Fungicide impairs silk production

Intensive use of agrochemicals in monoculture systems may be the cause of silkworm crop losses in Brazil.

By Peter Moon  |  Agência FAPESP – One of the problems caused by the use of pesticides is their effect on organisms other than those they are designed to combat. “The growing use of pesticides worldwide has caused environmental problems such as declines in populations of nontarget organisms. Because of its economic impact, the most notorious face of this story is the global mortality among bees used commercially for honey production and pollination services,” said Daniel Nicodemo, a professor at São Paulo State University’s School of Agrarian & Technological Sciences (FCAT-UNESP) in Dracena, Brazil.

Honeybees are not the only economically beneficial insects that are suffering from the intensive use of agrochemicals. “The silkworm is another managed insect whose performance may have been impaired by pesticide poisoning,” Nicodemo said.

The silkworm is the larva or caterpillar of the domesticated silkmoth Bombyx mori. The best-known silk is obtained from cocoons spun by larvae of the mulberry silkworm reared in captivity (sericulture). 

Disease, malnutrition and inappropriate management have a negative impact on the development cycle of the mulberry silkworm and can lead to the death of populations raised by silk farmers. Even in the absence of these factors, however, Brazilian silk farmers have reported a fall in the production of silkworm cocoons. 

An article published in the Journal of Economic Entomology tells how Nicodemo, Fábio Ermínio Mingatto, also a professor at FCAT-USP, and students investigated the effects of pyraclostrobin, a strobilurin fungicide widely used as a crop spray, on the mitochondrial bioenergetics of silkworms and their production of cocoons. The research was supported by FAPESP.

The researchers found that application of the fungicide to mulberry plants increased caterpillar mortality up to threefold and significantly reduced the size of the cocoons spun by surviving caterpillars, causing silk production losses.

Brazilian silk farmers are smallholders who grow mulberry plants to provide food for the silkworms, which feed solely on mulberry leaves. In this business model, the life cycle starts in a centralized textile facility, where moths of the species B. mori mate and lay eggs. After the eggs hatch, the larvae are fed on mulberry leaves and raised by silk farmers from the third instar (larval stage). 

“The silk farmer feeds the caterpillars on mulberry leaves for approximately 20 days,” Nicodemo explained. “The caterpillars then stop eating and, for three days, spin cocoons. They do this by secreting a gelatinous substance that solidifies in air to become a silk filament. Each cocoon is made of a single thread of raw silk, which can be as long as 1,500 meters.”

Three days later, the final molt from larva to pupa takes place inside the cocoon, which provides a vital layer of protection during this stage, when the caterpillar metamorphoses into a chrysalis and will eventually emerge as a moth. At this point, the sericulturists collect the cocoons and hand them over to the textile manufacturer, where the chrysalises are killed by submersion in boiling water before the moths emerge in order to avoid irreparable damage to the silk thread. While the cocoons are being cooked, the gummy substance called sericin that binds them softens and dissolves in the water. The thread is carefully unwound. Several threads are then reeled onto a spindle to produce a uniform strand of raw silk.

Most Brazilian silk farms are found in northern Paraná, but there are also sericulturists in São Paulo State and Mato Grosso do Sul. They are small isolated concerns surrounded by vast sugarcane, soybean or corn plantations where pesticides are heavily used.

“This is one of the possible causes of the silkworm crop losses,” Nicodemo said. When the big plantations are sprayed with insecticides, herbicides or fungicides, the wind often blows these agrochemicals into neighboring farms, where mulberry plants may be growing.

“Silk production depends mainly on the quality of the food given to the caterpillars,” Nicodemo explained. “The quantity of leaves produced by each plant is inversely proportional to their nutritional quality. Silkworms are usually fed on mulberry leaves harvested 90 days after the last pruning, even though their nutritional quality decreases significantly in the last few weeks of the plant’s development.” 

A typical silk farm is about three hectares in size, with mulberry planted throughout. Leaves are harvested monthly in each hectare by turns, so that plants pruned in January, for example, have until the end of March to produce new leaves, which will be harvested only in April, 90 days after the last pruning.

