The genetics of Spodoptera frugiperda

One interesting fact about Spodoptera frugiperda is that this moth might be actually 2 different species. It was first recognized by Dorothy Pashley in the 1980’s that the Fall ArmyWorm larvae can be found on different host-plants: corn, of course, but also rice and other grasses (Pashley et al. 1985, Pashley 1986 reviewed in [1]). They have since then be classified as host strains. So, while they are considered polyphagous, FAW larvae display a certain degree of preference in host range. Given that some plants are actually domesticated plants, such as corn, one hypothesis may be that FAW larvae are in the process of specialization to some plants, while the ancestral FAW being more generalist. This could be similar to what happens with the European Corn Borer (ECB) Ostrinia nubilalis, specializing to corn, except that the ancestral form of ECB, Ostrinia scapulalis, is not a generalist, but also a specialist, feeding on wild mugwort.

Analysis of genetic markers indicated that the 2 FAW host strains – the corn strain (C) and the rice stain (R) – while being morphologically indistinguishable from one another, are indeed well differentiated. The most used of such markers is COI. This mitochondrial gene encodes for the subunit 1 of the cytochrome c oxidase complex and is often used in genetic analyses. Because of its mitochondrial origin, DNA is found in 100 to 1,000 time more copies than the nuclear DNA, so it is easier to extract from specimens, especially dead and precious ones from museums. It was later found by Rodney Nagoshi and Rob Meagher, that other markers are associated to each of these FAW strains: the FR repeat, located on the W chromosome or the tpi gene, located on the Z chromosome. Both markers display consistent differences in sequences or in length between the strains.

[On a side note, here I will remind that sex determination in Lepidoptera depends on a ZW system. In this system, it is the male that is homogametic ZZ, while the female is ZW. Remember also that mitochondrial DNA is transmitted through the mother, as in any other sexually reproducing animal.]

The other interesting perk of the FAW strain genetic differentiation, is that it is not linked to geographical distribution of moths. That means, that whether you are in the USA or in Brazil, you will find FAW larvae in corn fields associated to genetic markers of the C strain and larvae in grass fields identifiable as the R strain. Both strains live in sympatry, that means, in the same geographical distribution. So, possibly, there should be substential gene flow between the strains. And indeed, hybrids have been reported. 15% of larvae found in corn fields could be hybrids.

So, while initial studies may indicate that FAW population is composed of two genetically distinct entities associated to different ecological behaviors (feeding preference), and thus might be « in the initial stages of speciation » (Pashney, 1988), subsequent models of speciation with gene flow advise us to take caution with this interpretation. That said, additional evidences suggested that maybe the 2 host strains might be « two sister species capable of hybridizing » (Pashney, 1988). First, there is limited viability of hybrids as well as a preferential direction of interbreeding. Second, a multi marker analysis of a large population sample of FAW, shows two distinct clusters, that are statistically assigned to two different species with divergence times dating back to 2.5 mya [2].

During our collections in Florida, we sampled 2 corn fields (Citra, FA and Tifton, GA) as well as a pasture grass field (Jacksonville, FA). Back in Montpellier, we spent the spring with a Master student, Rima, genotyping our collections. The method we chose was PCR-RFLP (or CAPS) on the COI gene, where the marker gene is first amplified by PCR and then digested with a restriction enzyme. The trick is that the restriction site on the marker gene coincides with a described SNP polymorphism. In the case of COI, the fragment amplified is 500 bp long. If digested, it is cut in a fragment of 450bp and another of 50bp. Our goal was to identified in our collection, individuals of the C strain and of the R strain in the same field. Citra was our most successful field so we started sampling this one.

Here’s what we obtained. We treated 33 individuals, only 1 of them was of the R strain. So, in this particular sweet corn field, 97% of larvae were of the corn strain. Strong association between genotype and ecology here. But it was not good for us, since we really wanted more R strain on corn. We turned to the Tifton samples where we had more luck. Out of 18 individuals tested, 4 were R strain. 22% of R strain in a corn field was approximately what we were expecting. We decided to go ahead and select the Tifton larvae for the rest of our transcriptomic studies. Just to be sure, we tested some individuals from Jacksonville. On this grass field, they were all of the R strain.

Each lane on this agarose gel represents an individual larva from each field. The dark band in the control is  a 550bp amplified DNA fragment from the mitochondrial COI gene. The Corn strains (C) contains a sequence polymorphism that allows this fragment to be digested by MspI into a 500bp and a 50 bp fragment. So it migrates lower on the gel. The Rice strain (R) DNA is not digested because its sequence is different.

I have to say, it was pretty amazing to observe by myself what I read in papers and theses. There really is consistent genetic differences associated with host plant in the Fall Armyworm.

With Rima, we also tested additional markers, to rule out any hybrids. That’s when things became strange. We chose tpi, a gene involved in sugar metabolism, located on the Z chromosome. This gene was more tricky to work with. Because it is nuclear, the DNA yield from sample extractions was lower than for mitochondrial COI, and the best SNP we found associated with a restriction site was for an enzyme called AvaII, which cost about 200 Euros for 10 µl of enzyme. We had just enough to perform a few tests. All 6 Jacksonville individuals showed no digestion, indicative of the R strain. So, no surprise here. But in Tifton samples, out of the 4 R strains for COI, only 1 was identified as R strain for tpi. All others were identified as C strain, raising the possibility that they might all be hybrids, except for one individual. Just to make sure, we used another marker, the FR repeat. R strains contain more copies of this transposon than C strain. But some of the hybrids turned out to be also R. And not the « pure » R strain individual. I had to check post facto the sex of these individuals. No male showed amplification of FR repeat. Only the females of the R strain. That makes sense if this repeat is located on the W chromosome, which is transmitted by the mother. Then, both mitochondrial and W chromosomes are of the maternal genotype.

To summarize our sampling, for all markers tested, 100% of individuals on grass field were of the R strain. almost 100% of individuals from Citra were of the C strain, and some individuals but one, of the Tifton site, were hybrids from a R strain mother. Which in turn indicates that R strain females are capable of laying eggs in corn field as well…

On the next note, I will describe what we found in term of concordance between field samples and laboratory samples at the transcriptional level.

[1] Current Status of Fall Armyworm Host Strains, Dorothy P. Pashley, The Florida Entomologist Vol. 71, No. 3 (Sep., 1988), pp. 227-234

[2] Phylogenetic molecular species delimitations unravel potential new species in the pest genus Spodoptera Guenée, 1852 (Lepidoptera, Noctuidae). Dumas P, Barbut J, Le Ru B, Silvain JF, Clamens AL, d’Alençon E, Kergoat GJ. PLoS One. 2015 Apr 8;10(4):e0122407


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