Tropical Tasar Silkworm Antheraea Mylitta Research Articles

Molecular markers for genetic analysis of Indian tropical tasar ecoraces and hybrid populations (Antheraea mylitta.d)

The tropical tasar silkworm Antheraea mylitta D., a wild sericigenous and polyphagous insect is cultivated in the dense, humid, tropical forests of eastern, central and southern India. Its primary food plants are Terminalia arjuna, Terminalia tomentosa and Shorea robusta. The improved varieties of these silkworms can be evolved by hybridization through backcross method, to improve the traits in the upcoming generation by selective parental selection. The present studies of genetic relations between Andhra local and Daba TV ecoraces is identified by DNA markers viz., simple sequence repeats (SSRs), most of which have produced polymorphic bands and based on phylogeny of tasar ecoraces using SSR markers, which further provides molecular evidence of the fact that climatic factors, the changes at DNA level and its wide range of distribution in varied geographic conditions would lead to genetic divergence ultimately leading to the formation of new ecorace.. KEYWORDS :Antheraea mylitta, tasar ecorace, microsatellites, phylogeny, genetic diversity, Breeding, Backcross

Cytochrome b gene as a potential DNA barcoding marker in ecoraces of tropical Tasar silkworm Antheraea mylitta Drury

BackgroundTropical Tasar silkworm Antheraea mylitta Drury plays a very crucial role in the economic development of the country employing around 9.76 million people in rural and semi-urban areas. Since the population of this species has spread over many states with varying environmental factors, the population has been isolated into many pockets, specifically adapted to that niche, and are called as “ecoraces”. Even though phenotypically these ecoraces may look similar to some level but the intra-generic genotypic variability between them is correlated to the economically important yield traits of their cocoons. The purpose of our study was to evaluate the effectiveness of Cytochrome b (Cyt b) gene of mitochondrial genome as a potential DNA barcoding marker for identification of these ecoraces as to are they really ecoraces, and attempts to establish the first country report of DNA barcode reference library of this silkworm species. Methods and resultsDNA was extracted from pupae of pure ecoraces collected from core ecozones. PCR amplicon was bead purified and further subjected to Sanger Sequencing. The parsimony tree was also created using the MEGA 11 software. The genetic distance, gene flow and relation among the ecoraces was evaluated. The conserved regions of Cyt b gene were identified along with the documentation of the mode and tempo of nucleotide variation and evolution. ConclusionThe present study confirms the efficiency of Cyt b gene as a potential DNA marker for identification of ecoraces of Tasar silkworm and is the first country report on barcoding of A. mylitta Drury. Apart from being used as an early-warning system for ecological degradation, this knowledge will help in assessing the evolutionary relationship of these economically important ecoraces and identify their position in the process of evolution aiding in the assessment of conservation priorities.

Temporal variability in foliar protein content and trypsin inhibitory levels in two host trees of tropical Tasar silkworm Antheraea mylitta, Drury

Aim: Variation in total soluble proteins and protease inhibitor levels were investigated to determine nutritional and plant defense status in different leaf types of Terminalia arjuna (Roxb.) Wight and Arn., and Schleichera oleosa (Lou.) Oken, two host tree species of the economically important, non-mulberry Tasar silkworm, A. mylitta. Methodology: Quantitative spectrophotometric assays based on diagnostic amidolytic substrates were conducted to assess serine protease inhibitor activities in young, semi-mature and mature leaf types. A microplate quantification assay for total protein estimations was used with leaf types sampled over a year. Efficacy of total trypsin inhibitor and chymotrypsin inhibitor activities detected in T. arjuna (a primary host plant) was further evaluated on gut extracts of fourth instar A. mylitta and Pieris brassicae L. (a Pierid pest of crucifers) to assess the physiological adaptation of larvae to dietary antifeedants. Molecular provenances with rbcL genes were obtained that are available as NCBI accessions #MN460810 for T. arjuna and #MT010554 for S. oleosa. Results: Intra-specific variations were evident in leaf phenology of two tree species. Generally, young leaf type of both tree species had high levels of total protein and trypsin inhibitory activities, while mature leaf type of T. arjuna had low total protein content and trypsin inhibitor levels. Mature leaf type of S. oleosa had low trypsin inhibitor levels during the months of July and August. Midgut proteases of A. mylitta and P. brassicae were significantly more susceptible (p≤0.05) to both trypsin and chymotrypsin inhibitors detected in young and semi-mature leaf types of T. arjuna than the mature leaf type. Interpretation: In this study, differential inhibition of digestive proteases in A. mylitta and P. brassicae by protease inhibitors from different leaf types of T. arjuna, suggested adaptation to dietary antifeedants. Such reports on nutritional quality, foliar antifeedants, phenology and host plant utilization are relevant for strategies to domesticate the tropical Tasar silkworm, A. mylitta. Key words: Antheraea mylitta, Protease Inhibitors, Terminalia arjuna, Total protein, Tree phenology

