On the Breeding of Bivoltine Breeds of the Silkworm , Bombyxmori L . ( Lepidoptera : Bombycidae ) , Tolerant to High Temperature and High Humidity Conditions of the Tropics

The hot climatic conditions of tropics prevailing particularly in summer are contributing to the poor performance of the bivoltine breeds and the most important aspect is that many quantitative characters such as viability and cocoon traits decline sharply when temperature is high. Hence, in a tropical country like India, it is very essential to develop bivoltine breeds/hybrids which can withstand the high temperature stress conditions. This has resulted in the development of CSR18 × CSR19, compatible hybrid for rearing throughout the year by utilizing Japanese thermotolerant hybrids as breeding resource material. Though, the introduction of CSR18 × CSR19 in the field during summer months had considerable impact, the productivity level and returns realized do not match that of other productive CSR hybrids. Therefore, the acceptance level of this hybrid with the farmers was not up to the expected level. This has necessitated the development of a temperature tolerant hybrid with better productivity traits than CSR18 × CSR19. Though, it was a difficult task to break the negative correlation associated with survival and productivity traits, attempts on this line had resulted in the development of CSR46 × CSR47, a temperature tolerant bivoltine hybrid with better productivity traits than CSR18 × CSR19. However, though, these hybrids are tolerant to high temperature environments, they are not tolerant to many of the silkworm diseases. Keeping this in view, an attempt is made to develop silkworm hybrids tolerant to high temperature environments.


Introduction
Silkworm breeding aims to achieve superior performances in respect of egg yield, cocoon raw silk yield, cocoon stability, and production followed by expansion to new areas besides others.Silkworm breeders continue to strive for an inherent gain in resistance by incorporating resistant genes into the genetic backgrounds of high yielding temperate bivoltines.Besides this, the cocoon crop stability also relies more on improving other production technologies which have to be explored.It is interesting to note that in inbreeding experiments, besides choice of parents, selection and inbreeding the hybrids are very important which have to be carefully executed since both inbreeding and hybridization are forms of nonrandom mating or selective mating, but operate in opposite ways.Inbreeding is a kind of genetic assortative mating as compared with phenotypic assortative mating in hybridization.The major effect of inbreeding which is most apparent in the reduction of mean performance of the population is in question.While gene frequencies do not change on the whole, genotypic frequencies do change towards the production of more homozygotes and fewer hetrozygotes.Thus, any change in the population mean as a result of inbreeding must be related to difference in genotype value between homozygote and heterozygote [1].
India enjoys the patronage of second position for the production of silk in the world next only to China.Sericulture in India is practiced predominantly in tropical environmental regions such as Karnataka, Tamil Nadu, Andhra Pradesh and West Bengal and to a limited extent in temperate environment of Jammu and Kashmir.The existing tropical situation provides scope for exploiting multivoltine Psyche × bivoltine hybrid at commercial venture as they are hardy and have tremendous ability to survive and reproduce under varied or fluctuating environmental climatic conditions.But its quality is at low ebb when compared to the existing international standard.
