The application of fungal entomopathogens, such as Metarhizium brunneum, has the potential to continue to impact targeted host populations through secondary cycling. In the present study, we examined the horizontal transmission of M. brunneum conidia from conspecific mycosed cadavers or soil on which the cadavers sporulated to Agriotes obscurus beetles. Close contact between the susceptible hosts and sporulating cadavers or contaminated soil was required for increased mortality of hosts and subsequent production of mycosed cadavers.

Wireworms (Coleoptera: Elateridae), the larvae of adult click beetles are subterranean and generalist crop pests that can be difficult to target. Targeting adult beetles, however, may be an effective method to lower wireworm populations. Metarhizium brunneum (Petch) kills click beetles but the mortality rate was expected to vary according to temperature. Using a thermal gradient plate to simulate daily oscillating temperatures for April, May, and June, the effectiveness of M. brunneum strains LRC112 and F52 in causing mortality to Agriotes obscurus (L.) and A. lineatus (L.) beetles was studied. Mortality was fastest in beetles exposed to June temperatures and slowest in those exposed to April temperatures, with differences among beetle species x M. brunneum strain combinations. Warmer temperatures resulted in more rapid mycelial outgrowth and conidiation in beetle cadavers, with only A. obscurus infected with M. brunneum LRC112 attaining near 100% conidiation. The number of degree days required to kill 50% of the beetles (LDD 50 ) was least for A. obscurus infected with M. brunneum LRC112 (176) followed by A. obscurus x M. brunneum F52 (212), A. lineatus x M. brunneum LRC112 (215), and A. lineatus x M. brunneum F52 (292). Hypothetical calculations showed that M. brunneum exposure earlier in the season resulted in a longer LT 50 but the earliest LT 50 calendar date. Later M. brunneum exposure dates resulted in lower LT 50 ’s, but later LT 50 dates. This conceptual work demonstrates the importance of considering daily temperature oscillations, seasonality, and degree days in predicting the efficacy of entomopathogens to manage agricultural pests.

1. Larvae of multiple click beetle species, i.e. wireworms, are important pests of agriculture worldwide, and are generally managed with insecticides. Conceivably, semiochemical-based management tactics that target male beetles and reduce the mating success of females, would reduce the formation of new larvae in the field. 2. Using two of the best studied species, Agriotes obscurus (AO) and A. lineatus (AL), we evaluated the ability of male beetles to find traps that simulate calling female beetles in field plots treated with various formulations and densities of pheromone-treated substrates. Four disorientation studies were conducted, and the response of wild and marked-released beetles inferred from frequent trap collections. 3. Beetle responses differed between male AO, female AO, and male AL. The presence of AO pheromone increased male AO movements, reduced captures in baited traps, and attracted wild AO beetles into the plots. By the final experiment, the pheromone-treated substrate effectively disoriented male AO for > 17 d. However, treatment with AL pheromone reduced male AO movements and/or repelled them from the plots. Female AO were slightly attracted to their own pheromone early in the season, but not thereafter. Treatment with AL pheromone attracted male AL into plots but did not increase their activity. Both AO and AL pheromone disrupted male AL behaviour, but less significantly than observed for male AO. 4. These results suggest pheromone-based click beetle mating disruption for wireworm management is feasible. However, further work is required to determine application rates and methods, and if this can be developed for other species.

Synthetic mulches used to warm soil, conserve moisture, and prevent weeds can be penetrated by soil-borne wireworms that seek fruit such as tomato, cucurbit, and melon resting on the surface. Fruit placed on 0.7mil biodegradable- and 0.9mil polyethylene mulches were readily penetrated by wireworms; 4mil and 6mil polyethylene significantly reduced penetration. Mulch penetrability implied their permeability to attractant carbon dioxide emitted by the fruit. The impenetrability of woven fabric implied physical exclusion since it is entirely permeable to CO2.

