Sterile culture of host insects

T. molitor larvae were successfully reared in sterile wheat bran substrates with 15% moisture content at 25°C under natural day light, and cultured for more than five generations before use for the tests (Figure 1e). The microbes on the larval surface were diluted from generation to generation, and the larvae were relatively sterile. The larvae used for tests were cultured on sterile wheat bran with 50% moisture content to investigate their sterility. T. molitor larvae were alive after 15 d, and no microbial growth was observed. This result suggests the absence of microbial contamination. Thus, the efficacy tests of M. anisopliae did not exhibit microbial interference.

T. molitor has a life cycle that consists of four stages, namely, egg (Figure 1a), larva (Figure 1b), pupa (Figure 1c), and adult (Figure 1d). They can complete their life cycle under desiccation stress, in which the larval stage exhibits relatively high desiccation endurance. The life cycle of T. molitor can encompass four months to several years, depending on the number and duration of the instars [11]. Under the experimental conditions, the larvae had 13 instars and pupated at 13th instar larvae. Figure 1b shows some larvae at various instars from third to 10th instar larvae.

Conidial germination rate of M. anisopliae isolates at different moisture levels

Conidial germination of all tested M. anisopliae isolates, namely, MAX-2, MAL-1, MAC-6, and MAQ-28, was positively influenced by moisture contents of the substrates (Figure 2). After 24 h of culture, no germination occurred in the substrates with low moisture contents (8% and 15%) for all the M. anisopliae applied treatments, and only MAX-2 had a poor germination rate of approximately 5% at 20% moisture level. The conidial germination rates of the isolates improved with the moisture levels, and reached 56% for MAX-2, 47.1% for MAC-6, 35.6% for MAL-1, and 23.4% for MAQ-28 at 35% moisture level.

Efficacy of M. anisopliae isolates against T. molitor larvae at different moisture levels

After 15 d of inoculation, the mortalities of T. molitor larvae reached 100% for all the isolates, except MAQ-28 (95% mortality) in the substrate with 35% moisture content. The efficacies between MAX-2 and other isolates showed no significant difference. However, the efficacies differed significantly between MAX-2 and other isolates at moisture levels of 8% to 30%. MAX-2 had the highest efficacy, whereas MAQ-28 had the lowest efficacy. MAX-2 maintained 100% mortality at 30% moisture level, whereas the efficacies of other isolates decreased. The mortalities for MAC-6, MAL-1, and MAQ-28 continued to decrease drastically with the decrease in moisture levels, and reached zero or close to zero at 8% moisture level. However, the mortality for MAX-2 slowly decreased with the decrease in moisture levels, and maintained medium mortality of 41% at 8% moisture level.

T. molitor larvae were healthy in control treatments with different moisture levels (8% to 35%) and continued their life cycle.

Infection characteristics of MAX-2 under desiccation stress

The efficacies of all isolates decreased with the decrease in moisture levels, but the efficacy of MAX-2 was less affected by desiccation stress (Table 1). The efficacy of MAX-2 was almost unaffected by the decrease in moisture levels?>?25%, and no statistical difference was observed among higher moisture levels from 25% to 35%. Its efficacy slowly decreased with the decrease in moisture levels < 25%, and a significant difference was observed among lower moisture levels from 8% and 20%. The efficacy of MAC-6 significantly differed among all moisture levels from 8% to 35%. The efficacy of MAL-1 significantly differed among higher moisture levels (from 20% to 35%), but no significant difference was observed between lower moisture levels (8% and 15%). The efficacy of MAQ-28 significantly differed among higher moisture levels (from 25% to 35%), but no significant difference was observed among lower moisture levels (from 8% to 20%). These results suggest that the four isolates with gradient descent efficacies also had gradient descent capacities against desiccation, and MAX-2 had significantly higher antistress capacity under desiccation stress than the other isolates.

MAX-2 caused similar symptoms to other isolates in the wet microhabitat (substrate with 35% moisture content; Figure 3a). T. molitor larvae exhibited bradykinesia, and the internodes of insects turned slightly brown in the early stage of infection (2 d to 3 d post-inoculation; Figure 3b). The internodes gradually became dark black, and the larvae died within the following 2 d (Figure 3c). White mycelia sprang up and gradually covered the cadavers approximately 10 d after inoculation. The conidia formed, and the larval surface turned green after another 1 d to 2 d (Figure 3d). The substrate also showed white mycelia, which gradually turned light green during the course of infection. This phenomenon suggests that new conidia formed and added to the initial inoculum concentration, thereby resulting in a large number of inocula around the larvae (Figure 3e).

