Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

Activity of Steinernema colombiense in plant-based oils.

Entomopathogenic nematodes (EPNs) are excellent biological control agents. Although traditionally EPN application targeted belowground insects, their aboveground use can be supported if combined with adjuvants. We hypothesized that EPN infective juveniles (IJs) could be combined with plant-based oils as adjuvants, without decreasing their efficacy against insect larvae under various scenarios. Specifically, our objectives were to evaluate the activity of Steinernema colombiense (Nematoda: Steinernematidae) when mixed with two plant-based oils (coconut and olive oils) and maintained at different temperatures and times, or combined with entomopathogenic fungi. First, we evaluated how these oils affected IJ survival and virulence against last instar Galleria mellonella (Lepidoptera: Pyralidae) larvae when maintained at five different temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days), using water as control treatment. Second, we evaluated virulence when combined with these two oils as well as with water (control) and combined with the entomopathogenic fungi (EPF), Beauveria bassiana (Hypocreales: Clavicipitaceae). Infective juvenile survival was higher in coconut than olive oil and water mixtures up to 7 days at 4°C. Conversely, olive oil supported higher larval mortality than coconut oil at 4 to 20°C and 14 days. Similarly, the number of days needed to kill insect larvae increased at extreme temperatures (4 and 24°C) after 14 days. Finally, the EPN + EPF combination showed an additive effect compared to EPN and EPF single treatments. Our findings indicate that our plant-based oil mixtures maintain viable IJs at moderate temperatures and up to 7 to 14 days, and can be used in single EPN mixtures or combined with EPF.Entomopathogenic nematodes (EPNs) are excellent biological control agents. Although traditionally EPN application targeted belowground insects, their aboveground use can be supported if combined with adjuvants. We hypothesized that EPN infective juveniles (IJs) could be combined with plant-based oils as adjuvants, without decreasing their efficacy against insect larvae under various scenarios. Specifically, our objectives were to evaluate the activity of Steinernema colombiense (Nematoda: Steinernematidae) when mixed with two plant-based oils (coconut and olive oils) and maintained at different temperatures and times, or combined with entomopathogenic fungi. First, we evaluated how these oils affected IJ survival and virulence against last instar Galleria mellonella (Lepidoptera: Pyralidae) larvae when maintained at five different temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days), using water as control treatment. Second, we evaluated virulence when combined with these two oils as well as with water (control) and combined with the entomopathogenic fungi (EPF), Beauveria bassiana (Hypocreales: Clavicipitaceae). Infective juvenile survival was higher in coconut than olive oil and water mixtures up to 7 days at 4°C. Conversely, olive oil supported higher larval mortality than coconut oil at 4 to 20°C and 14 days. Similarly, the number of days needed to kill insect larvae increased at extreme temperatures (4 and 24°C) after 14 days. Finally, the EPN + EPF combination showed an additive effect compared to EPN and EPF single treatments. Our findings indicate that our plant-based oil mixtures maintain viable IJs at moderate temperatures and up to 7 to 14 days, and can be used in single EPN mixtures or combined with EPF.

Patterns of Occurrence and Activity of Entomopathogenic Fungi in the Algarve (Portugal) Using Different Isolation Methods.

Entomopathogenic fungi (EPF) are distributed in natural and agricultural soils worldwide. To investigate EPF occurrence in different botanical habitats and soil-ecoregions, we surveyed 50 georeferenced localities in the spring of 2016 across the Algarve region (South Portugal). Additionally, we compared three EPF isolation methods: insect baiting in untreated or pre-dried-soil and soil dilution plating on a selective medium. We hypothesized that forest habitats (oak and pine semi-natural areas) and the acidic soil ecoregion may favor EPF occurrence. Overall, EPF species were present in 68% of sites, widely distributed throughout the Algarve. The use of selective media resulted in higher recovery of EPF than did either soil-baiting method. Contrary to our hypothesis, neither vegetation type nor ecoregion appeared to influence EPF occurrence. Traditional and molecular methods confirmed the presence of five EPF species. Beauveria bassiana (34% of sites), was the most frequently detected EPF, using pre-dried soil baiting and soil dilution methods. However, baiting untreated soil recovered Fusarium solani more frequently (26% of sites), demonstrating the utility of using multiple isolation methods. We also found Fusarium oxysporum, Purpureocillium lilacinum and Metarhizium anisopliae in 14%, 8% and 2% of the sites, respectively. Three abiotic variables (pH, soil organic matter and Mg) explained 96% of the variability of the entomopathogen community (EPF and entomopathogenic nematodes) in a canonical correspondence analysis, confirming the congruence of the soil properties that drive the assemblage of both entomopathogens. This study expands the knowledge of EPF distribution in natural and cultivated Mediterranean habitats.

