Novel biomarker technique used to control crop pests

Researchers used mitochondrial DNA as a marker to assess the effectiveness of biological control by wasps (photo: wasps of the genus Trichogramma/ESALQ)

A group of more than 30 wasp species in the genus Trichogramma are some of the most widely used insects in biological control programs deployed by major agricultural countries, including Brazil, to combat pests that attack sugarcane, corn, soybeans, cotton and tomatoes, among other crops.

Trichogramma spp. parasitize more than 200 species of lepidopteran pests, such as caterpillars that attack crops. However, assessing the field performance of these wasps is difficult because they are only about half a millimeter long.

Their small size, along with the fact that they are parasitoids, makes it impossible to mark them with photoactive dye before release into a plantation, for example, to find out later whether they have stayed there and are controlling the targeted pest.

Researchers at the University of São Paulo’s Luiz de Queiroz College of Agriculture (ESALQ-USP) in Brazil, in collaboration with colleagues at the University of California, Riverside in the United States, have managed to overcome this obstacle by using a molecular biology technique to assess the success of field releases of lines of Trichogramma pretiosum Riley, widely used in Brazil to control soybean and tomato pests.

The molecular biology technique was developed as a doctoral research project supported by a scholarship from FAPESP and supervised by José Roberto Postali Parra, a professor at ESALQ-USP. Its use in this study is described in an article published in the scientific journal PLOS ONE.

“The molecular biology technique we used to assess the field performance of T. pretiosum could perhaps be used for other species of Trichogramma,” said Aloisio Coelho Junior, first author of the article and the PhD student who developed the technique.

He told Agência FAPESP that the technique consists of using mitochondrial DNA fragments as genetic markers to differentiate lines of T. pretiosum and to detect which line displays the best performance in the field.

To develop the technique, the researchers sequenced mitochondrial DNA base pairs from lines of T. pretiosum and classified them using a method of laboratory analysis known as real-time PCR.

They found very clear differences between the lines in a fragment of the mitochondrial cytochrome c oxidase I (COI) gene, with three rare “types” that could be used as genetic markers.

Based on this finding, through a number of crossings, they created 45 different lines of the three mitochondrial types of T. pretiosum(15 of each type).

“We created pure lines marked with these fragments of mitochondrial DNA,” Coelho Jr. said.

Field test

The 45 lines were ranked best, intermediate and worst according to the mean offspring production and the proportion of female offspring. The insect’s performance in attacking crop pests is a reflection of these two factors combined.

To confirm the validity of using mitochondrial DNA fragments from cells of T. pretiosum as markers for the purpose of assessing its field performance, the researchers released lines in each of the three categories with different mitochondrial DNA profiles into a corn plantation and measured the extent to which they parasitized eggs of the flour moth Ephestia kuehniella, a common pest.

The moth eggs were distributed on cards around a small part of the plantation, and the wasps were released from its center. The different lines of T. pretiosum had previously been classified in the laboratory according to their reproductive characteristics, and each had a different mitochondrial profile.

One day after the release, they collected the cards with the moth eggs, took them to the lab and waited for the wasps that had parasitized the eggs to emerge.

Using real-time PCR, they analyzed the wasps’ mitochondrial DNA to identify the field release line to which the COI profile corresponded. In this way, they were able to determine which wasps parasitized the most moths and produced the most offspring in the field, for example.

“Mitochondrial DNA served as a kind of fingerprint in the case of these insects,” Coelho Jr. said.

The results of the mitochondrial DNA analysis showed that the line that had ranked best in the lab, in terms of fertility and the proportion of female offspring, also performed best in the field in terms of parasitism.

The researchers concluded that selection and performance measurement in the lab based on fertility and offspring sex ratio were good predictors of field success in Tpretiosum and that using mitochondrial DNA as a marker was a rapid and efficient technique for identifying lines of parasitoids.

“This biomarking technique could be very useful for selecting the best strains of Trichogramma by biofactories that produce parasitoids for biological control,” Coelho Jr. said.

Biofactories produce Trichogramma in moth eggs parasitized by the wasps on the basis of lineages selected for their successful parasitism in the lab. Without a marker, it is difficult to know whether a species of Trichogramma found in a particular plantation was released by the farmer or was already there. It is also impossible without markers to measure field performance against lab performance, let alone measure the parasitoid’s dispersal range, Coelho Jr. explained.

“This marking technique enables a number of experiments to be performed to answer these and other questions,” he said.

The article entitled “Laboratory performance predicts the success of field releases in inbred lines of the egg parasitoid Trichogramma pretiosum (Hymenoptera: Trichogrammatidae)” (doi: 10.1371/journal.pone.0146153) by Aloisio Coelho Junior et al. can be read in the journal PLOS ONE at