Geospatial mapping of ecoraces of tasar silkworm (Antheraea mylitta Drury) using remote sensing and geographic information system techniques

Tropical Tasar silkworm Antheraea mylitta Drury (Family: Saturniidae) produces Tasar silk which has very high demand globally and because of their wide range of distribution in tropical regions, they have adapted to different niches forming different ecoraces. In Odisha, alone seven types of ecoraces of A. mylitta have been reported, namely Modal, Sukinda, Nalia, Jata-Daba, Adaba, Umerkote, and Boudh. These ecoraces show differences in their phenotypes, genetic constitution, fecundity, voltinism, etc. In the past few decades, there is the substantial deterioration of habitats due to many anthropogenic activities. This has resulted in a large decline in wild Tasar cocoon production. Lepidopterans are accepted as ecological indicators of ecosystem health and it is apprehended that through anthropogenic and climatic factors this precious genetic resource, which took millions of years to evolve, may face the threat of extinction shortly if adequate conservation measures are not taken. This paper attempts the geospatial mapping of these ecoraces in the state of Odisha through RS and GIS tools, which will provide a greater scenario for their conservation

DETERMINATION OF TEMPERATURE SENSITIVE DIAPAUSE TERMINATION STATE OF DABA TRIVOLTINE ECORACE OF ANTHERAEA MYLITTA DRURY

The Daba trivoltine ecorace of tropical tasar silkworm Antheraea mylitta Drury is one ofthe most commercially exploited non-mulberry silkworms in tropical India. It is generally grown inthe latitude range of 20°N to 25°N in Eastern India, especially, in parts of West Bengal, Jharkhand,Orissa, Chhattisgarh and Andhra Pradesh. The pupae of this ecorace remain in facultative pupaldiapause state for five to six months (January-June). During this long period of diapause losses occurdue to un-seasonal and unsynchronised emergence of male and female moths which is estimated tothe tune of 25-30% leading to reduction of seed production in grainages. The losses become moreprevalent when dry conditions prevail soon after pre-monsoonal showers. This can be avoided byconsigning the pupae of diapause termination state to low temperature. In the present study, thespecific age of diapause termination state has been worked out on the basis of the presence ofhaemolymph biochemical constituents like glycerol, trehalose, glycogen, quantitative total proteinsand free amino acids and protein profile comparing them with non-diapausing pupae harvestedduring first and second crop. Among diapausing pupae, the trehalose concentration always remainedat its low level during diapause and an increase in concentration was observed at the fag end ofdiapause when pupae attained the age of 145 to 150 days. Contrary to this, the level of glycerol andglycogen was always higher through out the diapause period and a down surge in the concentrationwas noticed when pupae were 145 to 150 days old. The level of protein was higher in nondiapausinggeneration. However, the level of protein and amino acids showed a fluctuating trendthrough out diapause development. The haemolymph protein profile of the diapausing pupae showedthe presence of a diapause specific protein band of 16kD which remained in its full intensity till thepupae attained the age of 145-150 days, thereafter, its intensity went down and protein profile ofdiapausing pupae looked similar to non-diapausing pupae. Therefore, it is confirmed that the diapausetermination state in Daba pupae occurs when pupae become 145-150 days old. The diapausingpupae of this age can be further exploited by working out low temperature treatment schedule toavoid losses in grainages

Biochemical characterization of sericin isolated from cocoons of tropical tasar silkworm Antheraea mylitta raised on three different host plants for its prospective utilization

• Tasar food plants affect the chemical nature of sericin. • Higher C: N ratio and phenolic content was observed in Shorea robusta sericin. • Higher aromatic and hydrophobic amino acids were also detected in S. robusta sericin. • S. robusta sericin shows higher antioxidant potential and enzymatic inhibition. Sericin were isolated and characterized from tasar cocoons raised in three different food plants i.e. Terminalia arjuna, T. tomentosa and Shorea robusta for its applications. Their molecular composition, structure and physical nature were determined by elemental analysis, amino acid analysis, Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) analysis. These results show that tasar food plants influence the physical and chemical properties of sericin. Further, sericin isolated from cocoons of S. robusta food plant shows better antioxidant potential and inhibition of tyrosinase, elastase and glutathione-S-transferase activity than other food plants. This may be attributed to its amino acid variations and associated phenolic content. The present study appears to be useful in utilizing tasar sericin as a potential bio-molecule for its prospective utilization in pharmaceuticals and its associated fields.

