Proteins (Amino Acids) Needed for Silkworm Growth

Protein is the basic substance that constitutes silkworm body cells. Active substances such as enzymes, carriers and certain hormones are all proteins, which are indispensable substances for the maintenance of life processes. If the ingested protein exceeds the need to maintain tissue growth, renewal and repair, or when the energy storage material in the silkworm body is insufficient, part of it is oxidized to supply energy. Protein is also an important raw material for silkworm larvae to synthesize silk material and female moths to lay eggs.

Amino acid is a general term for organic compounds containing amino and carboxyl groups. It is a basic unit of biologically functional macromolecular protein and an important constituent of protein required for animal nutrition.

As the silkworm digests and decomposes the eaten protein into amino acids and then uses them, this article mainly describes amino acids.

According to the nutritional and metabolic functions of various amino acids, the L-amino acids required for silkworm growth can be divided into the following five categories:

1. Essential amino acids (group 1): arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine. 
2. Essential amino acids (group 2): aspartic acid, glutamic acid.
3. Semi-essential amino acid: Proline.
4. Non-essential amino acids (group 1): Alanine, Glycine, Serine. 
5. Non-essential amino acids (group 2): tyrosine, cystine, hydroxyproline.

1. Essential amino acids

The 10 essential amino acids in the first group are all necessary for the life activities of silkworms. During the 2 to 5th instar period, the feed for each instar lacks any of them, and the growth of silkworms is blocked, and they all die at the current instar and cannot enter the next instar.  The 5th instar silkworm cannot mature and form cocoons. Unlike other higher animals, the necessity for arginine and histidine does not decrease with growth and development.

It is generally believed that silkworms lack a synthesis system of related amino acids. Even if they can synthesize a very small amount of essential amino acids, they are far from meeting the needs of silkworms. The isotope tracer test showed that ¹⁴ C-labeled glucose, aspartic acid, and glutamic acid were given in the larval stage , and ¹⁴ C could bind to leucine, arginine, and proline in a very small amount. Adding indole lactic acid and phenylpyruvate to feed can partially replace the functions of tryptophan and phenylalanine in silkworm bodies. Isotopic tracing experiments have proved that arginine can be produced from citrulline in silkworms, but no radiolabeled arginine produces citrulline. Therefore, it is believed that the ornithine cycle of silkworms is not complete. There is a break between the acid and citrulline. By replacing arginine in the feed with citrulline, the silkworm can obtain a certain degree of growth. It can be considered that the in vivo supply route of the precursor substance citrulline does not actually exist.

It can be seen that the silkworm can synthesize essential amino acids when given certain precursor substances. The lack of a synthetic system for these precursors in the silkworm is the inherent reason for the essentiality of these 10 amino acids.

In the absence of essential amino acids, silkworms stop growing and tend to die, but the effects of their physiological and biochemical levels are still unclear. It is only known that when the fourth-instar silkworms are fed with feed lacking essential amino acids, the infiltration of isotope-labeled amino acids into tissue proteins is significantly reduced, and the composition of free amino acids in the blood is also significantly changed.

The metabolic pathways of essential amino acids in silkworms have not yet been fully ascertained. Biochemical genetic studies have found that the metabolites of tryptophan in silkworm are related to the synthesis of egg color and eye pigment. Tryptophan can produce kynurenine through formylkynurenine, and then produce 3-hydroxykynurenine from kynurenine, and then produce various eye pigments.

Phenylalanine can generate tyrosine under the catalysis of phenylalanine-4-hydroxylase. Tyrosine is oxidized by phenolase to produce dihydroxyphenylalanine (DOPA), which is further oxidized and deamination by DOPA to produce quinones (such as dihydroxybenzoic acid) that can harden the epidermis. This quinone reacts with epidermal proteins. Quinone tanning causes hardening of the epidermis. DOPA can also be oxidized to form o-quinone phenylalanine, and finally form 5, 6-o-quinone indole, and re-synthesize melanin from o-quinone indole. DOPA can also generate ether dopamine via dopamine.

