Sericulturists from the tropical countries like India relay on climatic conditions prevailing in the field and restrict themselves to rear silkworm breeds suitable for high temperature conditions. This prompts the commercial exploitation of multivoltine x bivoltine hybrids in India, as they are hardy and ably to survive and reproduce under fluctuating climatic conditions (Kumari
Performance of each organism is determined and affected by genotype, environment and interactions between them. Therefore, individuals with high genetic potential must be selected in order to improve the quantity and quality of silk production, besides improving environmental conditions (Nezhad
Euclidean or straight-line measure of distance is the most commonly used statistics for estimating genetic distance between individuals by morphological data (Mohammadis and Prasanna, 2003). Numerous studies represent the hierarchical clustering of different silkworm breeds based on their performances with respect to quantitative traits. The economically important quantitative traits like fecundity, larval weight, yield/ 10000 larvae by weight, yield/ 10000 by number, shell weight, cocoon weight, shell %, filament length, filament size, neatness, etc., have been traditionally used for characterization / diversity studies (Thangavelu
Silkworm breeds and thermal treatment
Twenty silkworm breeds consisting of two multivoltine (Nistari and Cambodge) and eighteen bivoltine breeds (CSR2, CSR17, S5, D13, 5HT, 8HT, BHR2, BHR3, D6(P), SK3, SK4, D6(P)N, SK4C, B37, S38, NN6D, ATR16, ATR29) were used for the study. Multivoltine breeds were considered as reference to compare with the performance of bivoltine breeds. Silkworm rearing was conducted following the standard method under recommended temperature and humidity conditions till 2nd d of 5th instar as suggested by Krishnaswami
During silkworm rearing, data on six quantitative traits were collected separately for control and each treatment batch (32 ± 1℃, 34 ± 1℃ and 36 ± 1℃). The traits like pupation percentage (%), larval weight (g), cocoon yield for 10,000 larvae by weight (kg), cocoon weight (g), shell weight (g) and shell percent (%) were noted. The overall mean of each trait was calculated for each breed at all four temperatures. Pupation percentage was estimated as live pupa present inside the cocoon during metamorphosis of larva into pupa expressed as a percent. The percentage change in the performance of the breeds in treated over control with respect to their pupation percentage was calculated. This genetic trait was considered as the measure of index of thermotolerance in silkworm. Percentage change / reduction in the pupation percentage was calculated as follows (Kumari
Hierarchical clustering was done by using the rearing data of twenty silkworm breeds at four temperatures. Euclidean distance was calculated as a measure of genetic diversity by using the rearing parameters of the twenty silkworm breeds at 25 ± 1℃, 32 ± 1℃, 34 ± 1℃ and 36 ± 1℃. Based on Euclidean distance dendrogram was constructed using Ward’s method with a bootstrap value of 10000. Analysis of variance was performed on pupation percentage, larval weight, cocoon yield for 10,000 larvae by weight, cocoon weight, shell weight and shell percent of silkworm breeds using temperature as factor. All the analysis was done with SPSS software package.
