Parasitoids used as biological control agents often parasitize more than a single host species and these hosts tend to vary in suitability for offspring development. The population dynamics of parasitoids and hosts may be altered by these interactions, with outcomes dependent on the levels of suitability and acceptance of both host species. Parasitism of individuals of an unsuitable host species may indirectly increase populations of a suitable host species if eggs laid into unsuitable hosts do not develop into adult parasitoids. In this case the unsuitable host is acting as an egg sink for parasitoids and this can reduce parasitism of suitable hosts under conditions of egg limitation. We studied parasitoid-mediated indirect interactions between two aphid hosts, Aphis glycines (the soybean aphid) and A. nerii (the milkweed, or oleander aphid), sharing the parasitoid Aphelinus certus. While both of these aphid species are accepted by A. certus, soybean aphid is a much more suitable host than milkweed aphid is. We observed a drastic reduction of parasitoid offspring production (45%) on the suitable host in the presence of the unsuitable host in microcosm assays. Aphelinus certus females laid eggs into the unsuitable hosts (Aphis nerii) in the presence of the suitable host leading to egg and/or time limitation and reduced fitness. The impact of these interactions on the equilibrium population sizes of the three interacting species was analyzed using a consumer-resource modeling approach. Both the results from the laboratory experiment and the modeling approaches identified apparent predation between soybean aphid and milkweed aphid, in which milkweed aphid acts as a sink for parasitoid eggs leading to an increase in the soybean aphid population. The presence of soybean aphids had the opposite effect on milkweed aphid populations as it supported increases in parasitoid abundance and thus reduced the fitness and abundance of this aphid species.

Experiment 1: Parasitoid-mediated indirect interactions between milkweed aphid and soybean aphid

Experiment 1.1: Parasitoid oviposition

In the no-choice assay, one leaf of soybean or milkweed (leaves of the two plants were chosen to be of similar size) was cut at the stem and separately placed into a tube (diameter: 2.5 cm, h: 7cm) with 1 cm of watered sand and closed by a perforated cap. Each leaf was then infested with 20 3^rd instar A. glycines (20Ag) or A. nerii (20An) on soybean and milkweed, respectively (n=25-27 replicates).

In the choice assay, one leaf of soybean and milkweed was cut and placed into the same tube in the same conditions as in the no-choice assay. The appropriate leaves were then infested with 10 aphids of A. glycines and A. nerii (10Ag+10An) (n=25 replicates) or 20 aphids of each species (20Ag+ 20An) onto their respective plant (n=25 replicates). The two densities enabled us to evaluate the impact of aphid abundance on the potential egg sink caused by the presence of the unsuitable host. One mated female parasitoid (between 24 and 48 hour-old) was then introduced into the tube of both the no-choice and choice assays for 24 hours.

After parasitoid removal in both no-choice and choice assays, aphids were frozen and later dissected at 40x magnification to record the number of eggs laid by the parasitoid into individuals of each aphid species. To determine whether these female parasitoids experienced egg limitation during the assays, 12 randomly chosen females from each of the four different treatments were frozen after they were removed from the arena and later dissected at 40x magnification to count the number of remaining eggs. To make sure there was no systematic bias in size of adults among the treatment, the right hind tibia length was also measured.

Experiment 1.2: Parasitoid developmental success and offspring fitness

In the no-choice assay, one plant of soybean (10 days old) or milkweed (21 days old) of approximately the same size was potted individually (depth: 13.5 cm, top diameter: 14 cm) and covered in a cylindrical plastic cover (diameter: 11 cm, h: 21cm) with several 3cm wide mesh covered holes cut in the sides and on the top. Potting soil was covered with white plaster to prevent the development of fungus gnats. The plants were then infested with 50 3^rd instar A. glycines (50Ag) or A. nerii (50An) on soybean and milkweed, respectively (n = 18 and 33 plants, respectively).

In the choice assay, single soybean and milkweed plants were potted together under the same conditions as in the no-choice assay. The appropriate plants were then infested with 25 aphids of each species (25Ag+25An) (n=27 replicates) or 50 aphids of each species (50Ag+50An) onto their respective plant (n=26 replicates). As in Experiment 1.1, the two different densities enabled us to determine the impact of aphid abundance on the potential egg sink caused by the presence of the unsuitable host. One mated female parasitoid (between 24 and 48-hours-old) was then introduced into the covered pots for 24 hours.

In both the no-choice and choice assays, plants were inspected 10 days later and all mummies were removed and isolated into 0.6 mL micro centrifuge tubes. The total number of mummies and emerged parasitoids were counted and the adult offspring sex ratios were recorded. To determine the effect of the treatments on egg limitation in A. certus, 46 (n for each of the 4 treatments was 11 or 12) were frozen after the 24 hours of the experiment and later dissected at 40x magnification to count the number of remaining eggs and measure the right tibia length.

Experiment 2: Impact of parasitism on milkweed aphid

In this experiment we evaluated mortality of the unsuitable host A. nerii induced by parasitism by Aphelinus certus using a combination of direct observation and rearing. To prepare for observations, a milkweed leaf was placed upside down within a clear plastic cube (1cm³) under a magnifying lens (8x). One second or third instar milkweed aphid was introduced onto the leaf within this cube using a fine brush. Five minutes later, one mated female parasitoid (between 24 and 48 hours old) was also introduced and observed until oviposition occurred (n=37). The same procedure was followed without introducing a parasitoid as a control to quantify background aphid mortality (n=33). Aphids parasitized or not were then placed separately onto milkweed leaves cut at the stem which were then introduced into a tube (diameter: 2.5 cm, h: 7cm) with 1 cm of watered sand at the base (as described above) and closed with a perforated plastic cap. The aphids were observed daily to record their longevity, fecundity and the period of time before the first nymphs were laid. In order to record daily fecundity, newly produced nymphs were removed every day.