What can vegetarian insects teach us about the importance of vitamin C in their development?

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Vitamin C or ascorbic acid is important to us for the health of our connective tissue, but why do vegetarian insects need ascorbic acid in food? They need her for another role. This molecule is essential for building their proper external skeleton

By Amos Navon, former senior researcher in entomology at the Agricultural Research Administration

The progeny larvae hatch from the eggs. Photo: shutterstock

Vitamin C or ascorbic acid is important to us for the health of our connective tissue, but why do vegetarian insects need ascorbic acid in food? They need her for another role. This molecule is essential for building their proper external skeleton. The effects of ascorbic acid deficiency in vegetarian insects are found only in the growth of the larva on a synthetic food substrate that does not contain this molecule. Man and a limited number of mammals and vegetarian insects, have lost the ability to synthesize ascorbic acid. For man, it is important that the food includes vegetables and fruits, and if necessary then also provide vitamin C as a dietary supplement.

Vitamin C in humans

The discovery of scurvy, a disease deficient in vitamin C, began in seafarers during the discovery of continents, mainly by Columbus, Vasco da Gama and Magellan. The seafarers’ food did not contain it and they did not know it was an ascorbic acid deficiency. The disease manifested itself in death from hemorrhages in the gums and the rest of the body, tooth loss and death from infections and starvation. Those who did not die suffered from a weakness that prevented them from functioning. It was then considered an occupational disease. Seafarers found that the natives in the countries they came to showed no signs of vitamin C deficiency because they were fed plant foods. Later we will learn the need to add citrus fruits to the food basket of seafarers. The British Navy has issued a regulation that must be equipped with transatlantic cruises with citrus fruits. This regulation allowed the UK to take over the continent of Australia and New Zealand. After many years of medical research, it turned out that scurvy is a disease of connective tissue damage and the role of ascorbic acid is to serve as a repellent molecule in the hydroxylation of lysine and proline – amino acids typical of collagen protein. Today in the diet in the western world, scurvy does not pose a threat to humans because our food contains excessive amounts of vitamin C.

The evolution of ascorbic acid intake in mammals

Over the years it has become clear that apart from humans, those who need ascorbic acid are human-like monkeys (primates), voles (rodents) and fruit-eating bats (there are other animals I will not mention here). Common to these animals is that the abundance of ascorbic acid in their natural food has led to a loss of the ability to synthesize this molecule. In the process of evolution it seems that the dependence on this ascorbic acid passed from primate apes to humans. This dependence occurred in the vultures because they were distinctly vegetarian while other rodents, for example mice fed on nuclei, did not need this molecule in their food. Mammals that depend on ascorbic acid in their food have lost the ability to synthesize the enzyme golonolactone oxidase, the last enzyme in the biosynthetic chain of ascorbic acid. This mutation probably occurred in the Middle Paleolithic period (several tens of thousands of years ago), when human nutrition was based on hunting and eating plants and fruits (hunter-gatherers). There is a possibility that the loss of the ability to synthesize ascorbic acid happened later, during the Agricultural Revolution (10,000 years ago) when man began to grow large domesticated farms that served as a food source for man and the vegetarian insect.

Research on ascorbic acid has been around for many years and on a huge scale. It is made by researchers from various fields in the natural sciences and medical sciences, and in the pharmaceutical industry. The basic study of ascorbic acid has come to the point of identifying the difference in the DNA sequence that encodes the formation of the final component in ascorbic acid biosynthesis.
The difference is in the genetic difference between the nitrogenous bases in the genome compared to a mouse that is not a consumer of ascorbic acid and the human:

CTTCTGGCTGCTGTTCAA mouse
TTTCTGACTCCTGTTTGC A person

Evolution and physiological influence on prodenia “

The association between larvae and higher plants began tens of thousands of years ago. Vegetarian insects have become pests in the agricultural revolution because the domestication of plants such as grains and legumes has allowed insects and humans to feed on annuals. The documentation of cotton as the surrogate of the Prodania is mentioned in papyruses from the second millennium BC. The name of this insect in English is The Egyptian Cotton Leaf worm. The damage from prodenia (a popular name) to cotton in Egypt is still a problem today. The desert locust, which is incapable of synthesizing ascorbic acid, is mentioned in the Bible as one of the plagues of Egypt, and is known as the most severe pest of agricultural crops. It is a desert locust. Among the above vegetarian moths, there is also the silkworm whose cultivation in nature and under artificial conditions is obligatory on the leaves of mulberry tree.

