The composition of the free fatty acids from Dendrolimus pini exuviae

Abstract

The pine moth Dendrolimus pini effectively resists many insecticides, but it can be controlled by the use of bioinsecticides such as entomopathogenic fungi. In the use of microbial agents for the biocontrol of D. pini, it is important to identify the cuticular lipids of this pest if we are to understand the factors responsible for the preferential adhesion or selective repulsion of entomopathogenic fungi that are potentially useful in biocontrol. In this work the qualitative and quantitative analyses of free fatty acids in two exuviae extracts (petroleum ether and dichloromethane) and two developmental stages (larval–larval and larval–pupal molts) were studied. The free fatty acid composition of the epicuticular lipids from exuviae of D. pini was characterized chemically using gas chromatography (GC) and gas chromatography–electron impact mass spectrometry (GC–MS). Structural analyses of the dichloromethane extracts from larval–larval exuviae (LLE) and larval–pupal exuviae (LPE) revealed that the carbon numbers for the major acid moieties ranged from C_8:0 to C_34:0. Only C_23:0 was not identified in the LPE extract. The relative contents of fatty acids in the extracts varied from trace amounts to 34%. The fatty acids extracted by dichloromethane were essentially the same as those in the petroleum ether extract. We also identified dehydroabietic acid in the exuviae of D. pini. The respective quantities of dehydroabietic acid obtained from D. pini LLE and LPE were 1763 ± 103 μg/g exuviae and 11521 ± 1198 μg/g of exuviae.

Introduction

The epicuticular surface of an insect is covered by a thin layer of lipids. They can prevent desiccation, ensure chemical communication between species and reduce the penetration of chemicals and toxins as well as infectious microorganisms (Buckner, 1993, Nelson and Blomquist, 1995). It is also possible that specific cuticular lipids play a role in prey recognition by specialized and predatory insects. Epicuticular lipids contains mainly free lipids consisting of aliphatic polar and non-polar compounds (Lockey, 1988). The major components of the cuticular lipid coating of adult insects are often hydrocarbons, among which methyl-branched alkanes are the most abundant (Lockey, 1988). Nevertheless, a wide range of more polar lipids have been identified as constituents of the cuticle, including fatty acids, alcohols, aldehydes, ketones, wax esters, esters of primary and secondary alcohols, and acylglycerols (Blomquist and Jackson, 1979, Buckner, 1993, Gołębiowski et al., 2007). The quantities and composition of free cuticular lipids can differ among insect groups, and sometimes within the developmental stages of the same species (Jackson and Blomquist, 1976). Surface lipids have also been useful to distinguish species within insect orders, particularly in the Diptera, Hymenoptera and Orthoptera (Nelson and Blomquist, 1995).

In recent years there has been a resurgence of interest in the use of fungi to control insect pests (Ferron, 1978, Ferron, 1985, Soper and Ward, 1981, Goettel, 1984, Khachatourians, 1986). Naturally occurring entomopathogens are important regulatory factors of insect populations. At present, several species of entomopathogenic fungi are employed as biological control agents of insect pests (Clarkson and Charnley, 1996, Khachatorians, 1996). The pine moth Dendrolimus pini L. (Lepidoptera: Lasiocampidae) is one of the most serious forest insect pests. The pine moth (or pine-tree lappet) is native to Europe but is also known from western Asia. Host plants include a wide range of conifers such as fir, cedar, juniper, spruce, pine, Douglas fir and larch. Larvae usually begin hatching in early September and are about 5 mm in length. Before winter, larvae usually molt two to three times. Pupation begins in May–June and lasts 4–5 weeks. Pupae are formed inside loose, partially transparent cocoons, and can be found in tree crowns, on the bark of trunks and on the understory vegetation (Koehler, 1961, Sliwa, 1992). Biological and chemical insecticides can be used to control the pine moth. The stabilizing influence of natural enemies in ecosystems has been reviewed by numerous authors (Szmidt, 1951, Szmidt, 1955, Głowacka-Pilot, 1974, Sliwa, 1992), but in the case of the pine moth there is lack of agreement about the relative importance of parasites, predators and microorganisms in its natural control. Koehler (1968) briefly summarized the theoretical basis for the use of biological methods in forest protection. The role of entomopathogenic bacteria and fungi in the regulation of pine moth populations in Poland has been studied extensively. Exposure of D. pini larvae to Conidiobolus coronatus, an opportunistic fungal pathogen with a wide host range, resulted in rapid insect death (Boguś et al., 2007). Cordyceps militaris is another entomogenous fungal species that can also be important in limiting pine moth populations. Infection of pine moth larvae with isolated fungi under laboratory conditions showed that Paecilomyces farinosus and Beauveria bassiana had the strongest pathogenic properties (Głowacka-Pilot, 1974).

