Introduction Among the diversity of insect-parasitic nematodes, entomopathogenic nematodes (EPNs) are distinct, cooperating with insect-pathogenic bacteria to kill insect hosts. EPNs have adapted specific mechanisms to associate with and transmit bacteria to insect hosts. New discoveries have expanded this guild of nematodes and refine our understanding of the nature and evolution of insect–nematode associations. Here, we clarify the meaning of “entomopathogenic” in nematology and argue that EPNs must rapidly kill their hosts with the aid of bacterial partners and must pass on the associated bacteria to future generations. Strangers, Acquaintances, and Enemies Nematode–arthropod associations are plentiful and range from beneficial to antagonistic [1], [2]. These associations have been divided into at least four categories: 1) phoretic (nematodes are transported by an insect), 2) necromenic (nematodes obtain nutrition from insect cadavers), 3) facultative parasitism, and 4) obligate parasitism (see Sudhaus 2008 for a more detailed breakdown [3]). It is thought that insect parasitism evolves in this sequence, with parasites evolving from non-parasitic insect associates (Figure 1A) [1], [3]. Nematodes also interact with bacteria in at least three ways: 1) trophism (nematodes eat bacteria), 2) parasitism (pathogens cause nematode diseases if not resisted), and 3) mutualism (nematodes and bacteria cooperate). Here, we consider entomopathogenic nematodes, which employ bacteria to kill insects. 10.1371/journal.ppat.1002527.g001 Figure 1 Evolution of nematode–insect associations and the entomopathogenic nematode life cycle. (A) The evolution of nematode–insect associations. Free-living: microbotrophic nematodes not known to associate with arthropods, vertebrates, plants, or fungi; only perhaps transiently associated with insects. Phoresy: a relationship where nematodes are adapted to use insects for dispersal or shelter but have no direct nutritional relationship to them. Necromeny: a relationship where nematodes are adapted to use saprophytic insect cadavers as a food resource but do not participate in insect death. Parasitism: a relationship where nematodes are adapted to use living insects directly for nutrition, likely inflicting some level of harm or even causing eventual death of the host. Entomopathogeny: a relationship where nematodes cooperate with insect-pathogenic bacteria to cause rapid insect disease and death and then feed and develop on the insect and bacterial resources. The distinction between parasitism and entomopathogeny is based on salient features including use of pathogenic bacteria and direction of selection (against virulence or avirulence), either making the nematodes more or less immediately harmful to their host. (B) The life cycle of entomopathogenic nematodes. The IJ stage is a developmentally arrested third larval stage and is the only free-living stage; all other stages exist exclusively within the host. EPN IJs carry symbiotic bacteria and search for potential insect hosts. They enter a host, gain access to the hemolymph, and release their bacterial symbiont. The symbiont plays a critical role in overcoming host immunity. The nematodes develop and reproduce in the resulting nutrient-rich environment until population density is high and resources begin to deplete, at which point new IJs develop and disperse, carrying the symbiotic bacteria to new hosts [5]. Entomopathogenic Nematodes The term entomopathogenic is widely used in parasitology and pathology, usually referring “to microorganisms and viruses capable of causing disease in an insect host” [4]. Nematodes in Steinernematidae and Heterorhabditidae associate with pathogenic bacteria to kill insect hosts, usually within 48 hours of infection. The hallmarks of this specific type of parasitism by nematodes, known as entomopathogeny, are 1) carriage of pathogenic bacteria by infective juvenile (IJ) nematodes (also known as dauer juveniles); 2) active host-seeking and -penetration by IJs; 3) release of the bacteria into the insect hemolymph; 4) death of the insect, and nematode reproduction and bacterial proliferation driven by cadaver-nutrient utilization; 5) reassociation of the pathogenic bacteria with new generations of IJs; and 6) emergence of IJs from the nutrient-depleted cadaver as they search for new insect hosts (Figure 1B) [5], [6]. Nematode parasites of this kind are known as EPNs. Recently, other nematode species have been shown to use pathogenic bacteria to parasitize insect