A novel wireworm ‘probe’ trap is described, characterized, and used in field trials to (i) determine effects of different spring tillage treatments on its efficiency capturing Agriotes obscurus L. Coleoptera: Elateridae wireworms; and (ii) assess its ability to predict crop damage. In pot trials, its attractiveness to other wireworm species was determined. In a forage/grass field, spring tillage treatments included: ploughing, rototilling, glyphosate-sprayed then ploughing, glyphosate-sprayed then rototilling, glyphosate-sprayed untilled, and untilled. The number of wireworms captured in tilled treatments increased until 20 October. The number of wireworms captured in untilled treatments remained low. Subterranean CO 2 levels in tilled treatments decreased after tillage and over the trapping period, suggesting the increase in captured wireworms occurred because trap CO 2 levels were not overwhelmed by soil levels. The decrease in subterranean CO 2 was less pronounced in untilled-glyphosate and relatively unchanged in untilled-no glyphosate, corresponding to the lower number of wireworms captured. In a separate trial determining the trap’s ability to predict crop damage, a 2 m-wide section was rototilled in grass/forage fields in the spring of Year 1. Probe traps assessed wireworm levels in August and October of Year 1 to predict crop damage for potato and corn planted in Year 2. The y -intercept of linear equations suggested that wireworms captured in October better-predicted potato damage and corn emergence although equations were significant only for August. October-captured wireworms ≤ 21 mm in length correlated better with crop damage than larger wireworms. Pot studies revealed the probe trap to also attract A. litigiousus , A. sordidus , A. brevis , and A. ustulatus .

Fungal entomopathogens can greatly reduce the fitness of their hosts, and it is therefore expected that susceptible insects will be selected to avoid exposure to pathogens. Metarhizium brunneum is a fungal pathogen that can infect Agriotes obscurus, which in its larval form is a destructive agricultural pest and is repelled by the presence of M. brunneum conidia. Due to the subterranean nature of larval A. obscurus, recent research has focused on tar-geting adult A. obscurus with M. brunneum. No-choice and choice behavioural assays were conducted to deter-mine if male adult A. obscurus avoid M. brunneum mycosed cadavers, or conidia applied to either food or soil. To further investigate the response of A. obscurus beetles to conspecific cadavers, the movement and behaviour of beetles placed at the centre of a semi-circular arrangement of mycosed or control cadavers was examined using motion tracking software. We found little evidence to suggest that A. obscurus male beetles avoid M. brunneum conidia or mycosed conspecific cadavers or alter their behaviour in their presence.

The success of autodissemination of entomopathogenic fungi depends to some degree on the transmission of infective conidia or spores from contaminated hosts to conspecifics. In the present study, we examined the potential for direct transmission of a lethal dose of Metarhizium brunneum (Petch) conidia between opposite sex pairs of Agriotes obscurus L in the laboratory. Male and female beetles were exposed to M. brunneum conidia prior to pairing with an uncontaminated opposite sex partner (‘indirectly exposed’ to M. brunnneum), 2, 21 and 45 h after M. brunneum exposure. Ninety-six percent of the female beetles that had been exposed to M. brunneum granules died by day 15, as compared to 59% and 53% of the indirectly exposed and control females. From 15 days after the beginning of the experiment, females indirectly exposed to M. brunneum had higher odds of dying at any point in time than unexposed females across all post-exposure time periods. There was no significant difference in the odds of dying between indirectly exposed males and their unexposed counterparts at any point during the experiment. When the female beetle was exposed to M. brunneum forty-five hours prior to pairing, the pairs engaged in fewer courtship interactions relative to the other treatment groups, however less than 50% of all the pairs engaged in courtship interactions. The low incidence of courtship interactions and limited transfer of conidia between A. obscurus beetle pairs suggests that negligible horizontal transmission of M. brunneum conidia may occur between conspecifics.

Autodissemination techniques can potentially be used to distribute insecticides, including microbial insecticides, to cryptic pests. This approach is reliant on the target insect either passing the pathogen passively to other insects or the pathogen cycling within the population after the initial host dies. Here we examine, in small scale experiments, whether male Agriotes obscurus click beetles passively transmit the spores of the fungus Metarhizium brunneum directly, or indirectly via the environment, and whether this is influenced by exposure to synthetic female pheromone. We found that the beetles did not avoid M. brunneum spores and that this behaviour was not affected by pheromone. Exposure to pheromone increased beetle movement and uptake of spores, but this did not result in an increase in infected beetles under our conditions. Beetles were able to transfer spores at high levels via environmental contamination. However, contamination of the environment declined rapidly after exposure to the spores. The results are discussed in the context of developing an autodissemination strategy for click beetles.

