Snyder 1. Matthew R. Snyder Biol. 862: Microbially Mediated Species Interactions May 18 th, 2011

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1 Snyder 1 Matthew R. Snyder Biol. 862: Microbially Mediated Species Interactions May 18 th, 2011 Symbiont-Mediated Resistance in Insects; An Annotated Bibliography of Research Concerning Microbially Mediated Defense Against Parasites, Parasitoids, & Pathogens in the Class Insecta Dion, E., F. Zele, J.-C. Simon, and Y. Outreman. (2011). Rapid Evolution of Parasitoids When Faced with the Symbiont-Mediated Resistance of their Hosts. Journal of Evolutionary Biology. No. 24, pp European Society for Evolutionary Biology. doi: /j x In this study, researchers studied the evolutionary effects on parasitoids of symbiont-mediated resistance by Acyrthosiphon pisum to the parasitoid, Aphidis ervi. They were able to take advantage of the parthenogenic life cycle of the aphid, allowing them to effectively rule out host evolution as the factor effecting results. They performed several experiments to determine what evolutionary effects arose and the level of selection efficiency. The researchers established lineages of aphids infected or not with Hamiltonian defensa (hereafter just SS because it is the only secondary symbiont in this study), and assayed the effect of infection on parasitoid resistance. After finding a significant effect they began a ten-generation experiment in which every generation of parasitoids was allowed to parasitize the different aphid treatment groups, and after each generation, the parasitoid s size and effective parasitism rate were measured. During this experiment, wasps were also split into groups that infected only infected or uninfected aphids for this experiment, but afterwards, a third experiment was conducted in which wasps from both groups were allowed to parasitize two clonal lineages of SS infected aphids to determine if the ability to use infected with SS aphids evolved over the course of the experiment. After only three generations, the effective parasitism rate among aphids infected with SS and those not infected became identical. And so it is not unexpected that researchers observed an increase in the ability for SS exposed wasps (exposed to SS infected aphids throughout the preceding experiment) to effectively parasitize a group of SS infected aphids that had not been subject to ten generations of parasitism. This shows that selection had a fairly high efficiency, occurring over ten generations or possibly fewer. No significant difference in parasitoid size was observed between the two treatments. Ferrari, Julia, Alistair C. Darby, Tim J. Daniell, H. Charles J. Godfray, and Angela E. Douglas. (2004). Linking the Bacterial Community in Pea Aphids with Host-Plant Use and Natural Enemy Resistance. Ecological Entomology, No. 29, PP C. The Royal Entomological Society.

2 Snyder 2 This very in depth paper, details a study that is in fact the combination of several experiments, and so the paper deserves a longer explanation of its content. Furthermore, specific methods only mentioned in other entries are explained in more detail here. In this paper, potential for predator and parasite resistance in the pea aphid, Acyrthosiphon pisum, harboring facultative bacteria was examined experimentally. 47 clonal lineages of aphids were used in treatments examining pea aphid traits. Several pea aphid traits were examined but only those relevant to the scope of this annotated bibliography, microbially mediated defense in insects, are mentioned here. The bacterial community in each of the aphids used in each treatment was assessed using terminal restriction fragment length polymorphism (T-RFLP). Using the Polymerase Chain Reaction, PCR, a portion of the 16S rrna was amplified from the gut contents of the pea aphids using universal primers. A terminal restriction enzyme was used to digest the amplified samples and the lengths of the fragments created were analyzed with an automatic sequencer, and verified by gel electrophoresis for a small number of the samples. Previous experiments have shown that this method can distinguish the common pea aphid symbionts, Burchnera, PASS(R), PAR, PABS(T), and PAUS(U) (as well as spiroplasma but in this study, due to very small amounts of spiroplasma present in the samples, this symbiont was disregarded). Aphidius ervi Haliday (here on A. ervi) parasitizes A. Pisum by ovipositing in the aphid nymph. The aphid eventually mummifies, becomes attached to the substrate, and a developed wasp emerges. The mechanism by which some aphids resist parasitism is unknown. Several replicate groups of 30 aphid nymphs each were exposed to a single wasp for 9 hours. The susceptibility was defined as the number of mummified aphids in the group after 14 days. Aphidius eadyi Stary, Gonzalez & Hall (here on A. eadyi) has a very similar method of parasitism, but aphid resistance is more complicated in this case. Some aphids mummify (no resistance) while other aphids either survive (completely resistant) or survive but show greatly reduced fecundity (partially resistant). However, the completely resistant and partially resistant clones were pooled, and so the analysis of susceptibility to A. eadyi was ultimately the same as that which was used for A. ervi. Finally, pea aphid resistance to the fungal pathogen, Pandora (Erynia) neoaphidis Humber (here on P. neoaphidis), was examined. P. neoaphidis infects adult aphids, causing them to mummify. Eventually the mummies burst during fungal sporolation. 20 adult aphids were exposed to fungal spores from two sporalating mummies for 90 minutes, and the % of individuals who exhibited sporolation within a 6-day period was used as a measure of susceptibility. Presence of PABS(T) was significantly associated with the resistance to parasitism by A. ervi but not when controlled for the non-significant effect of PAUS(U) and PASS(R) on resistance. Therefore, it can be determined only that the presence of accessory bacteria in general confers resistance but exactly which bacteria(um) has the greatest effect cannot be determined from this data. Resistance to A. eadyi was significantly associated with the presence of PABS(T), and the statistical significance of this result greatly increased when the effect of the aphid s host plant (one of the traits examined but not elaborated upon here) was controlled for. PAUS(U) was also found to be slightly positively correlated with resistance. Adding

