INTRODUCTION
Sericulture is the group of cultural and economic activities related to silk. It can be defined as the combination of the cultivation of a perennial plant and the rearing of an insect (Cifuentes and Sohn 1998). This agribusiness has three fundamental components for its success: forestry, with the cultivation of mulberry, livestock, with silkworm rearing, and industrial, with the transformation of the thread in the textile, cosmetic and medical industries (Meng et al. 2017).
There are several insects that produce silk or natural fibers. Bombyx mori L. is one of the most important species used for silk production in the world. Its rearing dates back more than five millennia and China is identified as its center of origin (Neog et al. 2015). Some factors involved in the process of sericulture are cultivation and processing of mulberry, climatic and environmental conditions, as well as the cleanliness and hygiene of breeding areas.
Based on the above, the objective of this paper was to describe the main methodological aspects of Bombyx mori breeding.
2. CHARACTERIZATION OF BOMBYX MORI
B. mori is an insect of Bombycidae family, Lepidoptera order and Insecta class. It is a domesticated insect, fully adapted to commercial breeding, which arises after many years of evolution and artificial selection. This species is not present in nature in a free state, as it has lost the ability to fly and survive under extreme environmental conditions.
2.1. Lifecycle
B. mori is a monophagous species, with complete metamorphosis, eating only mulberry leaves during its larval stage. The quality of leaves (Ravikumar et al. 2019) and the use of different mulberry varieties during worm feeding influence on larval growth and development (Adolkar et al. 2007 and Gangwar 2010), as well as on the quality of the obtained cocoons (Prieto-Abreu 2015).
This insect, in its adult stage, has no other mission than to perpetuate the species. Females lay 400 to 500 round eggs, although they can also be oval, flat, or ellipsoid. Its size varies from 1 to 1.3 mm long and 0.9 to 2 mm wide, depending on the breed.
During embryonic development, eggs undergo color changes, from yellow to dark and gray. Eggs laid by unfertilized females do not change color. Their hatching depends on climatic variables, such as temperature, relative humidity (Reddy 2001) and light intensity (Kogure 1933). Their shell is formed by a chitinous matter, with microscopic channels through which air enters. In addition, it has a tiny pore, known as micropyle, which is the place through which the larva emerges. The embryo completes its development in a period of 10 to 12 days before hatching (figure 1).
Newly hatched larvae are 3 mm long and covered with tiny black or dark brown hairs that give them the appearance of a hairy caterpillar. During their growth they change color, due to the development of the skin cuticle. Larvae undergo dormancy and exuviation process four times, from hatching to cocooning. Those who are between the first and third age or instar, are called young worms. The first instar normally lasts three to four days, the second around two or three, the third from three to four, the fourth from four to five and the fifth from eight to nine days. In total, larval stage lasts from 21 to 25 days (Chauhan and Tayal 2017).
The worms in the fifth instar ingest more than 88% of mulberry, and it reaches its maximum weight one or two days before starting to spin the cocoon. In addition, they rapidly develop the silk gland, which occupies up to 40% of their weight. When they finish their development and stop eating, larval integument appears transparent. At this stage, they are called mature worms, and begin to form the cocoon for two or three days (Cifuentes and Sohn 1998).
At the end of cocooning, this worm becomes a pupa. Generally, the female chrysalis is larger than the male. Later, from 10 to 15 days, and depending on the variety, chrysalises transform into a moth.
2.2. Classification of B. mori breeds
There are numerous breeds and varieties of B. mori that are distinguished by secondary characters. These characters include color, shape and size of cocoons, silk yield and quality, as well as differences in coloration patterns in worm skin. There are different characterization criteria for B. mori breeds, which include geographical origin, voltinism and moltinism.
2.2.1. Characteristics of breeds according to geographical origin
Prolonged geographic isolation in regions with different climatic conditions has produced a wide variety of B. mori breeds. According to the geographical origin, the silkworm is classified into Japanese, Chinese, European and tropical breeds.