Eighteen bivoltine and two multivoltine breeds were considered for the study. Variations in the larval marking, cocoon shape and cocoon colour were observed in experimental breeds. Among multivoltines, Nistari and Cambodge spun yellow coloured and oval elongate cocoons. Nistari larvae are marked and Cambodge larvae are plain. Among bivoltines, ATR16, B37, CSR2, CSR17, 8HT, S5, NN6D, S38 and SK3 larvae were plain and spun oval shaped cocoons. Other bivoltines
Overall performance of silkworm breeds under normal and high temperature treatments
Six important rearing traits
[Table 1.] Rearing performances of twenty silkworm breeds at 25 ± 1℃
Rearing performances of twenty silkworm breeds at 25 ± 1℃
[Table 2.] Rearing performances of twenty silkworm breeds at 32 ± 1℃
Rearing performances of twenty silkworm breeds at 32 ± 1℃
[Table 3.] Rearing performances of twenty silkworm breeds at 34 ± 1℃
Rearing performances of twenty silkworm breeds at 34 ± 1℃
[Table 4.] Rearing performances of twenty silkworm breeds at 36 ± 1℃
Rearing performances of twenty silkworm breeds at 36 ± 1℃
[Table 5.] Overall rearing performances of twenty silkworm breeds at four temperatures
Overall rearing performances of twenty silkworm breeds at four temperatures
[Table 6.] ANOVA on rearing traits with temperature as a factor
ANOVA on rearing traits with temperature as a factor
Percentage reduction in pupation percentage of treated groups over control
The negative sign in the percent changes over control in treated groups indicate a reduction over control and the value near to zero suggests a better performance of the breed. At all the treated temperatures, among multivoltines, the highest reduction in pupation percentage was in Cambodge against Nistari (Table 7). At 32 ± 1℃, 34 ± 1℃ and 36 ± 1℃ reduction in pupation percentage in Cambodge was 11.14%, 14.66% and 19.5% over control, respectively. Similarly, reduction in pupation percentage in Nistari was 9.08%, 10.13% and 15.04% at 32 ± 1℃, 34 ± 1℃ and 36 ± 1℃ over control, respectively. Among bivoltines, highest reduction of 65.15% in pupation percentage was demonstrated in CSR2 under 32 ± 1℃ and lowest reduction of 21.31% in SK4C. At 34 ± 1℃, B37 was highly affected by highest reduction in pupation percentage of 74% followed by CSR2 with 67.15%. At 36 ± 1℃, CSR2 had highest reduction in pupation percentage of 86.53% followed by B37 with 85.78%. BHR3 was more stable at 34 ± 1℃ and 36 ± 1℃ with lowest reduction in pupation percentage of 26.1% and 34.24%, respectively followed by SK4C with 26.7% and 35.76%, respectively (Table 7).
[Table 7.] Reduction in pupation percentage in silkworm breeds at high temperatures over control
Reduction in pupation percentage in silkworm breeds at high temperatures over control
Pairwise Euclidean distance was estimated between silkworm breeds based on their rearing traits under normal and treated temperatures. Using Euclidean distance as measure of diversity the dendrogram was constructed by Ward’s method. Twenty silkworm breeds were broadly divided into four clusters. Cluster I contained SK4C, BHR3, Nistari and Cambodge, which were thermo tolerant with pupation percentage of >70%. Cluster II was a large cluster with 8 silkworm breeds
The genetic distance ranged between 5.49 (between SK4C and BHR3) and 109.84 (between Nistari and CSR2) with an average of 36.74. In cluster I, four silkworm breeds were classified with an average genetic distance of 29.8 with 37.51 being highest between Nistari and SK4C and 5.49 being lowest between SK4C and BHR3. Similarly, in cluster II, eight silkworm breeds were divided with an average genetic distance of 17.63 with 30.92 being highest between CSR17 and NN6D and 9.78 being lowest between D6(P)N and NN6D. Silkworm breeds in cluster III were differentiated with an average genetic distance of 19.14 ranging from 10.58 between ATR16 and D13 to 28.21 between ATR16 and SK4. The two silkworm breeds, CSR2 and B37 formed cluster IV, which were distanced by a genetic distance of 31.38 (Supplementary material 1).
On increase in the rearing temperature above 25℃, all the breeds showed a decline in rearing parameters which is similar to that reported by Kumar
Abiotic and biotic factors have a considerable impact on the success of sericulture industry and thus they are of great importance (Kumar
It is a difficult task to assess thermotolerance trait in silkworms as it is governed by genetic and environmental factors (Kumar
One of the objectives of the silkworm breeder is to recommend stable breeds to the farmers for rearing under different environmental conditions. Silkworm breeds reared over a series of environments exhibiting less variation are considered stable. According to Allard and Bradshaw (1964), performance of the breed itself in a given environment indicates its superiority. As the majority of the economically important genetic traits of silkworms are qualitative in nature and phenotypic expression is greatly influenced by environmental factors such as temperature, relative humidity, light and nutrition (Pillai and Krishnaswami, 1980, Ramesha
The multivoltine breeds were more tolerant to high temperature than the bivoltines. It is well established that multivoltine breeds originated and reared in tropical countries are tolerate slightly higher temperature, as are cross breeds that have been evolved for a tropical climate (Ramesha
In this study, the highest overall means of larval weight and shell percent were noticed in CSR2, while, the highest overall mean of cocoon and shell weights were observed in CSR17. Highest cocoon yield per 10,000 larvae by weight was demonstrated in S5. The current study observed SK4C (73.34%) and BHR3 (73.05%) as two bivoltine breeds tolerant to high temperatures as they showed highest pupation percentage at all the temperatures tested. These breeds not only performed well under different high temperatures but also were stable with low percentage reduction in pupation rate over their controls indicating their ability to withstand high temperatures.