Trade-offs in the time and energy allocated to different functions, such as reproductive activities, can be driven by alterations in condition which reduce resources, often in response to extrinsic factors such as pathogens or parasites. When individuals are challenged by a pathogen, they may either reduce reproduction as a cost of increasing defence mechanisms or, alternatively, modify reproductive activities so as to increase fecundity thereby minimizing the fitness costs of earlier death, a behaviour consistent with the terminal investment hypothesis (TIH). The TIH predicts that individuals with decreased likelihood of future reproduction will maximize current reproductive effort, which may include shifts in reproductive timing. We examined how wild, adult female click beetles (Agriotes obscurus) responded after exposure to the fungal pathogen Metarhizium brunneum. Field-collected beetles exposed to a high concentration of M. brunneum died earlier and in greater numbers than those exposed to a low concentration. Using a multivariate approach, we examined the impact of pathogen challenge on lifespan and a suite of reproductive traits. Stepdown regression analysis showed that only female lifespan differed among the fungal treatments. Fungal-induced reductions in lifespan drove changes in the reproductive schedule, characterized by a decrease in preoviposition period. Moving the start of egg laying forward allowed the females to offset the costs of a shortened lifespan. These changes suggest that there is a threshold for terminal investment, which is dependent on strength of the survival threat. From an applied perspective, our findings imply that exposing adult click beetles to M. brunneum to reduce their population density might not succeed and is an approach that needs further investigation.

Elaterid female sex pheromone, while currently used for monitoring the adult life stage (click beetle), has only recently been explored as a potential management tool. Consequently, there is little understanding of how abiotic and biotic conditions influence the response of click beetles to the pheromone. We examined whether the response of male Agriotes obscurus L. (Coleoptera: Elateridae) beetles to a cellulose-based formulation of female sex pheromone (‘pheromone granules’) is influenced by air movement, presence of visible light, and month of beetle collection. In addition, we investigated the distance from which beetles were attracted to the pheromone granules. Click beetle response was determined by measuring movement parameters in free-walking arena experiments. The response to pheromone was not affected by the presence or absence of visible light. We found that beetles collected earlier in the season had increased activity and interaction with pheromone under moving air conditions, compared to beetles collected later. When controlling for storage time, we confirmed that individuals collected in May were less active than beetles collected in March and April. In the field, beetles were recaptured from up to 14 m away from a pheromone granule source, with over 50% being recovered within 4.4 h from a distance of 7 m or less. Understanding how abiotic and biotic factors affect pest response to pheromone can lead to more effective and novel uses of pheromone-based management strategies.

The biogeography and genotype diversity of Metarhizium species in northwestern North American soils was examined; 20 ecoregions were sampled, including 58 agricultural and 80 natural habitat subsites, and areas that were glaciated during the Pleistocene epoch. One hundred and twenty-nine isolates of M. brunneum, 26 isolates of M. robertsii, four isolates of M. guizhouense, one isolate of M. flavoviride, and 55 isolates of Beauveria were recovered. Metarhizium and Beauveria species were isolated in diverse ecoregions within the study area, but a trend for increased isolation of Metarhizium species in western regions of the study area was observed. Consistent with this observation, the prevalence of M. brunneum and M. robertsii decreased at higher elevations, and the opposite was true for Beauveria. Both M. brunneum and M. robertsii were more commonly isolated from agricultural and natural habitat subsites, and considerable genotypic diversity was observed in both habitats and within the same subsite. Metarhizium robertsii, but not M. brunneum, was more commonly isolated from nonglaciated locations; however, less diversity and richness was observed for M. brunneum recovered from glaciated versus nonglaciated locations consistent with insular biogeography. The study has implications for microbial control strategies in the region.

