Bacteria may be inconspicuous, but they led an agricultural revolution

In Europe, flour is the most important food ingredient, but for more than two hundred years, flour merchants have been troubled by a kind of big fat worm. They are the larvae of the Mediterranean flour moth, a full two centimeters long, quietly hiding in warehouses where flour is stored, devouring flour without restraint. They breed frantically in warm flour warehouses and bakeries, then emerge as adults, quickly mate and leave behind the next generation, then die - it takes only 7 weeks to complete a generation, and each female moth can lay nearly 600 eggs.

Eating the flour and leaving behind shed skins, corpses and excrement does not matter. For flour merchants, the real trouble is that during flour processing, their fat bodies go into the machines along with the flour, then jam the parts, causing machine damage or factory shutdowns - in an industrial society, affecting production really is the most headache-inducing thing.

Do not think flour production is a small business. This is a flour factory from the early last century. Image: The book of wheat - an economic history and practical manual of the wheat industry (1908)

Starting in 1879, these insects began to plague all of Europe, leaving flour merchants helpless. In 1927, humans finally succeeded in isolating Bacillus thuringiensis from the corpses of Mediterranean flour moth larvae that had died untimely deaths. This success made humans truly realize the important role of Bacillus thuringiensis, and humans finally did not let this bacterium slip through their fingers as they had several times before.

Bacillus thuringiensis under a 1000x optical microscope with Gram staining. Image: Dr. Sahay / wiki commons

A generous gift nearly missed

At the end of the 19th century, the Japanese had already dealt with this bacterium. At that time, Japan's silkworm industry faced an unprecedented massive blow - silkworms that looked healthy suddenly raised their front halves, stopped eating mulberry leaves, swelled at the thorax, and while convulsing spat out liquid, as if an alien were about to burst out of their chests at any moment. Within a few hours at most, these silkworms became completely paralyzed and suddenly fell dead, and not long after death their bodies became soft, quickly rotted, and released large amounts of dark brown liquid from within. Even silkworms lucky enough not to die developed very slowly. For a time, Japan's silk industry was shrouded in the shadow of death.

A Japanese silkworm farm at the turn of the 19th and 20th centuries, with workers preparing cocooning sites for silkworms about to spin silk. Image: Boston Public Library

In 1901, bacteriologist Shigetane Ishiwata isolated a rod-shaped bacterium from diseased, dead silkworms. Based on the symptoms, he named this bacterium Bacillus sotto. No one knows why Shigetane Ishiwata set this matter aside without continuing his research, and his discovery was never mentioned again.

10 years later, German microbiologist Ernst Berliner rediscovered this bacterium in the Thuringia region of the German Empire. In 1915, Berliner republished this species, naming it after the place of discovery and calling it Bacillus thuringiensis (B. thuringiensis), a name still used today.

A street scene in Erfurt, the largest city in the Thuringia region, in 1915. Image: Public domain / wiki commons

However, I strongly suspect that the person who originally devised the Chinese name for this species did not realize that the specific epithet is actually a place name, and so just followed the English pronunciation of thu-run-gien and rendered it as "Bacillus thuringiensis".

Unfortunately, although Berliner described Bacillus thuringiensis very carefully and even pointed out that these bacilli contain many protein crystal structures - these crystals are like regular rhombi and were named parasporal crystals - he actually lost the culture. This accident caused Bacillus thuringiensis to be shelved again, delaying things by another 10 years.

Bacillus thuringiensis under an electron microscope. The c in panel A corresponds to the crystal inside the cell. Image: Swiecicka I et al. (2008) Appl. Environ. Microbiol.74(4):923-930

A safe poison

With the success achieved in 1927, human research on Bacillus thuringiensis finally entered the fast lane. Soon, people discovered that Bacillus thuringiensis is highly specific - lepidopteran insects were its target.

At that time, gypsy moths were ravaging American forests, so the U.S. government naturally became the first to try it, and the results were outstanding. When Europeans saw this, they happily took this bacterium into commercial use as well.

