Methods

1. Introduction Black (Vincetoxicum nigrum) (Fig. 1) and pale (V. rossicum) (Fig. 2) swallow-worts are invasive weeds that have become an environmental and agricultural problem in the northeastern United States. Swallow-worts invade fields, lawns and gardens, smother nearby native plants, and are toxic to grazing animals (DiTommaso et al. 2005). Ultimately, the goal is to eliminate large infestations of swallow-worts by releasing specialized herbivores of the plants (Tewksbury et al. 2002). Currently, we are evaluating the European moth Hypena opulenta (Christoph) (Lepidoptera: Noctuidae) as a biological control agent against these weeds. H. opulenta was collected in 2006 from southeastern Ukraine and appears to be a specialist herbivore of Vincetoxicum spp. (A. Weed, pers. obs.). In this study, we evaluated how each weed and feeding treatment (potted plants versus excised leaves) affected larval performance of H. opulenta in order to determine the best rearing medium. We were specifically interested in testing whether plant or feeding treatment affected larval performance (total development time and mass gain) and final pupal weight. • Results • Average Larval Development Time • Host plant species (P = 0.447) and treatment (P = 0.587) did not significantly affect total larval development time (Figs. 7a and 7b). • Hypenaopulenta larvae took 15.1  1.06 (d  SD) days to reach the prepupal stage. Discussion The results demonstrated that the major influence on the larval development of Hypena opulenta was due to the feeding treatment, not host plant species. Feeding larvae excised leaves over potted plants did not affect the time needed to reach pupation, but did affect the ability of the larvae to gain weight during the 5th instar stage. Previous observations suggest that H. opulenta consumes 74% of the total leaf area during the 5th instar (A. Weed, unpublished data) and this study determined that over 70% of the total larval weight is also gained during this instar. From a lab rearing perspective, this is important because it confirms that rearing on excised leaf material is just as effective as rearing on potted plants. This saves time and the cost of sacrificing whole plants. The fact that larvae took longer to gain mass during their 5th instar stage on whole potted plants than on excised leaves has some ecological significance. For example, it is our impression that Vincetoxicum spp. are protected by toxic alkaloids that are induced by herbivory. By removing leaves from the intact plant such as in our excised leaf treatment, we have removed any potential inducible defense. The observation that 5th instars were able to gain weight significantly faster on excised leaves than those caterpillars reared on potted plants (at day 13) might suggest that the intact plants are responding to herbivory by producing defensive chemicals. Although our sample size was relatively small we still feel this is evidence enough to continue pursuing the influence of herbivory on the production of defensive chemicals by these plants. The weight of Lepidopteran female pupae is typically positively correlated with the number of eggs they are able to produce (fecundity) (Haukioja & Neuvonen 1985). Female pupal weight was greater when larvae were raised on Vincetoxicum nigrum than on V. rossicum, which mightsuggest that females raised on V. nigrum will be more fit. The observed difference in pupal weight due to feeding treatment strongly supports the use of excised leaves over whole plants for mass-rearing. Again, those caterpillars raised on excised leaves might produce adults that are more fit. Development of Hypena opulenta (Lepidoptera: Noctuidae) on two species of invasive Swallow-worts (Vincetoxicum)Alison Traver1, Aaron Weed2, and Dr. Richard Casagrande21Coastal Fellow, URI, 2Dept. Plant Sciences, University of Rhode Island A A A A Figure 7a. Average larval development time (d) per treatment (Ex. Lvs= excised leaves, PP= whole plant). Same letters indicate no significant effect at α = 0.05 level. Figure 7b. Average larval development time (d) per host plant (VN= Vincetoxicum nigrum and VR= V. rossicum). Same letters indicate no significant effect at α = 0.05 level. • Mass Gain • Mass gain was not significantly affected by host species (P = 0.242) and sex (P = 0.51). • Treatment had a nearly significant effect on mass gain (P= 0.064). • There was a significant day*treatment interaction (P = 0.0001). • Average mass at day 13, which