Insect species may be susceptible or resistant to fungal infection for a number of reasons (Vilcinskas and Götz, 1999). In many cases, fungistatic activities have been associated with insect cuticles. For example, the presence of antagonistic microbes, toxic lipids and cuticle phenolic compounds has been suggested as inhibiting fungal growth (Schabel, 1988, Fargues, 1984). The cuticle composition strongly influences conidia germination, resulting in the differential susceptibility of various insect species to a fungal pathogen (Boucias and Latge, 1988, El-Sayed et al., 1991, Wang and St Leger, 2005). Cuticular fatty acids have a profound effect on fungal spore germination and differentiation: they are either toxic or stimulatory to some pathogenic species (Gillespie et al., 2000). Determination of the cuticular fatty acid composition is therefore of great importance in understanding the background of insect susceptibility to fungal infection.

The lipid chemistry of the external waxy material of D. pini larvae has been described (Gołębiowski et al., 2008a). Analysis of the cuticular fatty acids extracted from D. pini larvae indicated a composition of C_16:0, C_18:0, C_18:1, C_18:2 and C_18:3, where C_18:1 was found to be dominant. In our previous work (Gołębiowski et al., 2008a) it was shown that the cuticular fatty acid profiles of D. pini and Galleria mellonella larvae (both species highly susceptible to C. coronatus infection) differ significantly from the profile of Calliphora vicina larvae (species resistant to fungal infection). The major difference is the presence of C_14:0, C_16:1 and C_20:0 in the cuticle of C. vicina. They are also absent in the cuticle of D. pini, whereas that of G. mellonella contains traces of them. Moreover, extracts from C. vicina contain traces of several short-chain saturated fatty acids absent in the larval cuticle of both lepidopteran species. Histological examination of D. pini, G. mellonella and C. vicina larvae exposed to sporulating C. coronatus colonies revealed the inability of C. coronatus conidia to germinate on the cuticle of C. vicina larvae, suggesting that the composition of fatty acids present in the C. vicina epicuticle is responsible for the remarkable resistance of this fly to mycosis (Boguś et al., 2007). In vitro tests on the effect of fatty acids on C. coronatus demonstrated that the presence of C_16:0, C_18:0, C_18:1, C_18:2 or C_18:3 in culture media inhibits fungal growth and reduces conidia production (Babiarz et al., 2001), whereas the virulence of fungal colonies cultivated in culture media supplemented with C_8:0, C_9:0, C_10:0, C_12:0 or C_14:0, all found in the cuticle of C. vicina larvae (Gołębiowski et al., 2008a), is attenuated (Boguś, unpublished).

In this study, gas chromatography (GC) combined with mass spectrometry (GC–MS) was used to analyze the surface fatty acid composition of D. pini exuviae. The molting process may affect insect resistance to pathogenic microorganisms and may therefore have an impact on the biological control of the insect (Watanabe, 1987). The interactions between the penetration of the larval cuticle by the fungus and the molting process of the larval host reduce the lethal effect of mycosis (Vey and Fargues, 1977). However, the elucidation of the surface hydrocarbon composition has been undertaken mainly with the use of whole juvenile or adult insects, and only a few reports are available on analyses done on larval exuviae shed at molting (Nelson et al., 1998, Buckner et al., 1999). The experiments described here were planned to fill this gap.