Japanese breeds have three larval markings: ocular, crescent, and stellar. Their larvae are generally strong and resistant under unfavorable conditions, but their larval period is long. They form cocoons with a peanut-shaped waist, usually white, although yellow and green are also present. It has a high tendency to produce double cocoons and short, thick threads.
Chinese breeds have an active diet, as well as rapid and uniform growth. Most of these breeds do not have larval markings, and are not sensitive to high temperature and humidity values. Their cocoons are elliptical or spherical, and their color is generally white, although golden yellow and pink are also present. The cocoon filament is thin and long, with good winding ability.
European breeds have large and heavy eggs, and worms with long bodies and light larval markings. Larval stage is very long, especially in the fifth instar, and they consume a large quantity of mulberry during this period. Cocoons are large and elliptical, with slight constrictions. They produce few double cocoons, most of them white in color, heavy shell and long filaments with good winding. They are difficult breeds to rear, due to their high sensitivity to unfavorable climate conditions and several diseases.
Tropical breeds originated in India and Southeast Asia. Their eggs are small and lightweight. Their larvae are tolerant to high temperatures and humidity. They have the smallest size among all breeds and produce small elliptical cocoons with green, yellow or white colors. The shell of cocoons is loose and light, with much lax mass of loose silk fiber, and thin filament. They produce few double cocoons, but the percentage of raw silk is very low (Cifuentes and Sohn 1998).
In Cuba, Chinese breeds and Thai poly-hybrids are the ones with the best performance. They have high hatching percentages and reduce the duration of larval stage, which makes it possible to carry out several rearing per year. They also allow better management of mulberry, by reducing intake days by larvae and the incidence of diseases, which generally increase in the last rearing ages (Pérez-Hernández 2017).
2.2.2. Characteristics of breeds according to voltinism
Voltinism is the genetic character that determines the number of generations in a year, under natural conditions. B. mori can be divided into three types of voltinism: univoltine, bivoltine, and polyvoltine. Univoltine silkworm breeds are capable of completing only one breeding generation under natural conditions in one year. Bivoltine silkworms are able of having two generations, and the polyvoltine ones can have three or more generations (Chauhan and Tayal 2017).
Voltinism depends on environmental factors, such as temperature, incubation photoperiod, and genes differentiated at different loci. It is closely related to geographical origin of breeds, since each region has its own environmental peculiarities. Generally, univoltine and bivoltine silkworms are dominant over polyvoltine. This characteristic is determined by the mother and segregation takes place in the third generation (Grekov et al. 2005).
Univoltine breeds are characterized by eating large amounts of mulberry, high productive yields with good quality silk, and by low resistance to stress and diseases. Polyvoltine breeds consume less mulberry, have low productive yields and are highly resistant. Bivoltine silkworms have intermediate characteristics to those mentioned above.
Diapause is an important mechanism in the lifecycle of various insects, since it allows the biological cycle to be synchronized with respect to seasons. This ensures food availability in the active stages of the animal. This phenomenon is hormonally regulated. During the embryonic stage, temperature and light affect the control of the secretory activity of the subpharyngeal ganglion. At high temperatures and long periods of light, secretion of the diapause hormone is promoted, while it is inhibited at low temperatures and short periods of light (Kobayashi 1990). If the ovaries that develop during chrysalis phase are affected by the diapause hormone, the female will produce diapausing eggs. Otherwise, it will lead to eggs without it. Diapause can be finished by acid treatment or egg hibernation (Figure 1) (Tzenov 2019).
2.2.3. Characteristics of breeds according to moltinism
Moltinism is the result of the interaction between various genetic constitutions and environmental conditions. This characteristic is related to the number of molts of larvae during their lifecycle. Depending on the number of molts, B. mori breeds can be classified as trimolters (M3), tetramolters (+M) and pentamolters (M5). Dominance relationships of M-alleles that control moltinism are M3> +M > M5. Some genes linked to sex are also related to the expression of this trait. The juvenile hormone of the winged body and the ecdysone of the prothoracic gland control the manifestation of these characters.