In the dry microhabitat (substrate with 8% moisture content; Figure 3f), MAX-2 exhibited medium efficacy (41% mortality), whereas the other isolates showed no efficacies or very low efficacies (< 5% mortality). Similar to the observations in the wet microhabitat, T. molitor larvae exhibited bradykinesia in the dry microhabitat, but the larvae exhibited local black patches on the cuticles 3 d to 4 d after inoculation (Figure 3g). The local black patches gradually extended to one to two somites, and the larvae became slower, died, and dried (Figure 3h). However, no mycelium or conidia emerged on the insect cadavers and substrates when kept in dry substrate all the time. This result suggests that the moisture level was too low to facilitate mycelial or conidial growth. However, when the cadavers were transferred to a moist filter, mycelium and conidia rapidly emerged on their surface within 2 d to 3 d (Figure 3j). In addition, few larvae completed exuviation and survived even when local black patches appeared on the shell (Figure 3i).

A valid laboratory bioassay system for evaluating M. anisopliae efficacy under desiccation stress

Water stress tolerance of fungal strains is usually evaluated using various salts to create different water potential scenarios. However, in testing the virulence of the strains, the salt can affect the life cycles of hosts. In this paper, a novel laboratory bioassay system was used to test the efficacy of M. anisopliae under desiccation stress on T. molitor larvae in dry substrate.

Extreme environmental trials of pathogens demand specific hosts. The yellow mealworm, T. molitor, is a freeze-susceptible, stored product pest. When provided with sufficient food supply, T. molitor larvae have low humidity tolerance and can survive under relatively xeric conditions because of their ability to metabolize water from ingested food [12].

Clopton et al. [13] sterilized adult and larval T. molitor by incubation at 36°C to 37°C for 5 d to eliminate the effect of existing gregarine infections on the tests. In the present study, the host insects were cultured and sterilized by generational dilution in sterile wheat bran substrates, and the insects were almost fully sterilized when given enough generation culture. This new method may provide host insects for strict experimental infections.

The efficacy of M. anisopliae under desiccation stress was tested in dry wheat bran substrate with initial moisture content of 8%. At this low moisture level, M. anisopliae was difficult to grow, but the isolate MAX-2 was still active, whereas the other isolates showed very low efficacy. This result suggests that the infection of sterile T. molitor larvae in wheat bran substrates with low moisture content could constitute a valid laboratory bioassay system to study M. anisopliae efficacy under desiccation stress.

Efficacy of M. anisopliae isolate MAX-2

This study demonstrated that M. anisopliae isolate MAX-2 had pathogenicity against T. molitor larvae in all the tested moisture levels, particularly lower moisture levels, and showed relatively high tolerance to desiccation stress.

Daoust et al. [14] indicated that the efficacy of M. anisopliae against insects depends on conidial germination. Conidial germination of all tested isolates in the present study showed a tendency to decrease with the decrease in substrate moisture content within the tested scope (8% to 35%). The mortality of larvae for the isolates in different moisture levels also showed the same tendency, which indicates the correlation between conidial germination and efficacy of M. anisopliae. However, the mortality for MAX-2 decreased much more slowly than those of the other isolates. At the substrate with 8% moisture, which was too low for M. anisopliae to facilitate germination, MAX-2 still showed medium mortality of 41% versus low mortality < 5% for the other isolates against T. molitor larvae. Howard et al.[15] observed that high virulence of M. anisopliae against mosquitoes is not significantly affected by low viability, and they deduced that the difference is possibly due to the different abilities of the fungal conidia to germinate on mosquito cuticles and the agar. Leger [16] also reported the existence of two diverse sets of selection pressures on Metarhizium spp., one for optimum characteristics for soil survival and another for virulence to insects. Although desiccation stress restricted the viability of MAX-2 on the substrate, this isolate might have more excellent ability to germinate and infect after attachment to the host by its own high trehalase activity, some other mechanism, or the induction of the hosts. Further studies on the possible mechanisms of water stress response and high efficacy for MAX-2 are recommended.

Sporulation of entomopathogenic fungi is significantly affected by moisture content, commonly between 1:0.35 and 1:0.60 (wet substrate: water) in mass production, of the solid substrate [17]. The optimum moisture levels of the substrate for M. anisopliae range from 57% to 58% [18]. In the present study, conidial germination and the efficacy of M. anisopliae were tested with a dry substrate at moisture levels from 8% to 35%, at which all isolates caused 100% mortality, except for MAQ-28 (95% mortality). The moisture contents of substrates decreased as water evaporated over time. To avoid contamination, the moisture levels were determined by testing the initial moisture contents of the substrates before inoculation. This study was conducted to test the efficacy of M. anisopliae under desiccation stress. The substrates become drier over the testing course, and the tested efficacies of the isolates might be slightly negative for the tested moisture levels.

