Rob Meagher and Rod Nagoshi were very kind to provide us some space in their lab, so we could process our samples at the end of each day.
Just a reminder that my main collection objective was to find L4 Spodoptera frugiperda larvae of both strains (more on that later) feeding on corn. If possible in the same field.
Finding both variants in a corn field didn’t seem to be a problem according to Rob. However, he was concerned about finding larvae of the right age. To identify the proper stage, I brought with me a chart (see pictures below) that we devised in Montpellier, and that we used to measure the width of the head capsule. Indeed, it is the toughest part of the caterpillar body and its only way to grow is during molting. Larvae of the same stage can vary a lot in weight and size but their head capsule should remain of the same width. Of course, people in Rob’s lab had also devised a similar chart based on head capsules and we could check that both matched.
The procedure in the lab was the following. From the cooler that we brought on the field, we would retrieve the plastic cups containing cut leaves and Spodoptera larvae. For each larva, we would measure the head capsule.
L4 larvae were killed quickly by cutting their head around the thoracic segments in order to severe the nerve ganglia. Then they were immersed individually in RNA later, in screw cap tubes. RNA later is supposed to preserve nucleic acids, especially RNA, of freshly collected tissues. If we were to measure gene expression, RNA integrity was to be of the utmost importance.
Older larvae were kept intact and killed in ethanol. Those we would bring back for Mylene and Sarah in Montpellier. They would perform a DNA extraction and perform a PCR to detect the presence of Densovirus -a small virus that is a pathogen of insects.
Younger larvae were kept in an incubator, always in plastic cups, feeding on cut leaves until they reach the right stage.
Obviously, we couldn’t perform this last step on the last day of the mission, so we avoided collecting very young stages from the Tifton field.
The last day was devoted to sorting the final collection samples, preparing and annotating the tubes and shipping them to France. International air shipment can sometimes be held up at customs and I didn’t want the samples to be warm too long, especially for RNA. So I wanted to use dry ice. Jean was kind enough to buys some for us on the campus while we were prepping the shipment box with Marie. Meanwhile, Gaël was collecting adult FAW the easy way… from the frozen collection of Rob and Rod. Indeed, the pheromone traps and light traps that we set up in Tifton didn’t yield any sample, which was unfortunate.
Finally, everything was bowed up, Fedex paper work, for shipment, for dry ice, and for customer were ready. Marie and I brought the package directly to the nearest FedEx office, which was only 20 minutes away, on the other side of the campus. Once there, however, they informed us that they weren’t allowed to take in packages containing dry ice. We had to go to the main office, which was North of Gainesville. Shortly after we left, my phone ran out of battery and Marie’s phone couldn’t be used for tracking our position. So we were in the middle of the afternoon traffic, with no map and in a remote industrialized neighborhood, bring to find the FedEx Ship Center. Fortunately, we remembered the address , we had a good sense of orientation and we were able to reach our destination, fill in the proper forms and ship our crate full go highly valuable biological samples. It only took us 2 hours and a half and, back in the USDA/ARS lab, Gaël was wondering what was happening (our phones were out).
This concluded our last day of work in Florida and I was happy I dedicated one full day, just for sorting the samples and prepare the shipment.