Considering these drawbacks, adoption of bivoltine sericulture became imperative and imminent considering its potentiality even under Indian tropical conditions.Keeping this in view, breeding experiments were initiated at Central Sericultural Research and Training Institute, Mysore to evolve hardy bivoltine silkworm races suited to tropical conditions for achieving the primary objective of establishing bivoltine hybrids as a concept among sericulturists.Accordingly, many productive and qualitatively superior bivoltine hybrids have been developed by utilizing Japanese commercial hybrids as breeding resource material [2].However, the hot climatic conditions prevailing particularly in summer are not conducive to rear these high yielding bivoltine hybrids throughout the year.It is as well-established fact that under tropical condition, unlike polyvoltines, bivoltines are more vulnerable to various stresses, that is, hot climatic conditions of tropics, poor leaf quality, and improper management during summer which are not conducive for bivoltine rearing.In order to select efficiently the breeds with high temperature tolerance, it is important to analyse the impact of high temperature on many silk yielding attributes of silkworm races and their heritability.
The success of sericulture industry depends upon several factors of which the impact of the environmental factors such as biotic and abiotic factors is of vital importance.Among the abiotic factors, temperature plays a major role on growth and productivity of silkworm, as it is a poikilothermic (cold blooded) insect [3].It is also known that the late age silkworms prefer relatively lower temperature than young age and fluctuation of temperature during different stages of larval development was found to be more favourable for growth and development of larvae than constant temperature.There are ample literature stating that good quality cocoons are produced within a temperature range of 22-27 • C and above these levels makes the cocoon quality poorer [4].However, polyvoltine races reared in tropical countries are known to tolerate slightly higher temperature [5], which is also true with crossbreeds, that have been evolved specially for tropical climate.
The continued efforts for the improvement of cocoon characters of domesticated silkworm were aimed at increased quality silk production.The main objective of silkworm rearing is to produce qualitatively and quantitatively superior cocoons, which in turn will have a direct bearing on the raw silk production.Therefore, it becomes imperative or essential to develop bivoltine breeds/hybrids which can with stand high temperature stress conditions.Sericulture, the viable agro-based industry aptly matches the socioeconomic backdrop of rural India.One of the main aims of the breeders is to recommend silkworm breeds/hybrids to farmers that are stable under different environmental conditions and minimize the risk of falling below a certain yield level.Silkworm breeds that are reared over a series of environment exhibiting less variation are considered stable.The climatic conditions prevailing in the tropics are most unpredictable and the problems of tropical sericulture are occurrence of aggravated silkworm diseases, unsuitable mulberry leaf for bivoltine silkworms, and lack of sustainable silkworm breeds for effective selection of desirable characters.In order to introduce bivoltine races in a tropical country like India, it is necessary to have stability in cocoon crop under high temperature environment.The prerequisite of summer hybrid is healthiness and adaptability to adverse conditions of high temperature, low food quality, relatively higher economic traits, with potential for increased cocoon production.
Considering the poor performance of productive bivoltine hybrids during summer season, emphasis was given to evolve bivoltine silkworm breeds suitable to tropical conditions for achieving the primary objective of establishing bivoltine sericulture with quality raw silk among sericulturists.Thus a compatible robust bivoltine hybrid, CSR18 × CSR19 was evolved from a Japanese hybrid, B201 × BCS12 under high temperature (36 ± 1 • C) and high humidity (85 ± 5%) conditions [6,7] by taking clues from earlier experiments conducted by Japanese experts [8][9][10].Though, this hybrid was authorized by Central Silk Board for commercialization, large-scale testing in the field is yet to take momentum due to its low productivity.Therefore, attempts are being made to develop bivoltine hybrids tolerant to high temperature conditions.