However, the nutritional value of the leaves peaks approximately 60 days after pruning. By 90 days, there are many more leaves, but they may be overmature and senescent. The use of pyraclostrobin is suggested to maintain leaf quality to the end of the 90-day period, which is important because leaf nutrients provide the proteins and other substances silkworms need to spin cocoons. The higher the leaves’ protein content, the more the caterpillars develop and the higher the quality and weight of their cocoons.

“Besides being used as a fungicide, pyraclostrobin also delays leaf senescence and strengthens oxidative stress tolerance in many crops,” Nicodemo said. “These effects could contribute to mulberry leaf quality enhancement and enable silk farmers to maximize production of high-quality leaves at pruning time. The goal of our research was to determine whether treating mulberry leaves with pyraclostrobin would improve cocoon quality. The results were the opposite of what we expected.”

A number of commercially available fungicides contain pyraclostrobin as well as other pesticides in their formulas. A commercial fungicide with pyraclostrobin as the active ingredient was chosen for the field experiment.

Respiratory chain inhibitor

The methodology used in the experiment consisted of treating a mulberry plantation with pyraclostrobin in three doses (100, 200 and 300 grams of active ingredient per hectare) applied as 1,000 liters of solution 60 and 75 days after each pruning. A control area was sprayed with water only. Leaf production, chlorophyll content, macronutrients and micronutrients were analyzed after each pruning. 

Mulberry branches from plants treated with the fungicide were fed to the caterpillars 15 and 30 days after application. Head and intestine mitochondrial bioenergetics were evaluated in vitro and in vivo, along with leaf consumption and caterpillar mortality.

Because mortality was very high when the caterpillars were fed leaves from plants treated with pyraclostrobin 15 days previously, the tests were continued using only leaves from plants treated 30 days before. In this case, the researchers found that at doses of 50 micromolar (µM) in vitro and 200 grams per hectare in vivo, pyraclostrobin inhibited oxygen consumption, dissipated membrane potential and inhibited ATP synthesis in mitochondria. ATP (adenosine triphosphate) is the nucleotide responsible for energy storage in cells. 

“Pyraclostrobin acted as a respiratory chain inhibitor, affecting mitochondrial bioenergetics,” Nicodemo said. “Thus, the main effect expected for fungi also occurs in silkworms.”

Mortality rates were statistically similar for the groups fed 60 days after pruning on leaves from untreated plants and plants treated with pyraclostrobin. However, mortality was 30.7% higher for the group fed leaves from plants treated with the highest dose of pyraclostrobin than for the control group, evidencing a dose-dependent effect.

At the end of the feeding period, the larvae were managed so as to be allowed to spin cocoons. One hundred cocoons were weighed per experimental group, and the silkworm pupae were then removed. At the end of the process, the cocoon shells were weighed separately. 

The results showed that the presence of the fungicide in leaves did not significantly affect their consumption by the silkworms but negatively affected cocoon weight when the dose of pyraclostrobin applied was 100 grams per hectare or more. The weights of the cocoons (-10%) and cocoon shells (-7%) were lower for caterpillars fed leaves treated with pyraclostrobin than for caterpillars fed untreated leaves. The dose of fungicide applied made no difference to cocoon quality.

According to Nicodemo, the toxicity of any substance is a key variable to determine its direct impact on the survival of individuals in a given species after exposure to a specific dose, concentration or both. 

“Nonlethality doesn’t mean the substance is harmless. Organisms may be harmed in various ways even if they survive,” Nicodemo said. “In our study, while silkworm mortality was relatively low when they were fed mulberry leaves that had been treated with pyraclostrobin 30 days previously, damage to head and intestine mitochondrial bioenergetics negatively influenced energy production by mitochondria as well as cocoon production.”

The article “Pyraclostrobin impairs energetic mitochondrial metabolism and productive performance of silkworm (Lepidoptera: Bombycidae) caterpillars” (doi: https://doi.org/10.1093/jee/toy060) by Daniel Nicodemo, Fábio Ermínio Mingatto, Amanda de Carvalho, Paulo Francisco Veiga Bizerra, Marco Aurélio Tavares, Kamila Vilas Boas Balieira and William Cesar Bellini can be read at: academic.oup.com/jee/advance-article/doi/10.1093/jee/toy060/4925588.