Bacillus thuringiensis on sheep blood agar medium. Image: PHIL / CDC / wiki commons

In 1938, France launched the first commercially applied Bacillus thuringiensis pesticide, Sporine. This pesticide was specifically used against those big fat worms that damaged the flour industry. By today's standards, this pesticide could simply be described as "crude" - it merely dissolved Bacillus thuringiensis in water.

Crude but effective, water-soluble formulations of Bacillus thuringiensis remained an important pesticide until the 1950s. Compared with chemical insecticides, Bacillus thuringiensis not only degrades easily, but is also nearly absolutely safe for other animals and plants except lepidopteran insects. Even today, when this thing is widely used in transgenic technology, some radical "environmental organizations" have only barely managed to find that when mice are fed Bacillus thuringiensis protein in "large doses" for "long periods", there may be a risk of liver damage.

In 1998, in Oregon, USA, an aircraft was spraying Bacillus thuringiensis over coniferous forest to deal with pest damage. Image: public domain

However, the same quality that makes it succeed is also its downfall: the disadvantage of Bt protein also lies in how easily it degrades. If the sun is a bit strong and ultraviolet light shines on it, the active ingredients in these pesticides become ineffective. Faced with longer-lasting, cheaper chemical insecticides, apart from a small number of farmers who insisted on so-called "green organic" production in the hope of obtaining higher product prices, other farmers basically switched to chemical insecticides.

In 1956, Canadian insect pathologist Thomas Angus first discovered that the insecticidal component of Bacillus thuringiensis was hidden in those parasporal crystals Berliner had found. These crystals are actually protein crystals, and Angus gave the proteins that make up these crystals a pleasant name: Bt toxin.

This name literally translates as "Bt toxin", but now it is generally called "Bt toxic protein" or "Bt protein". This protein has a very ingenious structure, so ingenious that it is itself non-toxic, so calling it a toxic protein really makes it bear an enormous undeserved blame.

Bt protein crystals inside Bacillus thuringiensis under an electron microscope. Image: Jim Buckman / wiki commons

Of course, although it is non-toxic, after it enters an insect's body, under alkaline conditions it is activated by special enzymes in the digestive tract of lepidopteran insects and becomes a toxic substance. The activated toxic substance destroys the cellular structure in the insect's gut, ultimately causing intestinal perforation and intestinal dissolution, with a very miserable death.

However, under acidic conditions, Bt protein is very unstable. When mammals eat this thing, it is mostly hydrolyzed in the stomach. Even if a very small amount of Bt protein fortunately enters the alkaline intestines, mammals lack the enzymes unique to insects that convert Bt protein into toxic substances, so these proteins ultimately cannot escape being digested or excreted.

The birth of a genetic engineering weapon

In the 1980s, research on Bacillus thuringiensis toxic proteins had already become very mature, and people finally discovered that Bacillus thuringiensis is actually an organism with a large number of subspecies, and each subspecies has different characteristics, produces different proteins, and may target different organisms.

The spatial structure of Bt protein. Image: Lucena WA et al. (2014) Toxins6(8):2393-2423

Because they exist in crystals, these proteins were also named Cry proteins, such as the most famous Cry1Ab secreted by the kurstaki subspecies. As more and more bacterial strains were tested, more and more Cry proteins were discovered, and the insecticidal range naturally became broader and broader, expanding from the earliest lepidopteran-specific killing to later coleopteran and dipteran insects, with more and more agricultural pests meeting their corresponding "weapons".

Commercial Bacillus thuringiensis tablets used to control mosquitoes. Image: Claus Ableiter / wiki commons

Because the proteins of these two families share a common point: they are deadly to their targets while safe and harmless to almost all other organisms, this made the concept of "safe and efficient insect-resistant crops" seem very attractive. In 1985, Belgians developed the world's first transgenic insect-resistant tobacco, and 3 years later, an American company also successfully developed the first batch of insect-resistant cotton.

In the early 1990s, China suffered an unusually severe cotton bollworm disaster: in 1992, cotton bollworms swept in, and that year 150,000 tons of insecticide were used to no avail. Radar was used to monitor the flight paths of the swarms, and traps were set midway to intercept them, yet the results were still very limited. According to conservative estimates, direct economic losses that year were at least 6 billion yuan, and total economic losses reached the level of tens of billions.