M3 breeds have a short larval stage and robust larvae. Larval body and cocoons are small and the cocoon filament is thin. Larval stage length, and larva and cocoon size of +M breeds are intermediate between M3 and M5. The M5 were induced by a natural mutation of the +M. They have a long-lasting larval phase, the size of larva and cocoon is large, with a very thick cocoon filament. These breeds are not very resistant to unfavorable conditions and are very susceptible to diseases (Cifuentes and Sohn 1998).
3. GENERAL REARING ASPECTS
3.1. Environmental conditions
Being a domesticated breed, B. mori is reared under captivity conditions, in facilities prepared for this activity. Growth and physiological indicators of this insect are affected by environmental conditions. The most important abiotic factors to control during worm rearing are temperature, relative humidity, ventilation, and lighting.
3.1.1. Temperature
Among the environmental factors that must be considered during larval rearing, temperature is probably the most important, as it is a poikilothermic animal, unable to regulate body temperature through internal mechanisms (Reddy 2001). Generally, early ages are more resistant to high temperatures, which increases survival and improves quality of produced cocoons. As temperatures rise, organic functions increase and, conversely, at low temperatures they decrease. The optimum temperature range for rearing larvae with high productivity levels varies between 23ºC and 28ºC (Parrey 2018 and Rahmathulla 2012). Table 1 shows the effects of some temperature values, other than the optimum, in B. mori breeding.
Temperature | Consequences | Authors |
---|---|---|
38 ºC | Decrease of cocoon formation time |
Rarnachandra |
36 ºC | Decrease of cocoon weight |
Kumar |
Decrease of larval stage duration |
Rahmathulla |
|
Decrease of cocoon quality and yield |
Chandrakanth |
|
Between 35 ºC and 36 ºC | Decrease of ingested and digested food |
Rahmathulla |
35 ºC | Decrease of weight of larvae and silk gland | Rahmathulla and Suresh (2013) |
Between 29 ºC and 30 ºC | Decrease of pupae formation rate |
Hussain |
Decrease of pupa stage duration | Tzenov (2019) | |
Decrease of pupae weight | ||
22 ºC | Increase of cocoon formation time |
Rarnachandra |
20 ºC | Increase of the amount of food digested by the silkworm Decrease of weight of cocoon and silk gland |
Rahmathulla |
Les tan 20 ºC | Decrease of physiological activities, specially at early ages | Rahmathulla (2012) |
18 ºC | Decrease of ingested and digested food | Rahmathulla and Suresh (2012) |
3.1.2. Relative Humidity
Temperature and humidity act synergistically on larval growth and development and on cocoon quality (Reddy et al. 2002). At early ages, larvae endure higher relative humidity values than adult worms, and vigorously grow under these conditions. Table 2 shows the effects of some humidity values on B. mori breeding, regardless of temperature.
Relative humidity | Consequences | Authors |
---|---|---|
98 % | Increase of cocoon formation time |
Rarnachandra |
95 % | Decrease of ingested food | Rahmathulla and Suresh (2012) |
55 % | Decrease of pupa formation rate Increase of larvae mortality |
Hussain |
Between 40 and 50 % | Decrease of ingested and digested food | Rahmathulla and Suresh (2012) |
40 % | Decrease of cocoon formation time |
Rarnachandra |
Decrease of cocoon and shell weight |
Rahmathulla |
3.1.3. Ventilation
Larvae have high oxygen requirements, especially when they are in their fourth and fifth age. Gases are produced during rearing, which modify air composition in the facilities. CO, NH3 and SO2 are some of the released gases in the facilities during rearing, due to the fermentation of animal and vegetable wastes, as well as the activity of humans (Rahmathulla 2012). Concentrations of 1% of CO2, 0.02% of SO2 and 0.1% of NH3 in the air are adequate limits for B. mori rearing (Singh et al. 2009). Young larvae are more susceptible to toxic gases, so artificial air circulation is sometimes useful (Grekov et al. 2005). Table 4 presents the effects of ventilation on B. mori rearing.
