Background

Beauveria bassiana is one of the commercially available entomopathogenic fungi (EPF), and a number of isolates with high virulence and broad host spectrum have been used to control agricultural and forest pests. Although the functional importance of genes in EPFs’ pathogenesis have been extensively studied, the precise ultrastructural mechanism of the fungal infection, particularly penetration of the host insect cuticles, is not well understood.

Results

In this study, we investigated the morphology and ultrastructure of the larval cuticle of the red flour beetle, Tribolium castaneum, after treatment with B. bassiana ERL1170 expressing an enhanced green fluorescent protein (Bb-eGFP). The Bb-eGFP showed high virulence against the larvae, with approximately 90% mortality at 48 h after treatment (HAT) and 100% at 72 HAT under our infection conditions. In these larvae, the regions of the body wall with flexible cuticles, such as the ventral and ventrolateral thorax and abdomen, became darkly melanized, but there was little to none melanization in the rigid dorsal cuticular structures. Confocal microscopy and transmission electron microscopy (TEM) indicated that germinated conidia on the surface of the larval cuticle were evident at 6 HAT, which formed penetration pegs and began to penetrate the several cuticle layers/laminae by 12 HAT. The penetration pegs then developed invading hyphae, some of which passed through the cuticle and reached the epidermal cells by 24 HAT. The larval cuticle was aggressively and extensively disrupted by 48 HAT, and a number of outgrowing hyphae were observed at 72 HAT.

To prepare the fungal inoculum, the fungal stock was cultured on ¼ SDA for 10 days and the harvested conidial suspension was adjusted with 0.03% siloxane solution as a surfactant to a concentration of 1×10⁷ conidia/ml. One hundred microliters of the conidial suspension were spread and cultured on ¼ SDA plates in Petri dishes (diameter 60 mm) for 7 days at 25 ± 1°C under a photoperiod of LD 14:10 h. Conidia were used for bioassay against T. castaneum after confirmation of the conidial formation (conidiogenesis) and removing hyphae by filtering. Larvae or mature adults (n = 20) were placed on a cultured plate for 10 min to expose them to the fungal conidia. The treated insects were transferred to a fresh plate with a layer of wet filter paper (200 µl of distilled water) and approximately 0.3 g of wheat flour was added to the plate as feed. Three replicates were applied as a treatment. Non-treated beetles served as a control, with the beetles being placed on the non-cultured agar plates. All the plates were kept in a plastic cage (30 × 20 × 15 cm³), which was layered with wet paper towels receiving ca. 10 ml of distilled water to achieve high humidity conditions. The plastic cage was covered with a lid and kept at a room temperature of 24 ± 2°C for 5 days. Living and dead beetles were counted daily, and fungal colonization and insect melanization were carefully investigated under a microscope.