Arginine forms ornithine under the action of arginase, and ornithine and α-keto acid undergo transamination to form various α-amino acids, such as alanine, glycine, and glutamic acid. The first two are the main amino acids for the synthesis of silk protein at the 5th instar. Finally, proline can be formed, which is an important source of energy metabolism in insects.

In silkworms, phenylalanine can be metabolized to tyrosine, and methionine is converted to cystine by cystathionine, and then further participates in metabolic conversion.

Aspartic acid and glutamic acid are the second group of essential amino acids in the silkworm, and they have different nutritional and physiological effects from the first group of 10 essential amino acids. The ant silkworms were raised with a feed consisting of the first group of essential amino acids and the semi-essential amino acid proline, and most of them died at the first instar stage. If aspartic acid and glutamic acid are further added, the growth and development will be significantly improved. When these two acidic amino acids are removed from the amino acid mixture at the same time, the growth of silkworms is significantly inhibited. But if any of them are added, the growth of silkworms will be roughly normal, and the effects of the two will be roughly the same.

Aspartic acid and glutamic acid can be converted into each other. They play the role of amino donor in the transamination reaction. This is the main reason why aspartic acid and glutamic acid are essential amino acids.

2. Semi-essential amino acids

There is only one semi-essential amino acid in the silkworm, proline, and the growth and development of silkworms are delayed when proline is not given. Silkworm body cannot complete the ornithine cycle. Ornithine produced by the metabolism of citrulline, aspartic acid and arginine can produce proline. Therefore, proline becomes the semi-essential amino acid of silkworm body. Experiments have proved that ornithine can be converted into proline in silkworms, and proper amount of ornithine can be added to the feed to replace proline.

The rate of metabolism from citrulline to proline varies with feed conditions. When fed with feed lacking arginine, the conversion of citrulline to arginine from silkworms is promoted. When fed with feed lacking proline, the conversion speed of silkworms from arginine and ornithine to proline was 3.3 times and 1.7 times that of the control, respectively. The presence or absence of arginine or proline in the feed becomes the decisive factor for arginine utilization or proline biosynthesis, indicating that nutritional conditions can regulate the metabolic rate of amino acids.

3. Non-essential amino acids

Lack of alanine, glycine, serine, cystine or tyrosine in the feed will not affect the growth of silkworms, so these amino acids are called non-essential amino acids. These amino acids can be transformed from essential amino acids in silkworms, and removing one of them alone will not significantly inhibit growth.

The first group of non-essential amino acids alanine, glycine, and serine can be converted to each other, and is easily generated by the transamination reaction between aspartic acid or glutamic acid and the corresponding keto acid.

In silkworms, alanine can be formed by transferring the amino group of aspartic acid or glutamic acid to pyruvate through the catalysis of glutamate and alanine aminotransferase. The activities of these two enzymes are higher in silk glands, digestive tract, and fat body. Although alanine can also be produced from glycine, its production amount is far less than the amount of serine produced from glycine. Alanine can also be produced from serine, and its production amount is also less than the amount of glycine produced from serine. In addition, under the catalysis of the digestive tract kynurenase, kynurenine can also be decomposed into aminobenzoic acid and alanine, but the amount of production is small.

The main synthetic pathways of glycine in silkworms are as follows. The activity of glycine aminotransferase is higher in the posterior silk glands and digestive tract, and lower in body fluids and middle silk glands. As an amino donor, alanine has the best effect, followed by glutamic acid and aspartic acid. Others such as valine, arginine, and histidine also have certain effects. Through the tracing test of larval injection of radioactive threonine, it was found that the ratio of ¹⁴ C bound to alanine, glycine, and serine in silkworm cocoon silk protein was 1.5:6:1. When ¹⁴ C-1-glycolic acid was injected into the larvae, the blood amino acids of glycine were labeled the most, followed by serine, and alanine was rarely labeled. Glycine can be detected in the cocoon to absorb a large amount of radioactive energy, but the radioactive energy in alanine and serine is very small.

Serine can be generated from 3-phosphoglycerate produced in the process of carbohydrate metabolism, 3-phosphohydroxypyruvate is generated by dehydrogenase, phosphoserine is generated by transamination, and serine is finally generated by the catalysis of phosphatase. Serine is easily produced from glycine. Serine can also be produced from threonine.