Materials and Methods
To initiate the breeding programme of high temperature (40 ± 1 • C) and high humidity (85 ± 5%), six bivoltine breeds three each of oval, namely, CSR18, CSR46, and CSR50 and dumbbell, namely, CSR19, CSR47, and CSR51 were found to be temperature tolerant and selected as parental breeds for breeding programme.In oval lines, CSR46 and CSR50 are characterized by plain larvae while CSR18 is characterized by marked and plain (sex limited) larvae where female is marked and male is plain, similarly in dumbbell lines, CSR19 is characterized by sex limited (marked and plain) larvae and CSR47 and CSR51 are characterized by marked larvae.
For the development of breeds through appropriate techniques breeding programme was initiated with an objective to introduce the bivoltine breeds/hybrids for high temperature, that is, 40±1 • C and high relative humidity, that is, 85 ± 5%.By utilising the breeding resource material three dumbbell lines, namely, HH8, HH10, HH12, tolerant to high temperature and high humidity conditions were developed, the parentage of which are depicted in Table 1.
Silkworm rearing was conducted following the standard method under the recommended temperature and relative humidity till 2nd day of 5th instar.During the process of breeding composite layings were prepared by utilized fifteen to twenty disease-free layings to ensure large population size with wide genetic base from F1 to F5, and progenies were raised by conducted mass rearing.Ten replicates of 100 larvae were counted after passing out 3rd moult and were kept in plastic trays and subjected for two different temperature treatments, that is, 40 ± 1 • C and 50 ± 5% RH and 40 ± 1 • C and 85±5% RH in SERICATRON.(Environmental chamber with precise and automatic control facilities for uniform maintenance of temperature and humidity) from 3rd day of 5th instar and were fed fresh mulberry leaves twice a day.From F1 to F5 continuously thermal exposure was given, the larvae selected for two different high temperature treatments were exposed six hours per day (10 AM to 4 PM) till spinning which is appropriate time for exposure to high temperature [7][8][9].As continuous exposure under high temperature conditions reduces quantitative traits drastically, recurrent backcrossing/outcrossing was given with one of the productive parental breeds as outlined in each breeding plan.From the base population of F1, larvae were also counted (300 larvae) and inbreeding was done for each breed and reared at room temperature up to F12, these room temperature reared batches were considered as control batches.
Cellular rearing was resorted to from F6 onwards to F12 with minimum five replications preceded by half sib/full sib mating for three different temperatures.At the time of brushing, the rich egg layings showing good hatching % were selected from each set of room temperature and two different high temperatures and reared.Owing the thermal effect in successive generations, it was observed that after 5th generation both qualitative and quantative characters have declined sharply.So the experiment was modified in such a way that with every alternate generation from F6 onwards to F12 both high temperature lines were brought to room temperature conditions and reared continuously till spinning to recoup the lost vitality under stress conditions.The breeding plans of the three dumbbell lines are depicted in Figures 1 to 3. RH the survival percentage ranged from 90.9 to 94.1% with the highest of 94.1% at F1 and the lowest of 90.9% recorded at F6.At 40 ± 1 • C and 85 ± 5% RH, the highest cocoon yield/10000 larvae for HH8 was observed in F9 (15.79 kg) and the least (14.37 kg) at F2. Similarly, at 25 ± 1 • C and 65 ± 5% RH, the highest for HH8 was observed in F6 (18.58 kg) and the least (17.23 kg) at F2.The highest cocoon weight (1.607 g) for HH8 was recorded at 40 ± 1 • C and 85 ± 5% RH in F7 and the lowest (1.562 g) at F7. Similarly, at 25±1 • C and 65±5% RH, the highest for HH3 was observed in F5 (1.904 g) and the lowest (1.727 g) in F3.The highest cocoon shell weight at 40 ± 1 • C and 85 ± 5% RH for HH8 was observed in F9 (0.342 g) and the lowest (0.316 g) in F2.Similarly, at 25 ± 1 • C and 65 ± 5% RH, the highest for HH8 was observed in F5 (0.440 g) and the lowest (0.379 g) in F3.The highest cocoon shell percentage at 40±1 • C and 85±5% RH for HH8 was observed in F9 (21.42%) and the lowest (20.26%) in F2.Similarly, at 25 ± 1 • C and 65 ± 5% RH, the highest for HH8 was observed in F2 (22.78%) and the lowest (22.19) in F1.Analysis of variance with regard to pupation rate recorded highly significant difference (P > .001)while Yield/10000 larvae recorded significant difference (P > .01)and fecundity recorded significant difference (P > .05) at 40 ± 1 • C and 85 ± 5% RH between generations.Similarly, at 25 ± 1 • C and 65 ± 5% RH, cocoon weight, shell weight recorded highly significant difference (P > .01)and Yield/10000 larvae recorded significant difference (P > .05)(Table 2).