3 Snyder 3 PABS(T) to this regression model greatly improved the fit of the model. However, since the opposite did not improve the predictive power of the PABS(T) model, in this case, PABS(T) can be identified as the bacteria which is most important in pea aphid resistance to attack by A. ealyi. Lastly, the data concerning fungal pathogen resistance is more confusing than enlightening. The data had to be analyzed as two separate experiments, one sample in 1999 and the other in In the 1999 group, all aphids from one host plant had full resistance and all possessed PAUS(U), but aphids from another host plant had no associated bacteria but still showed a variation in resistance. The 1998 group were from a different host plant not specified in the paper, but they did show a significant relationship between presence of PAUS(U) and resistance to the fungal pathogen. Given that the two experiments used samples of presumably different aphid lineages, which, it can be assumed, have some variation in genotype, and it is this effect of genotype that may be producing the confounding results. This is hinted at in the discussion. However, given that PAUS(U) was shown in both experiments to confer at least a conditional resistance to P. neoaphidis, further investigation of the compound effects of PAUS(U) and genotype is warranted. John Jaenike, Robert Unckless, Sarah N. Cockburn, Lisa M. Boelio, and Steve J. Perlman. (2010) Adaptation Via Symbiosis: Recent Spread of a Drosophila Defensive Symbiont. Science 329, 212. DOI: /science This study showed that spiroplasma bacteria are able to increase resistance, to some degree, to a nematode in Drosophila neotestacea. Unlike a lot of the studies out on parasite resistance via symbiosis, this study actually seems to at least partially nail down a mechanism by which the symbiont achieves this benefit to the host. It appears that spiroplasma infection results in a severely reduced size of the mother worm which infects the fly, which translates into many times fewer nematode offspring. Some genomic work presented in the study also attempts to shed light on the ecological story behind the spread of this beneficial symbiont. The authors claim that spiroplasma infection has been relatively stable in eastern US populations for some time, and has only recently started spreading west due to a preceding westward expansion of the nematode infection. This benefit, which outweighs the cost of slightly reduced fecundity as a result of spiroplasma infection, is a necessary precursor to equilibrium levels of infection within a D. neotestacea populations. Oliver, Kerry M, Jacob A. Russell, Nancy A. Moran, and Martha S. Hunter. (2003) Facultative Bacterial Symbionts in Aphids Confer Resistance to Parasitic Wasps. PNAS. Vol. 100, No. 4, pp February 18 th, This paper was the first to be published on the effects of secondary symbionts (SS) on parasitism of Acyrthosiphon pisum, the pea aphid, by wasps, Aphidis ervi in this