Tyrosine, cystine, and hydroxyproline are the second group of non-essential amino acids, and they can only be synthesized from specific precursor substances. Tyrosine is produced from phenylalanine, cystine is produced from methionine, and hydroxyproline is produced from proline. Nutrition test shows that cystine and tyrosine have the effect of saving methionine and phenylalanine respectively. If the amount of these two essential amino acids in the feed is insufficient, the effect of adding cystine or tyrosine is more obvious. It seems that cystine and tyrosine are indispensable amino acids. It shows that a considerable part of methionine and phenylalanine in silkworms is used to synthesize cystine and tyrosine.

There are more free cystathionine and lanthionine in the body fluid of the silkworm, and the content of both varies greatly with the development stage, and the lanthionine in the body fluid reaches more than 2mg/mL at the highest point. From the point of view of the total amount of free amino acids in the body fluid of the silkworm, there are also differences between males and females. Among them, lanthionine and cystathionine have the largest difference between males and females. Both components are more female. The retention rate of sulfur in silkworms is also higher in females (40%) than in males (25%). 60% of the sulfur content in female pupae is used for egg formation, and it is rarely excreted in moth urine, while nearly 50% of male pupae are excreted, indicating that the difference in sulfur-containing amino acid metabolism between male and female is related to egg formation. Female silkworms require more sulfur-containing amino acids than male silkworms, and their ability to decompose sulfur-containing amino acids is greater than that of females. The activities of cystathionine synthase and cystathionine in the adipose tissue of the silkworm are both female and male. The difference is that the former is smaller than the latter, especially in the middle and late stages of the fifth instar, indicating that the cystathionine in the adipose tissue The difference in etherase activity between male and female is one of the reasons for the difference in the content of cystathionine in body fluids.

The content of free sulfur-containing amino acids in body fluids also varies with different feed conditions. When methionine is added to the feed, the content of cystathionine increases significantly, while the content of lanthionine is only when cystamine is added to the feed. There is a significant increase when it is acid.

Tyrosine is converted from phenylalanine, and the release of tyrosine from the fat body into body fluids is controlled by ecdysone. The activity of phenol oxidase in body fluids changes with development, especially before and after pupal molting. Tyrosine and phenol oxidase play an important role in body wall fusion and melanin formation.

Alienated metabolism and excretion of amino acids

The silkworm belongs to the uric acid excretion type insect. The excess amino acids ingested from food, or the amino acids produced by the decomposition of protein in the body’s tissues, undergo deamination in the midgut epithelium, body fluids, fat body or other tissues to produce ammonia. Ammonia is toxic and must be removed quickly. Some are excreted through Markov tubes in the form of ammonium salts; some are stored in the body in the form of amides to provide amino groups when newly synthesizing amino acids; some can participate in the synthesis of purine compounds, and are metabolized by purine compounds to produce uric acid or urea. The activity of purine oxidase in silkworm is higher in fat body, followed by Markov tube and midgut. If the amount of defatted soybean meal is increased in artificial feed containing a certain amount of sugar, the enzyme activity of fat body, martens tube, midgut, etc. will increase correspondingly, and the excretion of uric acid will increase. Although increasing the addition of amino acids in the feed can significantly increase the excretion of uric acid, if sucrose is added to this feed, the increase in the excretion of uric acid is not large. It can be seen that maintaining a proper addition ratio of sugar and protein in the feed is beneficial to improve the nitrogen utilization rate of the feed. The urea content of mulberry leaf rearing silkworms is very small, and there is more than 10 times more urea in the artificial feed breeding silkworms than the mulberry leaf breeding silkworms. It is known that the mulberry leaf breeding silkworm has strong urease activity in the digestive tube, but the artificial feed breeding silkworm cannot detect the urease activity. If the mulberry leaf powder is added to the artificial feed, the urease activity can also be measured in the body fluid of the silkworm pupae. It shows that urease in mulberry leaves can be adsorbed through the digestive tube, and the accumulation of urea in the artificial feed breeding silkworm may be due to the lack of urease in the feed.