1a. Objectives (from AD-416):
Develop at least one population of F2-derived soybean aphid clones from crossing two soybean aphid biotypes. Test derived aphid clones on plants with a specific Rag gene that differentiates the parental biotypes to determine potential inheritance of virulence on that gene; Create reciprocal symbiotic and aposymbiotic clones of each soybean aphid biotype and then test them for virulence on specific Rag genes to determine the potential effect of biotype-specific endosymbionts on virulence.
1b. Approach (from AD-416):
Objective 1: Sexual forms of each soybean aphid biotype will be induced in controlled environmental chambers or naturally in the field inside field cages. Males and females (oviparae) of each biotype will be paired reciprocally in isolation inside plant cages in growth chambers, or field cages, allowed to mate and lay F1 hybrid eggs on buckthorn plants. Following a period of vernalization, F1 hybrid eggs will be induced to hatch inside growth chambers or naturally in field cages and F1 clones will be maintained in separate growth chambers. Sexual forms will then be induced from the F1 aphids as described above, allowed to mate and lay F2 eggs. Backcrosses (BC) will also be made between F1 hybrids and each biotype parent. F2 and BC eggs will be hatched and the fundatrices, or the aphids hatching from the eggs, will be collected and isolated individually to produce F2-derived clones. Each F2-derived and BC clone will be tested on the soybean plants with Rag genes that differentiate the biotype parents. Data on the proportion of clones that are virulent on the test resistant plants will be analyzed to determine the inheritance pattern of virulence. Objective 2: Aposymbiotic clones of each of the three soybean aphid biotypes that are free of secondary endosymbionts will be established by means of injections with the antibiotic ampicillin, that does not harm Buchnera, into individual aphids for multiple consecutive generations. After antibiotic treatment, aposymbiotic clones will be maintained for three generations prior to being used for any subsequent experiment. Aphids will be tested for the presence of secondary endosymbionts at each generation using specific primers to the 16S-23S ribosomal RNA intergenic region. Sister clones without ampicillin treatment will also be maintained. Established aposymbiotic clones of each biotype will be reciprocally injected with secondary endosymbionts from the other biotypes using hemolymph, which is insect blood containing the endosymbiont, to establish additional sister clones of each soybean aphid biotype infected with secondary endosymbionts from different biotypes. All nine possible symbiont-biotype combinations will be produced.
3. Progress Report:
The virulence of the putative 2 x 3 and 3 x 2 F1 hybrids on multiple soybean resistance sources was evaluated by conducting standard choice and non-choice tests in controlled environmental chambers and greenhouse conditions. Results of the choice and no-choice tests clearly indicated different patterns of virulence between the reciprocal hybrid aphids. The 2 x 3 F1 colonized plants with Rag1, but not Rag2, similar to its female parental biotype 2, whereas 3 x 2 F1 hybrid aphids colonized Rag2 genotypes but not Rag1, similar to its female biotype 3 parent. These results suggest that there is a significant maternal influence on soybean aphid virulence, possibly due to differences in endosymbiont composition or cytoplasmic genes between the parental biotypes that were transferred from the female parents to the hybrids, or sex-linked inheritance as found in the Hessian fly. Early spring of 2013, a collection of F2 progeny from the 2 x 3 and 3 x 2 F1 aphids from buckthorn plants inside field cages where the hybrid aphids mated and laid their eggs last fall (2012). We established colonies of 190 F2 clones from the 2 x 3 cross and 90 F2 clones from the 3 x 2 cross on Williams 82 soybean plants. A representative sample of aphids from each F2 clone is being collected for DNA extraction. A series of reciprocal crosses conducted between all three biotypes, during last fall (2012) in field cages were successful, with the exception of the cross between females of biotype 1 and males of biotype 3. F1 aphids from the successful crosses are being maintained on buckthorn inside the cages. Representative samples of aphids were collected from each F1 hybrid for DNA extraction. Various concentrations of ampicillin and tetracycline were tested, and we found that these two antibiotics do not affect the function of the symbiotic bacteria Buchnera. We have also tested injected individuals via PCR and found that we have decreased the concentration of Arsenophonus in the aphid, but have not completely eliminated it.