4 Snyder 4 case. This paper is also credited in many reviews as being the study in which the term symbiont-mediated resistance was coined. They take advantage of the parthenogenic life cycle of the aphid, showing the effects of resistance while being able to control any extraneous variability of results due to genetic variation among hosts. In this study they used one clonal lineage that was uninfected, split them into treatment groups and then injected them with aphid bodily fluids from donors either infected with one of three SS or not infected. They chose this method because any method using antibiotics to create control groups might remove other beneficial symbionts within the aphids, causing extraneous variability in results. For reasons unstated, the author is not concerned with this effect in later studies. The study shows that indeed infection of the aphid host by some SS does increase aphid resistance to their natural parasitoid. Sarratia symbiotica (R-Type or PASS) and Hamitonela defensa (T-Type or PABS) both confer resistance, while a third symbiont called U-type PUAS. The study also establishes that SS infection had no effect on the number of successful wasp ovipositions within a group of aphids, and so, narrowing the focus of any investigation Oliver, Kerry M., Nancy A. Moran, Martha S. Hunter (2005). Variation in Resistance to Parasitism in Aphids Is Due to Symbionts Not Host Genotype. Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 36 (Sep. 6, 2005), pp Published by: National Academy of Sciences Stable URL: Accessed: 04/04/ :48 The prior consensus, almost by default, was that variation in pea aphid, Acyrthosiphon pisum, resistance to parasitism by the wasp, Aphidus ervi, was due to variation in genotypes. Previous publications by this author had shown that two aphid secondary symbionts, PABS and PASS, confer partial resistance to attack by A. ervi. In this paper, the author examined multiple isolates of PABS, Hamiltonela defensa, (multiple genetic backgrounds) in a common aphid genetic background, as well as a single symbiont isolate in multiple aphid genetic backgrounds. Essentially he is trying to tease out the effects of varying genetic backgrounds amongst symbiont and host on the expression of resistance to A, ervi parasitism. All told, aphid genetic background had no statistically significant effect on resistance, but the symbiont isolate had a very large effect on the level of resistance conferred. However, the differences between symbiont isolates pale in comparison to the difference between aphids that are infected vs. uninfected with H. defensa. Therefore, variation in aphid resistance to this type of parasitism is most likely the result of heterogeneity of symbiont genotype. Oliver speculates that this heterogeneity may be due to the known bacteriophage associated with H. defensa. Oliver, Kerry M., Nancy A. Moran, Martha S. Hunter (2006). Source Costs and Benefits of a Superinfection of Facultative Symbionts in Aphids. Proceedings: Biological Sciences, Vol. 273, No (May 22, 2006), pp.

5 Snyder Published by: The Royal Society. Accessed: 04/04/ This study was published after the more comprehensive study by the same authors, Variation in Resistance to Parasitism in Aphids Is Due to Symbionts Not Host Genotype, but it seems more like a follow up to their first study. In their first investigation of aphid symbiont-mediated resistance to parasitoids, they found that two secondary symbionts (SS), the R-type and T-type, both conferred resistance to parasitism, and apparently did so by causing mortality of the wasp larva within the aphid host. In this study they create lineages with SS super infection, meaning the host possesses both SS, and assay them for resistance to the same parasitoid. They do find that super infection confers a level of resistance that is even greater than that achieved via infection by only one SS; however, screening of wild populations (as wild as an invasive species taken from alfalfa crops in Utah can be) of the same aphid species, using simple Diagnostic PCR showed that superinfection is very rare at least in the wild populations they sampled. The researchers also sent samples of aphids with one, both, or no SS of to a lab for Quantitative PCR analysis, combined with a melting curve analysis, to determine the identity and relative abundances of SS within uninfected and (super)infected aphids. Interestingly, they found that S. symbiotica densities within superinfected individuals increased dramatically. Given these results, they attributed an observed reduction in fecundity among superinfected individuals to interactions between multiple SS, and propose that such fitness reducing interactions may be the reason for the low observed incidence of superinfection in wild populations despite increased symbiont-mediated resistance. Osborne SE, Leong YS, O Neill SL, Johnson KN Variation in Antiviral Protection Mediated by Different Wolbachia Strains in Drosophila simulans. Accessed April 1 st, PLoS Pathog 5(11): e doi: /journal.ppat In this paper, researchers perform several experiments investigate whether certain Wolbachia strains, known to reduce viral loads and protect their host, Drosophila melanogaster, from mortality induced by Drosophila C virus (DCV) and Flock House virus (FHV), can provide the same benefits to Drosophila simulans. The researchers find that only certain Wolbachia strains, all from a clade of Wolbachia lineages called supergroup A, confer resistance to mortality induced by DCV or FHV. Interestingly, one Wolbachia strain that only delays rather than eliminates mortality, does so without any reduction in the viral accumulation compared to control flies. Each individual strain of Wolbachia has similar patterns of effects on mortality and viral load when the host is infected with either DCV or FHV. In each experiment, the D. simulans lab line is either naturally infected with a given Wolbachia strain or is artificially infected and assays are performed to ensure the infection has reached an equilibrium state. The uninfectedwith-wolbachia controls used in each experiment are derived by curing some of the infected flies of the Wolbachia infection.