Wireworms (Coleoptera: Elateridae) are serious agricultural pests, with soil-dwelling larvae attacking subterranean tissues of crop plants and their fruit when in contact with the soil surface. Researchers collect wireworms for laboratory experiments to study their behaviour and test pest control agents but frequently lose them to Metarhizium Petch (Ascomycota: Hypocreales: Clavicipitaceae) infection. We found latent M. brunneum infection in 13-100% of live, asymptomatic Agriotes obscurus and A. lineatus wireworms acquired from agricultural fields and in wireworms maintained indoors, indicating its enzootic presence. M. brunneum DNA in the wireworms maintained indoors sometimes exceeded 250pg/ug total DNA (0.025% of whole-sample DNA mass). Expressed as copies of M. brunneum DNA/g, unadulterated soil levels of M. brunneum ranged from 4037 in agricultural field soil to 721,538 in soil harbouring a wireworm collection indoors, with the prevalence of latently-infected live wireworm specimens being directly related to soil levels. M. brunneum levels in live wireworms, when regressed against relative levels of 394 bacteria species in the microbiome, were proportionally related to only four: Pantoea agglomerans, Pandoraea pnomenusa, Nocardia pseudovaccinii, and Mycobacterium frederiksbergense. All four of these bacteria have previously been reported to express antimicrobial mechanisms. Consistent with occurrences of disease immunity reported for other pathogen-insect pairs, symbiotic bacteria may be suppressing M. brunneum-induced wireworm mortality. This would help explain why wireworms commonly succumb to infection after being brought into sterilized conditions, as well as the sometimes limited efficacy of M. brunneum when using it as a pest control agent in the field.

Wireworms are a serious agricultural pest, with control efforts targeting soil-dwelling larvae almost exclusively. They appear yearly for a brief period as adult click beetles to mate and oviposit, and as adults, possess qualities that make them good candidates for an attract and kill control tactic: (i) susceptibility to certain entomopathogenic fungi; and (ii) attraction of males to female sex pheromones. To expand the range of wireworm control options, our study aimed to determine if banded applications of a new granular formulation of Agriotes obscurus L. (Coleoptera: Elateridae) pheromone would increase beetle mortality when applied with banded Metarhizium brunneum Petch (Ascomycota: Hypocreales: Clavicipitaceae) LRC112. Pheromone granules applied at 12.7 kg/ha (1 % wt/wt 1:1 geranyl hexanoate:geranyl octanoate) together with rice conidiated with 2 × 1014 conidia/ha of M. brunneum LRC112 reduced beetle recapture by 98.2 % compared to M. brunneum alone. A lower rate (2 × 1013 conidia/ha) of M. brunneum with pheromone granules reduced recapture by 82.6 % compared to the lower rate alone. A significantly greater number of beetles aggregated at pheromone bands to acquire lethal doses of conidia in as little as 6 h, with conidia dose remaining unchanged up to 54 h later. Conidia dose acquired by beetles corresponded to treatment and was positively related to total beetle mortality and speed of death. Attracting and killing click beetles might represent a new tactical approach to control wireworm larvae by reducing click beetle fecundity. We expect the pheromone granules to also to have utility for click beetle mating disruption.

Applications of Metarhizium brunneum Petch (Ascomycota: Hypocreales: Clavicipitaceae) isolate LRC112 conidia caused high mortality to Agriotes obscurus L. (Coleoptera: Elateridae) click beetles in field trials. Banded conidiated rice (4.4 × 1014 conidia ha−1) and conidia dust (5.0 × 1013 conidia ha−1) resulted in 93.3 % ± 7.3 and 91.3 % ± 3.0 mortality after 18 days, while aqueous conidia suspension spray (5.0 × 1013 conidia ha−1) with and without 80 g ha−1 spinosad resulted in 68.2 % ± 17.7 and 52.6 % ± 17.4 mortality. Differences in results between 2012 and 2013 were attributed to rainfall, with pronounced effects in 2012 (rain beginning 35 h post treatment) and minimal effects in 2013 (rain beginning at 4 h). In another field experiment, beetles dosed with 1.49 × 107 ± 5.08 × 106 conidia per beetle retained 4.6 % of conidia after seven days while conidia viability on beetle bodies remained unchanged. The results inferred opportunities for controlling click beetles using fungal entomopathogens, and for horizontal transmission of the inoculum.