Discussion
The breeding of silkworm since long has been aimed towards evolving of superior and hardy breeds either by means of selection alone or by combining outcrossing or backcrossing with selection in the subsequent generations.The final aim of the breeder is primarily to evolve a breed which can give rise to stabilized crops and secondly to improve both quantity and quality of silk [11].The breeding of silkworm races probably dates back to the beginning of the history of silkworm rearing, but it has made great progress rather recently [12].Sericulturally advanced countries like Japan has achieved remarkable progress by executing systematic breeding plans for the development of productive races.In silkworms, studies carried out for various characters have shown that the characters could be changed to suit the breeders choice, since selection for one trait has correlation with genetic change of other characters.The correlation for few traits is negative and for some it is positive [13][14][15][16].Therefore, during the course of breeding of new breeds, the breeder has to be aware of the response of certain characters in selection and its correlated changes with other economic traits.Inbreeding of hybrids to stabilize silkworm breeds which bred true is well documented [12,[16][17][18][19][20][21][22][23][24][25][26].Similarly, Kovolov [27] is of the opinion that improvement of silkworm races is possible by outbreeding with exotic races and improvement of cocoon quality by repeated backcrossing [28].
According to Allard and Bradshaw [29], performance of the strain itself in a given environment indicates its superiority.During evaluation, emphasis was given on the phenotypic expression of traits of economic importance under different temperature conditions.However, as the objective of the study was for greater viability and high productivity merits, equal importance was given on these two traits during selection of parents.The significant variations observed in the phenotypic manifestation for the traits analyzed can be attributed to the genetic constitution of the breeds and their degree of expression to which they are exposed during their rearing.Such variations in the manifestation of phenotypic traits of the breeds studied can be ascribed to the influence of environmental conditions.Variable gene frequencies at different loci make them to respond differently.The results are in line with the findings of [29][30][31][32][33][34][35][36][37][38][39].
In the present breeding programme, which envisages evolution of new hardy bivoltines, the aim was to develop more resistant bivoltines that can give rise to stable cocoon crops with better viability, even though productivity is low compared to the existing productive bivoltine breeds that are currently used in the field.In silkworms, the correlation for some characters is positive and for some is negative [15,16].Such a negative correlation is observed for the traits productivity and viability and hence the attempt made was to increase the viability of the developed breeds.Moreover as suggested by [40,41], the selection parameters were primarily aimed at improving the viability character such as yielded by number without sacrificing much of the productivity traits like cocoon weight, cocoon shell weight, and yield by weight.In addition, during later generations of inbreeding, selection was applied to select desired genotypes to improve the traits of commercial importance like viability and productivity as suggested by [42,43] to improve the yield of bivoltines.
The imposition of exposure to high temperature levels in 5th instar and the resultant low pupation rate could be attributed to the low feeding activity of the silkworm resulting in the physiological imbalance and poor health of the larvae and an increased number of nonspinning worms in the mountages.The work in [44] demonstrated that silkworms are more sensitive to high temperature during Psyche 4th and 5th instars.The productive bivoltine breeds are reported to be susceptible to high temperature; the authors of [8,45] noticed higher survival in the hybrids than the pure races under high temperature conditions.In the present investigation, when lines are exposed to high temperature continuously there is a drastic reduction in the pupation rate and cocoon traits.The work in [46] observed that at high temperature (35 • C) and low humidity (50 ± 5%) and high humidity (85±5%) conditions, pupation rate was drastically reduced in productive hybrid, CSR2 × CSR5.Such drastic change is usually obtained as it is low heritable trait in the silkworm and is prone to large variations in environment and management [47].The work in [48] observed that the pupation rate in Indian popular bivoltine breed, NB4D2, is significantly influenced by both low and high humidity.
Silkworm breed which are reared over a series of environments exhibiting less variation are considered stable.One of the objectives of the breeder is to recommend stable breeds to the farmers for rearing under different environmental conditions.Effect of high temperature and low humidity in terms of cocoon crop depends on several factors that operate within and outside the body of the silkworm.In the present study, it was observed that apart from the temperature, humidity also influences the productivity pattern in the silkworm and is in agreement with [49,50].It was also observed that the cocoon Yield/10000 larvae, cocoon weight, cocoon shell weight, and cocoon shell percentage were also low in the high temperature treated batches when compared to the batches reared under optimum rearing conditions.The work in [51] reported the deleterious effect of high temperature and high humidity on quantitative traits of parents, foundation crosses, and single and double hybrids of bivoltine silkworm breeds of Bombyx mori L.

Table 2 :
Generation wise mean performance for rearing of HH8 at two temperature conditions.

Table 3 :
Generation wise mean performance for reeling of HH8 at two different temperatures.Denote significant difference at 5%, 1% and 0.1% level, respectively.ns: Denote non significant.Values in parenthesis are angular transformed.

Table 4 :
Generation wise mean performances for rearing of HH10 at two temperature conditions.

Table 5 :
Generation wise mean performance for reeling of HH10 at two different temperatures.Denote significant difference at 5%, 1% and 0.1% level, respectively.ns: Denote non significant.Values in parenthesis are angular transformed.

Table 7 :
Generation wise mean performance for reeling of HH12 at two different temperatures.Denote significant difference at 5%, 1% and 0.1% level, respectively.ns: Denote non significant.Values in parenthesis are angular transformed.