Free radical scavenging potential of sericin obtained from various ecoraces of tasar cocoons and its cosmeceuticals implication

Tropical tasar silkworm Antheraea mylitta is a wild sericigenous insect which is distributed in different geographical regions and named as different ecoraces. In the present study, we investigated the molecular characterisation and cosmeceutical properties of sericin extracted from different ecoraces of tasar cocoons. The surface morphology and molecular weight of cocoons were determined by scanning electron microscope (SEM) and SDS-PAGE, respectively. Characterisation of sericin was performed by various methods such as FTIR, CHNS, TGA and amino acid analyzer. The anti-tyrosinase, anti-elastase, glutathione-S-transferase inhibition, free radical scavenging potential and inhibition of oxidative damages were measured in tasar ecoraces sericin. SEM images have revealed the removal of sericin from the surface of cocoons. SDS-PAGE of sericin depicted the presence of diverse molecular weight of proteins. Structural determination by FTIR revealed the presence of both α-helical and β-sheet structures. Thermal properties of sericin were studied by TGA which showed a 50% weight loss at temperature 410 °C–430 °C. Additionally, ecoraces sericin contains 17 amino acids in which serine, aspartic acid and glycine are predominantly present (55.68–59.61%). Further, anti-tyrosinase, anti-elastase, inhibition of glutathione-S-transferase activity, free radical scavenging potential and inhibition of lipid peroxidation were also observed in ecoraces sericin. Our findings suggest that the present study appear to be helpful in exploiting sericin as potential biomaterial in cosmeceutical and allied field.

Impact of High Temperature Gradients on the Physiolgical and Biomolecular Changes in the Tropical Tasar Silkworm Antheraea mylitta D

Impact of High Temperature Gradients on the Physiolgical and Biomolecular Changes in the Tropical Tasar Silkworm Antheraea mylitta D

Impact of High Temperature Gradients on the Physiolgical and Biomolecular Changes in the Tropical Tasar Silkworm Antheraea mylitta D

Impact of High Temperature Gradients on the Physiolgical and Biomolecular Changes in the Tropical Tasar Silkworm Antheraea mylitta D

Biochemical constituents in leaves of primary host of tasar silkworm Antheraea mylitta

The present study attempts to evaluate some important biochemical constituents of the three primary host plants, i.e., Terminalia tomentosa (asan), Terminalia arjuna (arjun) and Shorea robusta(sal) of the tropical tasar silkworm Antheraea mylitta Drury. The biochemical constituents, like protein, ascorbic acid, reduced glutathione (GSH) and level of lipid peroxidation were assayed. The results indicate that the concentrations of protein, ascorbic acid and reduced glutathione (GSH) are highest in sal followed by asan and are found to be the least in arjun. The level of lipid peroxidation was highest in asan and lowest in sal.

A first report of egg parasitism in the Tropical Tasar Silkworm Antheraea mylitta (Drury) occurring on cashew

The present study has documented egg parasitism in Tropical Tasar Silkworm for the first time. The parasitoid is Anastatus leithi (Walker) belonging to Eupelmidae family. It is a solitary koinobiont endoparasitoid, emerges out as adults from the tasar eggs in 17-20 days during August- September. The parasitism rate was recorded at 11.90 percentage.

Silk gland fibroin from indian muga silkworm Antheraea assama as potential biomaterial

There is an increasing demand for new versatile biomaterials. Silk is reported by many researchers as an ideal biomaterial for different biomedical applications. Most of the studies are carried out on mulberry silk Bombyx mori, however silk from Indian non-mulberry silkworms are relatively unexplored. In this report we fabricate 2D matrices from the regenerated aqueous silk fibroin protein of the glands of non-mulberry Indian muga silkworm Antheraea assama (assamensis). Its biochemical, biophysical characteristics and its cytocompatibility for biomedical uses are evaluated. The properties of this muga gland fibroin are compared with silk gland fibroin from non-mulberry Indian tropical tasar silkworm Antheraea mylitta and with the fibroin from the cocoon of mulberry silkworm Bombyx mori. The gland fibroin of Antheraea assama is observed to consist of two polypeptides of approximately 250 kDa each linked by disulfide bond. Fourier transformed infrared spectroscopy and X-ray diffraction studies indicate random coil structure of dissolved fibroin solution. The alpha-helical structure in 2D films changed to beta-sheets upon ethanol treatment, imparting crystallinity and insolubility. The fibroin film is found to be the least hydrophilic, followed by B. mori and A. mylitta silk. Biocompatibility of the films from all three species is investigated through the cell attachment and spreading study of MG-63 human osteoblast-like cells. The cytocompatibility of non-mulberry fibroin matrices are comparable with that of standard tissue culture plates. The results indicate that the non-mulberry Indian muga silk gland fibroin is also suitable matrix as a natural biomaterial for tissue engineering applications.