hosts. Two Oscheius species, Oscheius chongmingensis and Oscheius carolinensis, and Caenorhabditis briggsae have been identified as potential insect pathogens by baiting soil for nematodes using insect larvae as prey, a common approach used for finding EPNs [7]–[11]. All of these have been found to associate with insect pathogenic bacteria of the genus Serratia, while O. carolinensis may have additional associates [9]–[12]. O. chongmingensis and C. briggsae require their bacterial partners to cause host death, and to grow and reproduce within killed insects, and emerging dauer juveniles are associated with the vectored pathogen [10], [11]. Ongoing studies suggest that these species are EPNs, though their classification as entomopathogens has been contested both semantically and conceptually in the literature and scientific meetings (e.g., the November 2010 NemaSym NSF RCN meeting and the July 2011 Society of Nematologists meeting) [13]–[15]. History, Context, and Formal Criteria The term entomopathogenic first appeared in the nematology literature in reference to the bacterial symbionts of Steinernema and Heterorhabditis [16]. Bacteria are considered entomopathogenic when their LD50 is 95% to ∼10% [35], [36]. Together, these findings reveal that Steinernema and Heterorhabditis are highly adapted to entomopathogeny and showcase adaptations likely to emerge as a result of long-term commitment to the entomopathogenic lifestyle, even though the biological basis for their symbiotic association with bacteria differs significantly [5], [37]. The exceptions and differences that have been observed for all of these hallmark characteristics highlight why specializations should not be used to exclude newly described associations, and emphasize that applying observations from a few representative members to whole clades can be problematic. Indeed, few species in either genus have been thoroughly explored, and we caution against assuming a priori these specializations to be true of all or even most steinernematids or heterorhabditids (e.g., [38]). Classification of Newly Described Associations According to the standards we propose above, C. briggsae may not be an EPN. IJs recovered from dead insects seem able to reinfect new hosts but are less virulent in G. mellonella as a complex than injection of the bacteria alone, suggesting either inefficient release of the pathogen or some antagonism by the nematode vector. This may reflect that C. briggsae is somewhere between necromenic and entomopathogenic, that it is a nascent entomopathogen and not yet efficient, or that G. mellonella is a poor host. However, symbiont heritability has not been demonstrated, and the nature of C. briggsae's bacterial association remains unresolved [10], [11], [39]. Because C. briggsae has not met the suggested criteria, it should not be considered an EPN, facultative or otherwise, until heritability of the pathogenic bacteria is demonstrated and more is known about bacterial release and speed of host death. Our suggested criteria have been tested and met for both O. chongmingensis and O. carolinensis [9], [10], [12]. Therefore, these taxa should be considered EPNs even though further research is required to determine the nature and heritability of their bacterial associations, and whether they are obligate or facultative EPNs. Symbiosis and Entomopathogeny Nematode–bacterium partnerships that do not explicitly fulfill the requirements to be classified as EPNs are still of extraordinary interest since they may represent developing, nascent partnerships, but they should not be considered entomopathogens. Our understanding of parasitism and its evolution is continually refined as biodiversity is explored and ecology and evolution become increasingly emphasized among established and satellite model systems. We have suggested specific and restricted use of the term entomopathogenic in nematology, which will facilitate unambiguous communication. Among the 20 or more parasitic lineages of nematodes, entomopathogeny is a unique type of insect parasitism not found among vertebrate- or plant-parasitic nematodes. Recent work indicates that entomopathogeny has arisen at least three times within Nematoda, and that recently described species (O. chongmingensis and O. carolinensis) may represent nascent stages of EPN evolution. These developments emphasize the tremendous specialization exhibited by Heterorhabditis and Steinernema and increase their usefulness as models for the evolution of symbiosis and parasitism.
