocoon characteristics. All these mentioned Nistari strains exhibit phenotypic variations like cocoon weight, shell weight and markings on larval surface. With this background following study is proposed to study variation among strains of Nistari at biochemical (using enzymatic and non-enzymatic cellular components, molecular level (using microsatellite markers)) and repairing of DNA damage ability and along with the differences in yield traits of Nistari variants available in Institute’s repository.
Keywords: Sericulture; Nistari; DNA; Molecular markers; Coccon
Abbreviations: ROS - Reactive Oxygen Species; CAT - Catalase Activity; SOD - Superoxide Dismutase Specific Activity; TSS - Total Soluble Sugar Content; LPO - Lipid Peroxidation as a Function of Malonaldehyde Oxidation; APOX - Ascorbate Peroxidase Activity; H2O2 - Hydrogen Peroxide Titre
Introduction
Sericulture of eastern region is highly dependent on Nistari as it is the hardy polyvoltine breed which can survive extreme climatic conditions of this region. Nistari is used for producing cocoon on commercial scale in West Bengal and other states of eastern India. On the basis of acclimatization in different region of eastern India it has been called by area names such as Nistari-Batepur, Nistari-Chalsa, Nistari-Shivnivas and Nistari-Debra. Moreover there are other strains too named as Nistari-Plain, Nistari-Marked, Nistari-White and Nistari-Sex-limited [1]. All strains of Nistari reportedly conserved genes imparting thermo tolerance and disease resistant though the breed is not productive 2. Some strains of Nistari show better fecundity, larval and cocoon characters 3.Sericulture in Eastern India demands region and season specific breeds due to its fluctuating climatic conditions 4.Nistari plain reportedly having better survival and cocoon yield; while Nistari marked has high shell weight and shell ratio.
Chalsa and Debra have better performance over plain and marked strains of Nistari. Nistari Debra+P which is derived from the crossing of Nistari Plain and marked, has enhanced fecundity and yield parameters 3. Studies suggest that phenotypic variability among Nistari strains is due to environmental variability rather than genetic variability. But inadequate information is available on the reason of variation among Nistari strains found in Eastern India. To study the genetic variability among various available strains of Nistari molecular markers can be used. Reactive oxygen species (ROS) are produced by cellular metabolism and by exogenous agents in the cells. ROS include a number of chemical reactive molecules derived from oxygen, such as super-oxide anions, singlet oxygen, hydrogen-peroxide and hydroxyl radicals. Over production of ROS can cause oxidative damage of various bio-molecules, especially lipids and proteins. The lipid per oxidation induced cellular leakage due to free radicals is one of the major reasons of cell death .The DNA is also the major target of direct and/or indirect action of generated ROSs. The harmful effect of the free radicals can however, be blocked by antioxidant substances. Like many insects, during biotic and a biotic challenges imbalance between oxidant generation and antioxidant defese occurs in silkworm. Reactive oxygen species (ROS) and associated anti-oxidative enzyme system plays an important role in biotic /abiotic stress resistance in various insects. Various stress inducted ROS usually reacts with proteins, lipids and DNA, causing oxidative damage and impairing the normal functions of cells 4.Many researchers correlated some of the reactive oxygen species (ROS) associated enzymes like superoxide dismutase (ROS), ascorbate per oxidase (APOX), catalyse (CAT) and non-enzymatic component like H2O2 generations and lipid per oxidation. Besides, stress induced DNA damage is also considered as a major event during a biotic stresses. Though some strains of Nistari showed varying degree of biotic and stress tolerances, but information is lacking on the comparative analysis of these major anti oxidative groups among the available strains of Nistari. The relationship with radical scavenging potentials and oxidative stress induced DNA damage repair abilities of the available Nistari resources are also not known.
Objectives of Study
a. Assessment of variability of available ‘Nistari’ variants using ROS stabilizing enzymatic and non-enzymatic components of mulberry silkworm
b. Evaluation of stress induced DNA damage repairing ability of ‘Nistari’ variants
c. Assessment of genetic variability of ‘Nistari’ variants using Microsatellite markers.
Materials and Methods
Six strains (=variants) of poly voltine silkworm (Bombyxmori L.) breed Nistari were selected for the study. These six [Nistari strains- plain, marked, chalsa, Debra, white and Sex-limited Y (YSL)] strains were reared under recommended temperature and humidity. While, for different biochemical assessments, haemolymph of fifth instar (4th day) larvae was collected by pricking the 1st abdominal proleg of each larva using 1.5mL of sterile microfuge tube contacting 5-10mg of phenylthiourea. Subsequently, haemocytes were separated from haemolymph by centrifugation at 5000rpm for 10min at 4 °C and haemolymph plasma was stored at -35 °C for further use.
Results and Discussion
1.1. Assessment of Variability of Available ‘Nistari’ Variants Using ROS Associated Non-Enzymatic and Enzymatic and Components
Six strains of Nistari namely Plain, Marked, Chalsa, Debra+P, White and Y-Sex-limited are maintained in institute’s repository which were reared twice during January-February 2018 and April-May 2018 following standardized protocol. Haemolymph was collected from 5th instar larvae and store at -35 °C in 0.5mL aliquots for estimation / assessment of different parameters.
Nistari variants (=strain) originated from B.mori L. varied significantly for the haemolymph contents of H2O2, lipid peroxidation (LPO) and total soluble protein (TSS) as well as the activities of ascorbate peroxidase (APOX), catalase (CAT) and superoxide dismutase (SOD; Table 1). Variations of antioxidative enzyme activities in the tested strains of Nistari were generally much higher than the anti-oxidative non-enzymatic components and TSS. Among the enzymes, variation of APOX activity was maximum (5.4 fold), followed by CAT (3.5 fold); while SOD was the least variable (1.6 fold).Among the tested non-enzymatic components, variation of LPO was maximum (3.3fold).
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