An Examination of Possible Maternal Effects due to Parasite and Density Stress on the Mealworm Beetle, Tenebrio molitor by Maria C. Bennell A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Maria C. Bennell 2011 An Examination of Possible Maternal Effects due to Parasite and Density Stress on the Mealworm Beetle, Tenebrio molitor Maria C. Bennell Master of Science Department of Ecology and Evolutionary Biology University of Toronto 2011 Abstract Few empirical studies examine the influence that the maternal parasite environment can have on offspring fitness (maternal effects) in invertebrates. Several recent studies have found that mothers can adjust offspring phenotype to counter the negative effects of parasite infection. In this thesis I subjected the parental generation of the host species, Tenebrio molitor (Insecta: Coleoptera), to a high parasite, high density, or control treatment. Offspring were subsequently subjected to either the same stress, the alternate stress, or to the control, and fitness-related life history traits were measured in both generations. The results from this thesis do not support the hypothesis that T. molitor mothers influence offspring fitness in a positive way. Instead, maternal effects led to a reduction in offspring fitness under both types of stress. At least under some environmental conditions, females invest in their fitness at the expense of their offspring. ii Acknowledgments I would like to thank my supervisor, Rob Baker, for his expertise, knowledge, and financial support. Thank you Rob, your ability to turn my incoherent ramblings into coherent statements is truly a gift! Thank you to my other committee member, Locke Rowe, and my thesis defence examiners, Megan Frederickson, Maydianne Andrade, and Helen Rodd for their feedback and intellectual input. Helen, thanks for being such an amazing professor. You have had such a positive impact on my graduate and undergraduate experience. Thanks to Tonia Robb for help in the early stages of my project, and to Christopher Yourth for his help with my experimental design, getting my experiments started, and for his personalized stats lectures. Chris, I can‟t tell you how useful our chats about science and stats have been to me. This thesis would not have been possible without the help of many undergraduate laboratory assistants and volunteers, especially Ali Feroz and Melissa Apostoli for taking on so much of the stress in the lab. Thank you to my fellow graduate student friends of past and present, especially Mersedeh Safa, Meghna Roy, Dorina Szuroczki, Crystal Vincent, Brie Edwards, Caren Scott, Anna Price, Maggie Neff, Monica Granados, Meg St John, Bronwyn Rayfield, and Cameron Weadick. My experience at U of T has been so much brighter having known and worked with you. Thanks for all the chats, fun, food, and Rum Wednesdays. I‟m going to miss you all terribly. I would like to thank my parents, Bernard and Christina Bennell for their unbelievable support, and especially for feeding me as I wrote my thesis because I would never have survived through this on take-out. Thanks to my siblings, Sean and Kevin Bennell, and Marissa Largo for their encouragement, and to our family cat, Lulu, for all the fuzzy breaks (yes, I did thank my cat). To my best friends Barb Colonna and Kate Rogucka, thanks for your unwavering faith in iii my abilities. Last but not least, thank you to Ramtin Samie for motivating me and for keeping me smiling. I couldn‟t have done this without you. iv Table of Contents Thesis Abstract………………………………………………………………………… ii Acknowledgements……………………………………………………………………. iii Table of Contents……………………………………………………………………… v List of Tables…………………………………………………………………………… viii List of Figures………………………………………………………………………….. ix List of Appendices……………………………………………………………………… x Introduction………………………………………………………………………….... 1 Importance of Parasite-Mediated Maternal Effects……………………………. 3 Immune Priming within a Single Generation…………………………………... 4 Trans-generational Parasite Studies……………………………………………. 5 Current Study…………………………………………………………………... 7 Methods………………………………………………………………………………... 9 Host System…………………………………………………………………….. 9 Parasite System…………………………………………………………………. 11 Appropriateness of System……………………………………………………... 12 Parasite Manipulations………………………………………………………….. 13 Clearing Existing Gregarine Infections………………………………… 13 Parasite Source Populations…………………………………………… 13 Parasite Infection Procedure.................................................................... 14 Experimental Design……………………………………………………………. 15 Grandparental generation (F-1)………………………………………… 15 Maternal Generation (F0)………………………………………………. 17 Offspring Generation (F1)………………………………………………. 