Stimulus | Treatment | Consequences | Authors |
---|---|---|---|
Variations of gas concentrations | [CO2]>1% | Delays larva growth |
Grekov |
[CO2]=10% | Produces vomits in larvae | ||
[CO2]>10% | Produces death of larvae | ||
[NH3]: between 0.5% and 1% | Produces death of larvae | ||
Air speed variations | Between 0.1 m/s and 0.5 m/s (in the fifth instar) | Decrease of larval mortality Increase of ingested and digested food Increase of cocoon weight and their formation speed | |
1 m/s (in the fifth instar) | Rahmathulla (2012) | ||
1.5 m/s | Cifuentes y Sohn (1998) |
3.1.4. Lighting
Larvae are photosensitive and tend to move towards dim light. They do not tolerate high light intensities or complete darkness. Young worms have positive phototropism and adults show negative phototropism. For this reason, larvae should not be directly exposed to light (Grekov et al. 2005).
Larvae optimally develop at light intensities between 15 lux to 20 lux. Young worms, subjected to periods of 16 hours of darkness and eight hours of light, have better survival. The same occurs for adult worms, subjected to periods of 16 hours of light and eight hours of darkness (Rahmathulla 2012). Table 4 shows the effects of lighting on B. mori rearing. The optimal environmental conditions for silkworm rearing are presented in table 5.
Stimulus | Treatment | Consequences | Authors |
---|---|---|---|
Photoperiod variations | LL | Increases the weight of cocoons, shell and pupae |
Janarthanan |
Increases the amount of M5 |
Singh |
||
OO | Increases the amount of M3 | Morohoshi and Takahashi (1969) | |
Decreases the weight of cocoons and shell Decreases the weight of female pupae |
Janarthanan |
||
12L12O | Decreases the weight of cocoon shell of male individuals |
Janarthanan |
|
Light intensity variations | 0.1 lux (0.002) | Induces diapause in few individuals | Shimizu (1982) |
5 lux (0.095) | Prevents diapause induction | ||
Increase of light intensity | Increases duration of larval, pupa and adult stages Decreases the weight of cocoon and pupae Decreases the number of eggs | Suji and Bai (2011) | |
Decrease of light intensity | Decreases duration of larval, pupa and adult stages Increases the weight of cocoons, pupae and shell Increases the number of eggs |
Notes: LL: light for 24 h, OO: darkness for 24 h, 12L12O: 12 h of light and 12 h of darkness, M3: trimolters, M5: pentamolters
Environmental factor | I Instar | II Instar | III Instar | IV Instar | V Instar | Cocoon formation |
---|---|---|---|---|---|---|
Temperature | 28 ºC | 27 ºC | 26 ºC | 25 ºC | 24 ºC | 25 ºC |
Humidity | Between 85 and 90 % | 85 % | Between 75 and 80 % | Between 70 and 75 % | Between 65 and 70 % | 70 % |
Lighting | 8L16O | 16L8O | ||||
Light intensity from 15 lux (0.285) to 20 lux (0.38) | ||||||
Ventilation | [CO2] =1%, [SO2] =0.02%, [NH3] =0.1%, vaire=1 m/s |
8L16O: eight hours of light and 16 hours of darkness
16L8O: 16 hours of light and eight hours of darkness
vaire: air speed
3.2. Nutritional requirements
Each age has specifications for its diet, so it requires different types of mulberry leaf. Leaves located in the upper part of branches are recommended for feeding young larvae, as they are more tender and have high contents of water, protein and carbohydrates. The amount of food required by larvae increases during growth.
It is very common to underfeed larvae, which results in a longer duration of the larval cycle, problems in spinning and low weight and lower quality cocoons. On the other hand, excess feeding results in the accumulation of plant material in the brood beds, which ferment and produce toxic gases.
