DOI: 10.5061/dryad.hmgqnk9k1
title: Insecticidal activity of Beauveri bassiana ERL1170-egfp against Tribolium castaneum

description: 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.
description: 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 1×10<sup>7</sup>
 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 <em>T. castaneum</em>
 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 cm<sup>3</sup>), 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.