John Chaston The Secondary Phase of Photorhabdus luminescens TT01 Is Closely Tied to the Sexual Form of Heterorhabditis bacteriophora Byron Adams, Microbiology and Molecular Biology Heterorhabditis bacteriophora and Photorhabdus luminescens are a symbiotically associated nematode/bacterium pair that act together as potent insect killers and have been cultured en masse for distribution as an insect control agent in agricultural settings. Heterorhabditis is a nematode species that acts as a vector to penetrate the insect exoskeleton through existing openings where it subsequently releases Photorhabdus, the bacterial symbiont that it carries in its gut, into the haemocoel of the invaded insect. The bacteria produce a variety of proteases and toxins that digest the insides of the insect into nutrients they use as they rapidly replicate. The nematode then feeds on the proliferating bacteria until all available nutrients are used up. In an unknown process the nematode eventually “saves” some of the bacteria, upon which it has been feeding, in its gut and leaves the nutrient depleted cadaver in search of a new insect host. My original intent was to generate mutant bacteria by using a special protein/DNA complex called a transposase that would randomly integrate itself into the bacterial genome and prevent proper expression of any gene it interrupted in the process. These mutants would be subsequently screened for symbiotic ability by exposing sterile nematode eggs to a monoculture of the mutant bacteria; however, the protocol that I used did not work with this taxa of bacteria, and I was unable to generate any mutants. My proof-of-concept work performing symbiosis assays was quite instructive, however, and by carefully noting interactions between the nematode and bacterium under normal conditions I designed several assays that allowed me to propose a mechanism for the interaction between the two organisms. There are several unique features of the Heterorhabditis/Photorhabdus system. First, the nematode Heterorhabditis has a life cycle that involves both a sexual and an asexual life form. The sexual form is only found inside of a parasitized insect host during the process of infection while several generations of reproduction occur inside the insect host. However, it is the asexual form that is released from the host and that subsequently penetrates into a new insect host when found. The life cycle follows a very specific timeline, where the asexual nematode enters and produces asexual progeny. These asexual progeny produce sexual progeny for one generation, and then asexual progeny are produced during several more rounds of reproduction until all available nutrients in the insect cadaver have been exploited. The process by which the nematode “decides” to go through a sexual life cycle for one generation as yet is unknown, but there is a clear evolutionary benefit to the process in that it prevents genetic bottlenecking that would normally occur in an entirely asexual species by allowing for sexual recombination of alleles. Interestingly, this sexual period of reproduction does not occur when the nematode is cultured in vitro. The bacteria also have an interesting dual-life cycle and exist in two specific phases, creatively termed the primary and secondary phases. The primary phase has been classically characterized as the “useful” phase because the asexual form of the nematode (which is the free living form and as such has been subject to more scientific investigation) requires the primary phase of the bacterium for growth and reproduction – in the absence of the bacteria in primary phase the nematode survives for a brief period of time on food stores inside its body and subsequently dies. In the laboratory the primary phase often switches to the secondary phase, thus far observed to be an irreversible process, a phase for which no specific function has been described. My observations regarded the interactions between the two organisms and their different life cycles (asexual vs. sexual and primary vs. secondary). I designed several assays that allowed me to observe the effects of the nematode on bacterial nematode phase switching and the effects of the bacterial phases on nematode growth and maturation. The assays with their results are shown in the table below: The effect of the primary phase of the bacterium on the growth and Primary phase kills and solubilizes sexual form within 48 maturation of both the sexual and asexual forms of the nematode hours while asexual form survives. No maturation of either form observed The effect of the secondary phase of the bacterium on the growth Secondary phase kills and solubilizes asexual form within and maturation of both the sexual and asexual forms of the 48 hours while sexual form survives. No maturation of nematode either form observed The effect of soluble proteins produced by the primary phase of Primary phase proteins kill and solubilize sexual form the bacterium on the growth and maturation of both the sexual and within 48 hours while asexual form survives. No asexual forms of the nematode maturation of either form observed The effect of soluble proteins produced by the secondary phase of Secondary phase proteins kill and solubilize asexual form the bacterium on the growth and maturation of both the sexual and within 48 hours while sexual form survives. No asexual forms of the nematode maturation of either form observed The effect of the sexual from of the nematode on phase switching Sexual form stimulates rapid conversion of primary to of the primary to the secondary phase secondary phase. The effect of the asexual from of the nematode on phase switching Asexual form prolongs the persistence of bacteria in of the primary to the secondary phase primary phase. The effect of gut proteins from the sexual form of the nematode on Sexual form proteins stimulate rapid conversion of primary phase switching of the primary to the secondary phase to secondary phase. The effect of gut proteins from the asexual form of the nematode Asexual form proteins prolong the persistence of bacteria on phase switching of the primary to the secondary phase in primary phase. From these observations I concluded that the secondary phase of Photorhabdus luminescens TT01 was closely tied to the sexual form of the bacteria. In order to confirm this expectation, I infected a number of insects with the nematode/bacterium complex at the same time and extracted bacteria from these cadavers at 12 hour intervals following infection. If the secondary phase were tied to the sexual form of the nematode, it would be expected to be found inside the insect host only during the period during which the sexual form predominates inside the host. As expected, the primary phase was found at all times in the cadaver except during a brief period 4 ½ to 5 days following infection, during which the sexual form of the nematode was present. These observations suggest a reason for the evolutionary preservation of a phase of the bacterium that has classically been described as useless to the nematode. However, as my results above indicate, I was not able to observe the development of the nematode from its juvenile form to a more advanced stage before all nematodes were killed in broth culture. New assays need to be developed that simulate an in vivo system more closely and would therefore more appropriately represent what occurs inside the insect cadaver. My results however, clearly suggest an alternative to the null hypothesis that has prevailed during the last 25 years of work on this system that only the primary phase is required for nematode growth and development. Further investigation of the interaction between the nematode and bacteria will lead to a greater understanding of the mechanism of symbiosis in the field of “pure” science and are important to the applied science of creating a more perfect bioinsecticide.
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