The artificial diet is used in countries where mulberry is not grown throughout the year. This type of diet includes formulations with nutrients such as: amino acids (Meeramaideen et al. 2017), sterols (Ito et al. 1964), fatty acids, vitamins (Etebari and Matindoost 2005, Kanafi et al. 2007 and Kumara and Kumar 2016) and minerals (Horie 1995 and Ito 1967).
3.3. Diseases and pests
B. mori has a very weak immune system to fight pathogens, which is associated with its domestication (Tayal and Chauhan 2017). Common diseases in B. mori are classified as infectious and non-infectious. The infectious ones are produced by pathogenic microorganisms and protozoa. These infections appear in larvae, pupae, and adults. Non-infectious diseases are produced mainly by arthropods, toxic substances and physiological disorders (Guo-Ping and Xi-Jie 2011).
3.3.1. Fungi
Among the most common fungal diseases affecting larvae are muscardine (Nirupama 2014) and aspergillosis (Kawakami 1982). Infected larvae lose their appetite, become inactive, and develop oil spots or black marks without clear border on their body surface. They die due to the secretion of aflatoxins by the fungus. After death, larvae become flaccid and gradually become harder as the fungus begins to grow in the larva. If it is muscardine, the body of the larva will appear white, while it will appear green in aspergillosis. The latter occurs, generally, in early ages, and causes the death of the larvae without obvious morphological symptoms (Tayal and Chauhan 2017).
3.3.2. Bacteria
Seven bacteria pathogenic to B. mori have been identified, of which five cause considerable losses in cocoon production. These bacteria are Streptococcus bombycis, Staphylococcus sp., Serratia marcescens Bizio, Micrococcus sp., Pseudomonas sp., Bacillus sp., Bacillus thuringiensis (Bacillus sotto Ishiwata) (Tao et al. 2011 and Ayoade et al. 2014). The predisposing conditions for the larvae to become infected with these bacteria are high temperature and humidity, and low-quality mulberry. These diseases are called flacherie, as the body of the dead larva is weak and the shell opens easily. The larvae infected by these bacteria become lethargic, lose appetite and their growth is retarded. They may present symptoms of vomiting, diarrhea and production of chain-type feces. Also, the cephalothoracic region becomes translucent. There are several studies on the incidence of affectations produced by some of these virus-associated bacteria (Tayal and Chauhan 2017).
3.3.3. Viruses
Diseases caused by viruses usually appear in young with poor diet or low-quality food. These diseases include nuclear polyhedrosis and cytoplasmic polyhedrosis (Tayal and Chauhan 2017), and those caused by densovirus (BmDV) (Guo-Ping and Xi-Jie 2011 and Sharma et al. 2020).
Nuclear polyhedrosis has an incubation period of five to seven days. This is the disease that most affects sericulture (Gani et al. 2017). High temperature and relative humidity favor the multiplication of this pathogen. This disease affects more frequently in later ages, which could be related to higher pathogen load and poor hygienic conditions during rearing. At the early stage of disease, larvae do not show symptoms of infection. Diseased larvae begin develop swollen intersegmental region and the integument becomes fragile. The latter breaks easily and spills a white fluid with high pathogen load. Pollution agents are the remains of dead larvae (Tayal and Chauhan 2017).
In 2016, the incidence of nuclear polyhedrosis in larvae reared in Cuba was reported for the first time. This study has a positive impact on the management of this viral infection during B. mori rearing, to avoid its transmission and great economic losses in Cuba (Martínez-Zubiaur et al. 2016).
Cytoplasmic polyhedrosis is orally transmitted, but the greatest infection occurs in brood beds. Young larvae are the most susceptible, especially if they are reared at extreme temperatures (high or low). Infected larvae stop feeding and their development is delayed. When opening the body of the larva, the midgut is white. Also, excretions become soft and whitish (Sharma et al. 2020).
