1. Experimental Manipulation of Mode Bull et al. (1991) used E. coli and filamentous phage f1 A gene conferring ampicillin resistance was inserted into the phage genome In a Vertical transmission treatment, phage was forced to pass from mother to daughter cell. In a Horizontal transmission treatment, phage was forced to pass from infected to uninfected hosts. Horizontal TreatmentVertical Treatment Jim BullIan MolineuxBill Rice pilated host phage with extra gene infected amp R host growth with amp dilution growth with amp heat kill hosts introduce fresh hosts growth with amp amp

2. Evolution à la Mode Bull et al. found that phage evolved under vertical transmission were more benevolent to their host– the growth period of infected host was significantly shorter. The phage in the vertical transmission treatment reduced its genome drastically. Further study revealed that changes in both the host and the phage were responsible for the benevolent coexistence under vertical transmission. HorizontalVertical infected host growth period (min) H H P H PP t0t0 t1t1 H P H H P Take 3 minutes to talk to your neighbor about the following: How might you experimentally test how a “vertically evolved” phage competes against a “horizontally evolved” phage? What competition conditions would be interesting to vary? (Hint: think of conditions that would favor benevolence or virulence– e.g., host density, host susceptibility, etc.)

3. The Evolution of Virulence Lecture Outline Introduction to virulence theory Transmission mode experiment Transmission timing experiment Metapopulation experiment Summary

4. The Case of the Gypsy Moth Etienne Leopold Trouvelot, an amateur entomologist, brought eggs of the Gypsy moth back from Europe in the 1860’s. Trouvelot soon noticed that some of the larvae from his backyard had escaped into nearby woods. Within two decades, the first local outbreak of gypsy moth occurred. Preventative measures (such as removal of egg masses and burning infested vegetation) were attempted and eventually abandoned. Currently, the gypsy moth has spread over a good portion of the northeast region of the US. Trouvelot Trouvelot’s house in Medford Gypsy moth removing eggs burning vegetation Gypsy moth range (circa 2007)

5. A Weapon against the Gypsy Moth A viral agent, nuclear polyhedrosis virus, has been used to control high density outbreaks of the gypsy moth– earning the virus the nickname “Gypcheck” The virus enters the catepillar through the gut (when it consumes infected vegetation) and a few weeks later ruptures the catepillar’s cells, releasing the virus into the environment. Here we have a system that is extremely important from a management perspective; however, we also have the opportunity to explore evolutionary phenomena (e.g., evolution of virulence) within this insect-virus community. host pathogen aspen killed by gyspy moth healthy catepillar infected catepillar

6. Experimental Manipulation of Timing Cooper et al. (2003) performed an experiment in which the timing of transmission of NPV between catepillars was manipulated. In an Early treatment, the virus was transferred from infected living larvae to uninfected larvae after 5 days. In a Late treatment, the virus was transferred from infected living larvae to uninfected larvae after 9 days. Vaughn CooperPaul Ewald Early Treatment Late Treatment inclusion body uninfected larvae infection incubation (5 days) separation incubation (9 days) separation initiate next cycle

7. The Importance of Timing (Scenario 1) Cooper et al. found that virus transmitted early evolved relatively higher virulence (higher mortality of the larval host) than virus transmitted late. Let us assume that more virulent virus mutants can out-compete less virulent viral strains within the host: - Scenario 1: Virulent mutants that kill their host between days 5 and 9 are selected against in the Late treatment Early Treatment Late Treatment only less virulent virus is available for next transfer… more virulent virus is available for next transfer…

8. The Importance of Timing (Scenario 2) Cooper et al. found that virus transmitted early evolved relatively higher virulence (higher mortality of the larval host) than virus transmitted late. Let us assume that more virulent virus mutants can out-compete less virulent viral strains within the host: - Scenario 1: Virulent mutants that kill their host between days 5 and 9 are selected against in the Late treatment - Scenario 2: More benevolent mutants (e.g., that exhibit restraint in use of host resources) experience a founder event (due to the viral bottleneck) and then inhabit a host that is more likely to live 9 days in the Late treatment. Early Treatment Late Treatment more benevolent virus is more likely to be transferred… more benevolent virus could be picked…

9. The Importance of Timing - Scenario 1: Virulent mutants that kill their host between days 5 and 9 are selected against in the Late treatment - Scenario 2: More benevolent mutants (e.g., that exhibit restraint in use of host resources) experience a founder event (due to the viral bottleneck) and then inhabit a host that is more likely to live 9 days in the Late treatment. After several cycles of selection, virus from the Early treatment was significantly more virulent than virus from the Late treatment, but not significantly different from the ancestor. This suggests that Scenario 2 played a part (note that the scenarios are not mutually exclusive). EarlyLate Ancestor

10. Resource Management by a Pathogen Cooper et al. found that while the (more virulent) Early virus was more productive after 5 days, the (less virulent) Late virus was more productive after 9 days. This suggests a tradeoff between short-term and long-term productivity. More virulent virus might compete better and produce more in the short- term, but (due to quick consumption of its host) its long-term prospects are not so pleasant. Less virulent virus employs a prudent strategy with regards to its host’s resources– this may favor better long-term exploitation (in terms of host survival and viral yield). If the more virulent type is a better competitor, then the virus is playing a “tragedy of the commons” Early Late dead alive more virulent ancestral more benevolent