Infection characteristics of MAX-2 under desiccation stress

M. anisopliae invades and infects the body of an insect by direct penetration of the cuticle or breathing apertures, ingestion into the digestive tract, or wounds [19]. The infected insects lose their appetite and exhibit somewhat sluggish behavior. Some changes in color might be observed shortly before death. At high humidity, the hyphae emerge through the cuticle and form a hyphal layer on the surface of the insect, and the conidium then emerges after death [20, 21]. The outward signs of infection on T. molitor larvae inflicted with M. anisopliae isolate MAX-2 under desiccation stress differed from those in the wet microhabitat. The treated larvae showed dark black internodes and fungal growth after death in the wet microhabitat. However, local black patches appeared on the cuticles and the cadavers dried, and no fungal growth after death was observed under desiccation stress. This phenomenon was possibly due to the possible production of defense measures by the larvae against a finite number of conidia, which had contact with the larvae in the dry microhabitat. Insects usually activate polyphenol oxidase and melanize their cuticles when wounded or infected with microbial pathogens to heal wounds or prevent microbial intrusion [22]. The local black patches on T. molitor larvae in the dry microhabitat could come from their own polyphenol oxidase activity or resistance to other pathogens. This phenomenon was supported by the few larvae that survived and exuviated, leaving the shell with local black patches (Figure 3i). The wet substrate allowed the production of mass mycelia and conidia, which added to the initial inoculum concentration and increased the penetration efficiency. In addition, the mycelia and its toxin released in the substrate could be ingested by the larvae. The larvae had little chance to protect against invasion, and no local black spots were found. This observation was supported by the high mortality in the wet microhabitat for all isolates. Whether the different symptoms suggest diverse infection mechanisms to T. molitor larvae is worthy of further investigation.

Efficacy of M. anisopliae isolate against pests under desiccation environment

As an alternative to chemical control, the use of fungal insecticides for the biological control of insect pests has attracted significant interest. However, entomopathogenic fungi have not achieved wide-scale use in agriculture in spite of their apparent efficacy in small-scale field trials, mainly because they require high humidity and temperature to grow and disperse. M. anisopliae is a common soil-borne entomopathogenic fungus that is found worldwide, and environmental factors affect its persistence and activity. Moisture level is a major factor that affects the ability of fungi to survive, propagate, and infect and kill their host [23]. The field moisture level usually does not satisfy the requirements for germination and growth of M. anisopliae[24]. Studies on drought tolerance, which is a key part of stress tolerance, are important for the use of fungi in biocontrol [5, 25]. Our results indicate that M. anisopliae isolate MAX-2 maintained high efficacy under desiccation stress, and exhibited great potential for development.

The isolate was obtained from Shangri-la in Yunnan, China. This region is at high altitude with an extensive annual arid period, high UV radiation, and dry and windy weather. The fungi might have developed desiccation tolerance to adapt to the extreme environment, such as low humidity. The tolerance of this fungus to other stressors needs further investigation. The characteristics of MAX-2 provide genetic resources of resistance, and indicate the potential of developing a biopesticide from the fungal isolate for managing pests under desiccation stress.

Solid substrates

Wheat bran substrates with different moisture levels were used in this study. The substrates were sterilized at 121°C for 20 min. Sterile wheat bran without water was used as a dry substrate to test the efficacy of M. anisopliae under desiccation stress. The moisture contents of substrates were adjusted by adding a certain amount of water and heating 5 g of the sterilized substrate at 100°C for 4 h. Moisture content was then calculated using the dry and initial weights. Moisture content of the dry substrate was determined to be 8%. The gradient of the substrates from the initial moisture content was adjusted to 15%, 20%, 25%, 30%, and 35%.

Sterile culture of host insects

T. molitor larvae were selected as host insects because they can remain active under desiccation stress, and are easily reared under laboratory conditions. Such conditions are convenient for testing the virulence of fungal pathogens under desiccation stress.

To eliminate the effect of some possible microbes, we cultured the host insects under sterile conditions. T. molitor larvae were washed in sterile water, and the water on the surface was absorbed using sterile filter papers. The cuticles of the larvae were wiped carefully with 75% alcohol cotton balls for seconds and transferred to sterile filter paper to dry in air for 5 min. Sterilized larvae were reared, incubated, and subcultured in sterile glass jars containing the wheat bran substrate with 15% moisture content.

Screening of MAX-2 with the capacity of infecting under desiccation stress