Biochemical Performance and Quantitative Assessment of F1 Hybrid of Two Ecoraces of Tropical Tasar Silkworm Antheraea Mylitta Drury (Lepidoptera: Saturniidae)

Antheraea mylitta Drury is basically a crossbreeding species, as such it seems to be potentially a good material for the exploitation of heterosis. In the present study F1 hybrid of wild ecorace Laria (L) and semi-domestic Daba (D) was raised and evaluated for various quantitative traits and biochemical parameters during larval stage. Improved fecundity (<TEX>$+18{\pm}1.8%$</TEX> and higher egg hatching rate (<TEX>$+10.96{\pm}1.3%$</TEX>) was recorded in the F1hybrid (<TEX>$L{\times}D$</TEX>). Biochemical parameters studied in the hemolymph, midgut and fatbody of the larva showed significantly higher (P<0.05) total proteins and carbohydrate concentration besides digestive enzyme activity. Correspondingly SDS-PAGE revealed more number of protein bands in the hemolymph sample of F1s, ranging between 29 kDa to 66 kDa compared to parental lines. The present study demonstrates the positive heterosis effect in the F1 hybrid of Laria <TEX>${\times}$</TEX> Daba. Biochemical analysis indicates that, there is possibilities of exploitation of hybrids with specific parents targeted for desirable commercial traits (silk yield and fecundity). Moreover, most of these biochemical parameters can be used as markers to analyze the genetic improvement in the tasar silkworms.

Effects of temperature on modulation of oxidative stress and antioxidant defenses in testes of tropical tasar silkworm Antheraea mylitta

High temperatures are known to cause physiological stress in organisms. This is often associated with enhanced generation of reactive oxygen species (ROS) leading to oxidative damage. The commercially important tropical tasar silkworm Antheraea mylitta has to endure high summer temperature before egg production on the onset of monsoon. In this study the status of pro-oxidants and antioxidants was studied in the testes of male pupae of tasar silkworm A. mylitta under thermal stress condition. Further, to find out the impact of temperature on physiological activity, oxygen consumption rate was measured. The result indicated higher level of thiobarbituric acid reactive substances (TBARS, as an index of lipid peroxidation) and total hydroperoxides in the male pupae exposed to high temperature (40±1°C). Similarly, it was found that increased levels of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), ascorbic acid (ASA) and low molecular thiols (L-SH) in testes are more prominent in high temperature rather than in moderate temperature (35±1oC) suggesting the activation of physiological mechanism to scavenge the ROS produced during stress. Further more, the order of higher level of oxygen consumption rate was observed as high temperature (40±1°C) > moderate temperature (35±1°C) > control groups (28±1°C). Oxygen consumption rate was positively correlated with oxidative stress and antioxidant defence indices. We infer from these findings that the testes of A. mylitta pupae modulate testicular antioxidant responses to thermal stress.

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F2 progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16905 bp showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F2 progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16826 bp). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk.

Studies on the High Temperature Induced Stress on the Biochemical Profile and Fecundity of Daba and Laria Ecoraces of Tropical Tasar Silkworm Antheraea mylitta Drury (Lepidoptera: Saturniidae)