corresponded to the 5th instar, was significantly greater for caterpillars reared on excised leaves (0.121g  0.018) than those reared on potted plants (0.092g  0.009) (P < 0.0001) (Fig. 8a). Figure 1. Vincetoxicum nigrum (black) plant, flower and seed pod Figure 2. Vincetoxicum rossicum (pale) plant and flower A A A A A A ♀ ♂ • Major Conclusions • Larval development of Hypena opulenta is the same on each weed • Larvae raised on excised leaves developed faster during the 5th instar • Caterpillars raised on excised leaves produced females that are potentially more fit than those raised on whole plants • Plants may have an inducible defense that inhibits larval development A A A A A B B B A A A A A A A A A A A A A A A A A Figure 8b. Average mass gain of females per treatment. Figure 8c. Average mass gain of males per treatment. A Figure 3. Distribution of V. nigrum Figure 4. Distribution of V. rossicum A A A A Methods Development of Hypena opulenta larvae was monitored on excised leaves (Ex.Lvs.) or whole potted plants (PP) of Vincetoxicum nigrum (VN)and V. rossicum (VR). A newly emerged larva (Fig. 10a) was added to either a potted plant (Fig. 5) or held in a 16-oz. plastic cup lined with moistened filter paper containing an excised leaf (Fig. 6). We set up five replicates of potted plants and ten replicates of excised leaves for each plant species. Whole plants were kept individually in 45 cm3 cages and plastic cups were held on trays. The experiment was conducted in our quarantine laboratory under (16:8) h photoperiod at 24°C and 60% relative humidity. Larvae were weighed on emergence, six days after emergence, and then every other day until pupation. Pupae (Fig. 10b) were then sexed and weighed. New leaf material was added when necessary and potted plants were watered every 3 days. The influence of host plant and feeding treatment on total larval development time (time from emergence to pupation) and pupal weight was analyzed by a Student’s t-test. The influence of host plant and feeding treatment on mass gain was analyzed by a repeated measures ANOVA. Statistical significance was determined at α = 0.05 level. Figure 8a. Average mass gain of both sexes per treatment of H. opulenta. Letters in figures 8a-c indicate results of Tukey’s test between the daily mass for each treatment. (Ex. Lvs= excised leaves, PP= whole plant) • Pupal Weight • Host plant did not significantly affect pupal weight over both sexes (P = 0.416), but there was a significant effect due to feeding treatment (P = 0.00048) (Fig. 9a). • Pupae of insects raised on excised leaves (0.104g  0.007) weighed more than pupae of insects raised on potted plants (0.087g  0.013) (Fig. 9a). • Pupal sex significantly affected pupal weight (P = 0.012) (Fig. 9b). • Pupae of females reared on V.nigrum weighed more than female pupae reared on V.rossicum(P = 0.0009), but males had similar weights regardless of host (P = 0.2903) (Fig 9c). Figure 10a. Larva, 5th instar Figure 10b. Pupa Figure 10c. Adult References DiTommaso, A., F. M. Lawlor, and S. J. Darbyshire. 2005. The Biology of Invasive Alien Plants in Canada. 2. Cynanchum rossicum (Kleopow) Borhidi [= Vincetoxicum rossicum (Kleopow) Barbar.] and Cynanchum louiseae (L.) Kartesz & Gandhi [= Vincetoxicum nigrum (L.) Moench]. Canadian Journal of Plant Science 85:243-263. Haukioja, E. & Neuvonen, S. 1985. The relationship between size and reproductive potential in male and female Epirrita autumnata (Lep., Geometridae). Ecological Entomology 10(3): 267-270. Tewksbury, L., R. Casagrande, and A. Gassmann. 2002. Swallow-worts. Pages 209-216 in R. V. Driesche, Lyon, S., Blossey, B., Hoddle, M., Reardon, R., editor. Biological Control of Invasive Plants in the Eastern United States. USDA Forest Service Publication FHTET-2002-04. A A A A A B B B Acknowledgements We would like to thank Lisa Tewksbury, Arielle Chaves, and Ryan Vazquez for laboratory assistance. The Coastal Fellows Program is supported by the University of Rhode Island Offices of the President and Provost and by the College of the Environmental and Life Sciences. Figure 9a. Average pupal weight of both sexes per treatment. Different letters indicate significant difference at α = 0.05 level. Figure 9b. Average pupal weight per sex. Letters indicate a significant difference at α = 0.05 level. Figure 9c. Average pupal weights between sexes per host plant. Letters indicate results of t-test conducted between hosts for each sex. Figure 5. Potted Plants Figure 6. Excised Leaves