Under the crisis, in 1995 China began planting transgenic cotton produced by an American company, and in 1997 began planting it in North China. The effect could almost be called immediate, and cotton yields gradually returned to normal. Today, the cotton in China's cotton fields is almost all transgenic insect-resistant cotton.

A cotton field about to welcome its harvest. Image: Kimberly Vardeman / wiki commons

Of course, besides providing timely help, transgenic insect-resistant cotton also brought an unexpected additional benefit - lower costs. When insect-resistant cotton was first introduced to China, this characteristic had already shown itself. The pesticide cost required for insect-resistant cotton is far lower than for non-transgenic cotton, the number of applications is reduced, labor use is reduced, and at the same time yields increase - overall, technological progress has given humanity enormous convenience and economic benefits.

The rising double helix

However, an awkward reality is that in nature, wherever there is a spear, a shield will inevitably arise. Just like antibiotics encountering drug resistance.

According to strict planting standards, when planting transgenic crops, a certain amount of non-transgenic "refuges" without insect resistance must also be planted. They will inevitably be riddled with holes by various pests, but their existence can effectively reduce selection pressure on the species and prevent resistance from appearing too early.

The role of insect refuges is to allow more insects without resistance to survive. Image: syngenta-us.com

However, in many underdeveloped regions and areas with tight agricultural land around the world, "clever" farmers intentionally or unintentionally choose to ignore this regulation. It may seem to bring higher returns from their land, but more and more agricultural pests, under extreme selection pressure, develop resistance early, catching farmers and seed companies off guard.

Of course, surrendering without a fight is not humanity's character either. People soon discovered that the weapons Bacillus thuringiensis provides to humanity are not limited to the Cry family. Scientists discovered another kind of protein in the israelensis subspecies of Bacillus thuringiensis. Unlike Cry proteins, they are distributed in the cytoplasm rather than in crystals, and so are called Cyt proteins.

Cyt proteins are very special. Although they can, like Cry, bind to the cell membranes of insect intestinal cells and throw the insect's system into complete chaos, they have no real kinship with the Cry family proteins, and their specific mechanisms of action are also different.

Comparison of pest resistance between transgenic peanut leaves (left) and ordinary peanut leaves. Image: Herb Pilcher, USDA ARS / wiki commons

The principle of action of Cyt proteins is to bind with phosphatidylethanolamine on insect cell membranes. They do not need enzymes and simply and crudely dissolve their cells directly. If anything deserves to be called a toxin, Cyt is actually more fitting. Their selectivity is achieved through another pathway - the proportion of phosphatidylethanolamine on the cell membranes of dipteran insects exceeds half, so they are easily directly torn apart by Cyt, while in lepidopteran insects that Cyt cannot kill, as well as in the cell membranes of us vertebrates, phosphatidylcholine is dominant - therefore, although Cyt can occasionally bind to our cell membranes, it is still safe for us.

With two sets of "antibiotics", we seem to have a way to do something about resistant pests. Just like in a hospital, when encountering a resistant bacterium, as long as the drug is changed, the board can be reshuffled. Of course, Cyt still has shortcomings. Being powerless against lepidopteran pests will directly affect its scope of use.

An experimental field of transgenic corn in Ohio, USA. Image: Lindsay Eyink / wiki commons

In addition, reducing the emergence of resistant pests through two different resistant crops also involves correspondingly complex procedures, and there is no way to stop "clever" farmers and "clever" seed companies from abusing them in combination, leading to the birth of a group of "super pests" resistant to both Cry and Cyt.

Now, in the search for new Crys and Cyts, seed companies seem to have encountered a bottleneck as well. Facing possible "super pests", it seems hard to say that transgenic crops have great prospects.

We sometimes feel that we have conquered nature, but in this contest with nature, we still have a long way to go.

This article is the 148th article in the 4th year of Species Calendar, from Species Calendar author @C.CristataX.

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Bacillaceae Bacillus subtilis