Tropical tasar silkworm Antheraea mylitta Drury experiences extreme temperature stress conditions during its life cycle particularly during diapauses and first crop. The present study witnessed the impact of high temperature on some biochemical profiles and egg production (fecundity) of semi-domesticated Daba and Shorea robusta (Sal) based wild ecorace Laria during seed cocoon (pupa) preservation. Cocoons of Daba and Laria were treated with high temperature at <TEX>$40^{\circ}C$</TEX> for 10 days in a BOD incubator. The protein profile and carbohydrate content in the hemolymph and fat body and total haemocyte count (THC) in the hemolymph of pupa were investigated. Further, the fecundity and fertility of egg was assessed. Significant increase in the protein concentration was observed in the hemolymph with reduction in the fat body (p<0.05). The difference in protein concentration was highly significant between the semidomesticated Daba and wild ecorace Laria (p<0.05). High pupal mortality (20%) and reduced fecundity (10-15%) in Daba was noticed compared to wild Laria. Also an increased THC (>28000) was recorded in Laria. The study infers the potentials of wild ecoraces in sustaining the extreme temperature conditions and need of adopting suitable package of practices for the preservation of diapause seed cocoons during extreme summer conditions. There is possibility to introgression thermal stress resistant traits in the semi-domesticated races of tasar silkworm by resorting to conventional breeding plans with wild races and keeping the thermal stress induced response as markers.

Study of Phenotypic Variability in Silk Gland Characters in Three Ecoraces of Tropical Tasar Silkworm Antheraea mylitta Drury

Study of Phenotypic Variability in Silk Gland Characters in Three Ecoraces of Tropical Tasar Silkworm Antheraea mylitta Drury

Diapause Specific Expressed Sequence Tags of Antheraea mylitta Drury

Daba bivoltine ecorace of tropical tasar silkworm Antheraea mylitta Drury undergoes facultative pupal diapause and shows different type of voltinism. During course of its long pupal diapause, erratic, unseasonal and unsynchronised emergence of adults is noticed and losses of seed stock range in between 10 - 30%. In order to avoid all these problems, proper understanding of induction, maintenance and termination state of diapause of this economically important insect is felt essential. The presence of diapause specific expressed sequence tags (ESTs) through PCR clones of Hsp70, Hsp23, hexamerins and PCNA genes have been reported in the present study. The ESTs obtained form the primers of Hexamerins were only seen when pupae were 65 and 165 days old. ESTSs obtained form the primers designed form Hsps 70 sequences were up regulated during early (D0), middle (D75) and late age (D135 to D165) of diapause period. The presence of Hsp23 was obtained during preparatory phase of diapause (IV instar) and pupae of early and mid aged diapause period (D0 to D75) and late age of diapause (D135 to D165). ESTs of Hsp22 were seen during preparatory phase of diapause (IV&V instar), throughout diapause period and even after diapause period was over. ESTs of Hsps90 were seen during preparatory stage (IV instar) and middle and late age of diapause period. Est of PCNA were down regulated throughout diapause period, their up regulation was seen at the time of diapause termination. Another group of ESTs obtained from different sets of Hsps 70 primers were up regulated intermittently through out the diapause period. Hsps90 were upregulated during middle and late age of true diapause period. At the fag end of true diapause period, ESTs disappeared when pupae became older than 165 days as no ESTs were seen when pupae were 195 days old indicating the actual age of diapause termination. It was also evidenced by the up regulation of PCNA.ESTs whose concentration remained very low through out the diapause period but its intensity increased at 195 days which further increased at 210 days. The pupae of Daba BV of 195 days and older can be further exploited for low temperature treatment to delay the moth emergence in adverse summer season so as to produce dfls matching with the actual cropping schedule.

Non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices for potential biotechnological applications

This study reports a novel biopolymeric matrix fabricated by chemically cross-linking poly (vinyl alcohol) with silk sericin protein obtained from cocoons of the tropical tasar silkworm Antheraea mylitta. Glutaraldehyde was used as a cross-linking agent with hydrochloric acid acting as an initiator. The matrices were biophysically characterized and the cytocompatibility of the matrices was evaluated for their suitability as biomaterials. The surface morphology was assessed using atomic force microscopy while the changes taking place after cross-linking were confirmed by Fourier transform infrared spectroscopy. The enhanced thermal stability of the constructs was assessed by thermogravimetric and differential scanning calorimetry. Fourier transform infrared spectroscopy analysis showed that sericin was chemically cross-linked with poly (vinyl alcohol) using glutaraldehyde. Silk sericin protein demonstrated a favorable effect on animal cell culture by successfully improving the adhering and spreading of cells on the poorly adhering surface of poly (vinyl alcohol). Confocal microscopy revealed cell spreading and actin filament development in sericin/poly (vinyl alcohol) hydrogel matrices. These findings prove the potential of non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices to be used as biocompatible and biopolymeric material for tissue-engineering and biotechnological applications.

Application of Parental Selection for Productivity Improvement in Tropical Tasar Silkworm Antheraea mylitta Drury-A Review

Application of Parental Selection for Productivity Improvement in Tropical Tasar Silkworm Antheraea mylitta Drury-A Review