In order to study the effects of ascorbic acid on the vegetarian insect it was necessary to assemble an artificial food substrate that does not contain even traces of this molecule. The number of entomologists who have done this is not large, and I am among them. In order to study the effects of ascorbic acid deficiency in Prodania, Spodoptera littorualis (scientific name), I assembled a synthetic substrate based on agar-agar gel. It contained vitamin-free casein, soy hydrolyzate, sucrose, cholesterol, wheat germ oil, B-group vitamin mixture, choline chloride and salt mixture. The source of the larvae for research on ascorbic acid was the mass growth of the insect on an artificial food soil that contained ascorbic acid.

Physiological and biochemical studies

The study was done as part of my doctoral dissertation and in a sabbatical year with the late Prof. Herbert Lipka at the University of Massachusetts-Boston, funded by the Israel-USA Foundation for Agricultural Research (BARD). The findings were published in the leading journals of insect physiology and biochemistry.

Physiological effect of ascorbic acid deficiency in the synthetic food substrate, the prodenia larva served as a model insect of ascorbic acid dependence on food. Initially, under conditions of deficiency in this molecule the larva grew slowly and its weight was smaller. He is still growing on the reserves of ascorbic acid he had in the swamp. In the last stages of the larva a dramatic process takes place. The larva fails to lower the larva. In an extreme situation, the old head box does not fall out of the head forward but remains attached to the head box and also the skin of the head does not fall out of the body, as seen in the picture below.

Figure 1. A prodigal larva that grows due to a complete deficiency of ascorbic acid. 1- The old head box 2- The new head box. 3 The larval skin of the larva that remains close to the body. (Last larval stage). Photo: Amos Navon
Figure 1. A prodigal larva that grows due to a complete deficiency of ascorbic acid. 1- The old head box 2- The new head box. 3 The larval skin of the larva that remains close to the body. (Last larval stage). Photo: Amos Navon

Failure to remove the body’s excretory skin will prevent the larva from clinging to the surface on which it is supposed to stand. In the least extreme situation, the damage is “postponed” to the embodiment stage. As shown in Figure 2.

Figure 2. Defective embodiment in the pupa of Prodania. 1 Abdominal joint exposed due to failure to stretch the wing pockets. Photo: Amos Navon
Figure 2. Defective embodiment in the pupa of Prodania. 1 Abdominal joint exposed due to failure to stretch the wing pockets. Photo: Amos Navon

The biochemical effect of ascorbic acid

Insects do not have connective tissue that is internal tissue in mammals, but rather rigid sclerotial armor that in the vegetarian larva will exist mainly in the head box and the pupa box. The shedding is done under the supervision of the youth hormone and the shedding hormone. In the healthy larva, a distance is created between the old cuticle and the one that develops beneath it, and the space between the two cuticles is filled with an excretory fluid that is secreted from the epidermal cells. The function of this fluid is to digest the chitin and protein in the old cuticle using enzymes. The larva excretes the fluid by ingestion to regain the enzymatic breakdown products of chitin and protein. Also, the larva uses the discharge liquid as a hydrostatic force in rolling up the discharge skin.

Ascorbic acid and the enzyme phenol oxidase are present in the effluent and their full function requires further study. Ascorbic acid has a role in the sclerotization process in that it controls the rate of sclerotene formation. The lack of this recycling molecule, will cause early sclerotization and its biological expression will be the failure of the larva to expand the head box and wing pockets in the pupa. In the larva, the jaws attached to the smaller head box will press on the swallowing house. As a result, the upper jaws will be pushed forward and the larva will not be able to bite into food. Due to the technical difficulties in operating the jaws, the larva will go into a state of starvation where the body components needed to perform the full abduction will be missing, and the larva will die of starvation within a day or two. Due to a partial deficiency of ascorbic acid in the food substrate, the embodied larva will not be able to expand its wing pockets to normal dimensions (Figure 2). The damage to the moth that emerges from the pupa will be manifested in damaged wings in such a way that it will not be able to fly and reproduce.

Ascorbic acid in vegetarian insect – Summary

The number of insect species in the world is estimated at 1-2 million. Not everyone was discovered. Of these, vegetarian insects make up about half a million species. Therefore, the “evolutionary accident” of ascorbic acid dependence in the vegetarian insect constitutes a minority among the insect world. In my study it was discovered by deliberately growing the larva on a synthetic food substrate devoid of this molecule. The process of abstinence constitutes an “Achilles’ heel”, both in those with full incarnation (holometabolic), for example in Prodania, and in those with semi-incarnation (hematabolic), which also includes the desert locust. It is also present in the arthropod system, in which the number of species is lower than in the insect class. It should be noted that insect infestation is one of the most complex processes in the animal kingdom. In humans the growth is done by the addition of cells in the body including connective tissue however in the insects, the larva must shed several times to increase the body volume until incarnation. This is done while operating a complex enzyme system and high energy capacity and performing in a very short time, to avoid madmen. The compensation for this is the huge reproduction rate of insecticides that consume most of the agricultural pests.

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