20 Overview of Statistical Analyses……………………………………………….. 22 Maternal Generation (F0) Analyses…………………………………….. 22 Offspring Generation (F1) Analyses…………………………………….. 23 Statistical Analyses…………………………………………………………….... 24 Larval Development…………………………………………………….. 24 Survival…………………………………………………………………. 24 v F1 Larval Growth……………………………………………………….. 24 Timing of Eggs………………………………………………………….. 25 Adult Female Death…………………………………………………….. 26 Total Egg Number………………………………………………………. 26 Egg Size…………………………………………………………………. 26 Results…………………………………………………………………………………... 27 Larval Development…………………………………………………………….. 27 F0 Larval Development…………………………………………………. 27 F1 Larval Development…………………………………………………. 27 F1 Larval Development: Parasite Analysis……………………………... 28 F1 Larval Development: Density Analysis………………………………. 28 Survival………………………………………………………………………….. 29 F0 Survival……………………………………………………………….. 29 F1 Survival……………………………………………………………….. 29 F1 Survival: Parasite Analysis…………………………………………… 29 F1 Survival: Density Analysis……………………………………………. 30 Larval Growth…………………………………………………………………… 30 F1 Larval growth………………………………………………………….. 30 F1 Larval Growth: Parasite Analysis…………………………………….. 31 F1 Larval Growth: Density Analysis……………………………………... 31 Reproduction Analyses…………………………………………………………... 32 Timing of Eggs…………………………………………………………………… 32 F0 Timing of Eggs………………………………………………………… 32 F1 Timing of Eggs: Parasite Analysis……………………………………. 33 F1 Timing of Eggs: Density Analysis…………………………………….. 33 Adult Female Death……………………………………………………………… 33 F0 Adult Female Death…………………………………………………… 33 F1 Adult Female Death: Parasite Analysis……………………………… 33 F1 Adult Female Death: Density Analysis……………………………….. 34 Total Egg Number………………………………………………………………... 34 F0 Total Egg Number…………………………………………………….. 34 vi F1 Total Egg Number: Parasite Analysis………………………………… 35 F1 Total Egg Number: Density Analysis…………………………………. 35 Egg Size………………………………………………………………………….. 36 F0 Egg Size……………………………………………………………….. 36 F1 Egg Size: Parasite Analysis…………………………………………… 37 F1 Egg size: Density Analysis…………………………………………….. 37 Discussion………………………………………………………………………………… 38 Parasite Stress…………………………………………………………………….. 39 Impact of Parasites within a Single Generation of Hosts………………….. 42 Trans-generational Impact of Parasites on Hosts………………………… 43 Limitations of my Findings………………………………………………... 45 Density Stress…………………………………………………………………….. 46 Impact of Poor Environment………………………………………………. 47 Increase in Fecundity…………………………………………………….. 49 Mothers Faced with Different Types of Environmental Stress do not Adjust Offspring Phenotype for the Future Environment in Similar Ways………………………………………………………………. 51 Literature Cited…………………………………………………………………………… 54 vii List of Tables Table 1: MANOVA summary table for the offspring generation larval development. Comparison of parasite and control treatments…………… 62 Table 2: Repeated measures ANOVA summary table for the offspring generation larval growth. Comparison of parasite and control treatments………………………………………………………………… 62 Table 3: Repeated measures ANOVA summary table for the offspring generation larval growth. Comparison of density and control treatments…………………………………………………………………. 63 Table 4: Repeated measures ANOVA summary table for the offspring generation timing of eggs. Comparison of parasite and control treatments…………………………………………………………………. 63 Table 5: Repeated measures ANOVA summary table for the offspring generation timing of eggs. Comparison of density and control treatments………………………………………………………………… 64 viii List of Figures Figure 1: Experimental design predictions of the interactions between the maternal (F0) and offspring (F1) generations……………………… 65 Figure 2: Gregarine infection procedure………………………………………………….. 65 Figure 3: Experimental schedule………………………………………………………….. 66 Figure 4: Analyses, including treatment combinations F0D F1P and F0P F1D……………. 67 Figure 5: Analyses, excluding treatment combinations F0D F1P and F0P F1D…………… 68 Figure 6: Effect of stress history (F0) on offspring development time for parasite vs. control treatments………………………………………… 69 Figure 7: Effect of treatments on larval survival in the maternal and offspring generations……………………………………………………… 70 Figure 8: Effect of time and treatments on offspring larval growth………………………. 71 Figure 9: Time for offspring exposed to current stress treatments (F1) of control vs. density to lay 50% and 100% of their eggs…………………… 71 Figure 10: Effect of treatments and body size on offspring total egg number for control vs. density treatments…………………………………………. 72 ix List of Appendices Appendix A……………………………………………………………………………….. 73 1: Means, standard errors (s.e.), and sample sizes (n) for all F0 generation analyses…………………………………………………….. 73 2: Means, standard errors (s.e.), and sample