6 Snyder 6 Scarborough, Clair L., Julia Ferrari, H. C. J. Godfray. (2005) Aphid Protected from Pathogen by Endosymbiont. Brevia. Science. Vol December 16 th, doi: /science Previous studies of the three most common secondary symbionts (SS) of Acyrthosiphon pisum, the pea aphid, have found that the R-type and T-type confer resistance to parasitic wasps, but the U-type SS had not been previously found to result in any fitness benefits for the host, making the reason for its persistence among aphid populations a mystery. This study comes after a very comprehensive study by Ferrari et al, 2004, which investigated the potential for aphid SS to confer resistance to fungal infection, but for reasons elaborated upon in the annotation for that study, the data collected fell short of providing any clear insight into U-type SS infection benefits. This study, which shares several authors with the formerly mentioned study, clears up this issue, showing that infection with U-type, Regiella insecticola, certainly does provide symbiont-mediated resistance to the parasitic fungus, Pandora (Erynia) neoaphidis. A relatively simple experiment was conducted in which infected and uninfected with U- type SS aphids were exposed to P.neoaphidis infection using the same methods of creating genetically identical infected and uninfected aphid lineages found in Ferrari et al, While successful fungal infection was not reduced, sporolation in the infected aphids was dramatically reduced among U-type infected aphids. This corresponds to a slightly lower but still significantly reduced mortality among U-type infected aphids. Given that P. neoaphidis is one a very common fungal pathogen among many aphid species, these results seem to provide ample evidence that persistence of the U-type in similar frequencies as T&R-types within aphid populations is likely due to increased fitness as a result of symbiont-mediated resistance. Teixeira L, Ferreira A, Ashburner M (2008) The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biol 6(12): e doi: /journal.pbio In this experiment researchers investigated the ability of Wolbachia to confer resistance to several viruses that infect Drosophila melanogaster. They found that wolbachia infection, at least by certain strains, confers resistance to the RNA viruses Drosohila C. Virus, Nora Virus and Flock House Virus. However, wolbachia had no effect on a considerably more rare DNA virus called Insect Iridescent Virus 6. Seems to me they did a lot of really in depth work, wrote a paper that is really hard to understand, and in the end these fairly simple results are what s really important here. They added to the growing body of research shedding light on why wolbachia is so common among all insects. They also showed with their controls that the method of creating an infected isoline, treating some animals with antibiotics, and subjecting the groups to a viral treatment is an effective way to study these phenomenon while ruling out variable genetic backgrounds as the factor effecting results.

7 Snyder 7 Xie J, Vilchez I, Mateos M (2010) Spiroplasma Bacteria Enhance Survival of Drosophila hydei Attacked by the Parasitic Wasp Leptopilina heterotoma. PLoS ONE 5(8): e doi: /journal.pone In this relatively simple study, researchers compared successful parasitism by the parasitoid wasp, Leptopilina heterotoma, in Drosophila hydie that were either uninfected or artificially infected with spiroplasma, a small bacteria from the wall-less group called mollicutes. They found that spiroplasma infection resulted in a dramatic increase in fly survival after successful wasp oviposition. Given that there was no observed drop in the number of successful oviposition, the resistance to this parasitoid that is gained from spiroplasma infection must be the result of a mechanism that takes place after successful wasp attack. This benefit could explain why spiroplasma is persistent in many Drosophila populations, but more investigation in this area will probably follow these very descriptive, recent, and preliminary results.