This study showed entomopathogenic fungus M. anisopliae SD-3 was highly virulent to R. ferrugineus, a serious pest of various palm species. Under the premise of harmless effect to environment or non-target organisms, biological control with pathogenic fungi would offer long-term insect control (Khetan 2001). The pathogenic fungi seem like to survival and distribute with the R. ferrugineus in its dark and humid surroundings. M. anisopliae is applied as conidia or mycelia in various formulations. By way of making the insects infected through the induction of a fungal epizootic, new conidia and viable cells are produced to spread to the health insects and thus achieve the control effect (Genthner et al. 1997). After the series of adhesion, prepenetration growth, penetration into the host, and settling down of the pathogen in the host, the insects would be infected by M. anisopliae (Lattanzio et al. 2006).
Most entomopathogenic fungi enter in the host by penetrating through the host cuticle. In the course of fungal infection, the fungi are adsorbed on the host cuticle in the first step before penetration (Urquiza and Keyhani 2013). Dong et al. (2009) proposed adhesion to occur at three successive stages: (1)adsorption of the fungi propagules to the cuticular surface; (2)adhesion or consolidation of the interface between pre-germinant propagules and the epicuticle; (3)fungi germination and development at the insect cuticular surface, until appressoria are developed to start the penetration stage. Infection will proceed after a successful penetration being achieved. In addition, we found intersegmental membrane and hair was the first spot invaded by M. anisopliae when R. ferrugineus larvae were infected by this entomopathogenic fungus (Fig. 1). This could be attributed to relatively thin chitin layers in the intersegmental membrane, which favored infection by germ tubes. During the cultivation of dead larvae on moist sterile filter paper, white hypha first occurred at the intersegmental membrane and hair associated with most abundant conidia, as well as longest germ tubes and hyphae. In addition, germination of conidia was first observed in the intersegmental membrane. Together these observations indicated that intersegmental membrane and hair was the weak point subject to M. anisopliae infection.
Histological section showed that before R. ferrugineus larvae died due to infection, associated body tissues were affected to different degrees, leading to obvious lesions. Direct death reason of the host remained unclear. It was likely that larva body tissues were seriously damaged due to infection by M. anisopliae, preventing normal larva physiological activities. Along with mycelia growth, larva bodies (muscles, fat, tracheaes digestive tube) were occupied by a large amount of fungal hypha (Fig. 2), which exhausted nutrients and impeded fluid circulation, leading to physical starvation and metabolic disorders. Thus, hypha invasion could cause body tissue failure of larva, which died due to the incapability of normal physiological activities.
Another reason caused larva death might be physiological and biochemical changes in infected larva body. Sloman and Reynolds (1993) suggested mycotoxin as the true reason for the death of insect infected by many imperfect fungi. Such toxin not only inhibits the immune function of host, but also affects associated central nervous system and has partial pathogenic effect, thus promoting the death of host. However, Wang and You (1999) indicated the uncertainty in functional mechanism of such toxin during relevant infection process. These authors only found pure toxin caused immune pressure, muscle paralysis and malpighian tubule damage in the host. Further study is needed to demonstrate whether mycotoxin produced by M. anisopliae was the primary reason caused the death of R. ferrugineus.
We used SEM and frozen section to observe the fungal infection. We also first applied frozen section to study histopathological mechanisms of M. anisopliae SD-3, and developed a set of methods suitable for studying high-fat larvae. In contrast, research of insect pathology has mainly employed traditional paraffin section, which usually takes 3–5 days for sample preparation and pre-treatment. By comparison, frozen section only requires 30–60 min for sample processes. This contributed to experiment effectiveness and reduced impacts of prolonged fixation on samples.
When collecting fresh samples of histological sections, caution must be taken to avoid water. Temperature is the most important factor for frozen section. As larvae contain substantial fat, high temperature may cause adhesive due to incomplete freeze. On the other side, low temperature may increase the fragility of samples which are easy to break during sectioning. Based on our observations in this work, we suggest rapid freeze at −28 ± 1 °C and section at −22 ± 1 °C yield optimum results.