Scavenging behavior and interspecific competition decrease offspring fitness of the entomopathogenic nematode Steinernema feltiae.

Entomopathogenic nematodes (EPNs) are well-studied biocontrol agents of soil-dwelling arthropod pests. The insecticidal efficiency of EPNs is modulated by food web dynamics. EPNs can reproduce in freeze-killed insect larvae, even in competition with free-living bacterivorous nematodes (FLBNs) in the genus Oscheius. The objective of this study was to assess the efficiency of EPNs as scavengers when competing with free-living saprophagous nematodes and fungi, and to determine the possible impact on subsequent EPN offspring fitness. Live and freeze-killed larvae of Galleria mellonella were used to evaluate the reproduction rate and progeny fitness of two EPN species, Heterorhabditis bacteriophora and Steinernema feltiae, applied individually or combined with the FLBN species Oscheius onirici or Pristionchus maupasi, or Aspergillus flavus, an opportunistic saprophytic fungus. We hypothesized that (1) EPN scavenging behaviors previously observed (for H. megidis and S. kraussei) apply to other EPN species, (2) infective juveniles (IJs) emerging from freeze-killed larvae will display reduced pathogenicity and reproduction, and (3) fitness reduction will be amplified by exposure to other organisms competing for the resources. The reproduction rate of S. feltiae was lower in freeze-killed larvae than in larvae infected and killed by the nematode, whereas H. bacteriophora failed to reproduce as a scavenger. The S. feltiae F1 IJs that emerged from freeze-killed larvae exhibited lower pathogenicity rates than IJs resulting from entomopathogenic activity, and also lower reproductive rates if they experienced high FLBN competitive pressure during development. This study illustrates that scavenging is a suboptimal alternative pathway for EPNs, especially in the face of scavenger competition, even though it provides a means for some EPN species to complete their life-cycle.

Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control.

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.

Reproductive efficiency of entomopathogenic nematodes as scavengers. Are they able to fight for insect's cadavers?

Entomopathogenic nematodes (EPNs) and their bacterial partners are well-studied insect pathogens, and their persistence in soils is one of the key parameters for successful use as biological control agents in agroecosystems. Free-living bacteriophagous nematodes (FLBNs) in the genus Oscheius, often found in soils, can interfere in EPN reproduction when exposed to live insect larvae. Both groups of nematodes can act as facultative scavengers as a survival strategy. Our hypothesis was that EPNs will reproduce in insect cadavers under FLBN presence, but their reproductive capacity will be severely limited when competing with other scavengers for the same niche. We explored the outcome of EPN - Oscheius interaction by using freeze-killed larvae of Galleria mellonella. The differential reproduction ability of two EPN species (Steinernema kraussei and Heterorhabditis megidis), single applied or combined with two FLBNs (Oscheius onirici or Oscheius tipulae), was evaluated under two different infective juvenile (IJ) pressure: low (3IJs/host) and high (20IJs/host). EPNs were able to reproduce in insect cadavers even in the presence of potential scavenger competitors, although EPN progeny was lower than that recorded in live larvae. Hence, when a highly susceptible host is available, exploiting cadavers by EPN might limit the adaptive advantage conferred by the bacteria partner, and might result in an important trade-off on long-term persistence. Contrary to our hypothesis, for most of the combinations, there were not evidences of competitive relationship between both groups of nematodes in freeze-killed larvae, probably because their interactions are subject to interference by the microbial growth inside the dead host. Indeed, evidences of possible beneficial effect of FLBN presence were observed in certain EPN-FLBN treatments compared with single EPN exposure, highlighting the species-specific and context dependency of these multitrophic interactions occurring in the soil.