Classification of silkworm breeds based on six rearing parameters at high temperatures
In the current study, the genetic relationship between 20 silkworm breeds was determined by using rearing data of silkworm breeds at four different temperatures. Based on the data Euclidean distance was estimated and dendrogram was constructed using Euclidean distance and Ward’s method. The lowest genetic distance was 5.49 between SK4C and BHR3. These two breeds had the highest pupation percentage at each high temperature as well as overall temperatures tested. They were developed by screening each generation at high temperature conditions and selection criteria for further generation was based on heat-tolerant traits (Moorthy
Cluster analysis revealed that SK4C and BHR3 (>70%) breeds were grouped together with the multivoltines (>80%), which had highest pupation percentage at all the tested high temperatures including overall means. Similarly, CSR2 (42.02%) and B37 (43.53%) showed an overall mean of lowest pupation percentage and high percentage reduction in pupation rate over control. Thus, CSR2 and B37 form a cluster. The clustering pattern and genetic distance suggests that the breeds were primary grouped based on their ability of thermotolerance epresented by pupation percentage. However, in cluster II, the silkworm breeds had pupation percentage between 60-70% except for CSR17, which had a pupation percentage of 57.86%, typical for cluster III (i.e. 50-60%). This clearly indicates the role of rearing traits other than pupation percentage in classification of silkworm breeds because CSR17 though had a pupation percentage of cluster III members; it shared very nearer values for other rearing traits with the members of cluster II. Therefore, it was grouped in cluster II.
In this study, the breeds reared in Southern India (S38 and D13) clustered with the breeds reared in Northern India (SK4, D6(P), SK3 and ATR16) to form cluster III. According to Chatterjee and Datta (1992) for the silkworm, domestication has played a major role in genetic diversification. As sericultural regions of the world have different climatic conditions, physiological diversification has also been influenced by agro-climatic factors. Thus, given geographic isolation and limited cultural exchange, some breeds of South India and North India inclusive in cluster III may have acquired similar genetics due to similar selection pressures. The above results revealed that the inclusion of silkworm breeds of the same region in different clusters clearly indicate the presence of considerable genetic diversity among the population used in this study. All these cases indicate that clustering on the basis of estimates of phenotype does not always reflect geographical distance, as has also been pointed out by researchers working on the clustering of plant materials (Harlan, 1971). Likewise Spagnoletti and Qualset (1987) pointed out that geographical position does not correspond with the phenotypic grouping for the origin of spike characteristics in Duram wheat. Kumaresan
Current study reveals a negative relationship between the tested temperature and the rearing parameters of silkworm breeds. SK4C and BHR3 with high pupation percentage at three different high temperatures and less percentage reduction in pupation percentage over controls, were identified as high temperature tolerant and stable bivoltine silkworm breeds over a range of 32 to 36℃. So far in silkworm, phenotypic studies to identify high temperature tolerant bivoltine breeds were carried out with a single high temperature treatment. In the present report, the high temperature tolerant bivoltine breeds were identified based on their rearing performance at three treated temperatures, which a breeder can rely on. Therefore, these bivoltine breeds can be recommended as potential breeding material for the development of thermotolerant silkworm breeds/hybrids. Furthermore, this study also suggests that Euclidean distance estimated on the basis of rearing data of silkworm breeds under high temperature conditions can be an effective approach in classifying the silkworm breeds based on their capacity of thermotolerance. Based on the Euclidean distance 20 silkworm breeds were divided into 4 clusters, which represented one each of thermotolerant, thermo susceptible and two clusters of moderately tolerant silkworm breeds. The utilization of breeds from different clusters for breeding will increase heterosis. These results will enhance the knowledge of the silkworm breeders in developing thermotolerant breeds or hybrids with maximum heterosis.