BmDVs are classified into type I and type II, according to their genetic complexion. Larvae are orally infected with these viruses. This virus attacks the columnar cells of the epithelium of the digestive tract of larvae. These cells undergo nuclear hypertrophy as the virus multiplies. Anorexia and lethargy followed by flaccidity and inhibition of metamorphosis are the most common symptoms (Chao et al. 1985). Those larvae associated with BmDV begin to show a whitish color, followed by progressive paralysis. Death occurs between 2 and 20 days, depending on the BmDV strain that caused the infection, age of larvae, and viral load. In addition, larvae show diarrhea and turn dark brown when they die (Gupta et al. 2015).
3.3.4. Protozoa
Nosema bombycis Nageli produces the most dangerous disease in sericulture, which is known as pebrine. It is a disease that is transovarianly or orally transmitted.
Infected larvae show no visible symptoms until the disease is very advanced. Once infected, they delay their growth, consume little food, the rearing loses uniformity and some larvae do not complete their molt. Black spots may appear on the body of larvae, but this does not serve as an indicator, as it does not always occur. The infected chrysalis turns dark and start to swell. However, if the infection is mild, it is not visually detected at this development stage. Infected moths have deformed wings, as well as poor egg production with a high rate of infertility. The presence of the disease cannot be determined only from external symptoms alone. Laboratory studies are required to identify the presence of this pathogen.
For diagnosing pebrine, in addition to observing the previously described symptoms, it is advisable to open the wall of larvae body and examine the silk gland. The presence of white and milky abscesses is an indicator of this disease. The definitive diagnosis of pebrine can be stated when pebrine spores are detected under the microscope, in eggs, first instar larvae, and hemolymph (Tayal and Chauhan 2017).
3.3.5. Pests
Some flies (Cifuentes and Sohn 1998) and wasps are among the main pests that attack B. mori larvae and lay their eggs. When hatching, larvae of these insects feed on the parasitized worm until they provoke their death. Other insects have diseases in common with B. mori, so their entry into the rearing facilities should be avoided. There are also some coleopterans that feed on the pupa inside cocoons and make holes in them (Tayal and Chauhan 2017). Other animals that should not enter the breeding facilities are toads, frogs, lizards and ants. In the case of mammals, such as dogs and cats, they can carry diseases to which the worm is susceptible (Pescio et al. 2008).
3.3.6. Poisoning
Poisoning is produced when the worm is in contact with certain substances, due to its high sensitivity to chemicals. It appears due to mulberry tree contamination, direct application in the breeding facilities or environmental contamination (Guo-Ping and Xi-Jie 2011). Insecticides cause rapid death of larvae, with symptoms of tremors, vomiting and wrinkled bodies. The worm quickly moves to both sides until it ends in a U-shape. Larvae may not die, but it will form a poor-quality cocoon. When they are affected by herbicides or fungicides, symptoms are mild. Larvae can even survive, but their growth is delayed, they do not feed and their development is poor. This produces poor quality cocoons, and even pupa death within them (Rodríguez-Ortega et al. 2012).
3.3.7. Disease Control
There are no curative measures for rearing these worms, always working with the prevention of diseases (Bebitha et al. 2016). There are two ways to control infectious diseases of B. morilarvae. The first consists of maintaining a pathogen-free environment, before and during each rearing. The second is to prevent the entry of pathogens into the facilities (Watanabe 2002). It is almost impossible to create completely pathogen-free environments in rearing areas. However, the use of preventive measures such as those mentioned above, reduces its quantity to tolerable levels (Tayal and Chauchan 2017).
CONCLUSIONS
B. mori rearing is mainly influenced by the interaction of four elements: larvae, environment, food and diseases. B. mori breeds differ in terms of characteristics of interest for sericulture industry development. Voltinism, moltinism and geographical origin, as well as the shape and color of cocoons, are some of these characteristics. Small variations in environmental conditions affect rearing yield, cocoon quality and B. mori lifecycle. The continuous change of these conditions during seasons makes it necessary to strictly control them in the breeding facilities. Inadequate feeding of larvae affects their growth and the production of cocoons and eggs.
Diseases need certain environmental conditions to thrive. The factors of interest in the host are related to their level of resistance to diseases and their nutritional status. Prevention is the most effective strategy to combat the diseases that affect B. mori.