The Composition of the Cuticular and Internal Free Fatty Acids and Alcohols from Lucilia sericata Males and Females

GC, GC–MS, and HPLC–LLSD analyses were used to identify and quantify cuticular and internal lipids in males and females of the blow-fly (Lucilia sericata). Sixteen free fatty acids, seven alcohols and cholesterol were identified and quantitatively determined in the cuticular lipids of L. sericata. Cuticular fatty acids ranged from C6 to C20 and included unsaturated entities such as 16:1n-9, 18:1n-9, 20:4n-3 and 20:5n-3. Cuticular alcohols (only saturated and even-numbered) ranged from C12 to C20 in males and C10 to C22 in females. Only one sterol was found in the cuticular lipids of both males and females. 23 free fatty acids, five alcohols and cholesterol were identified in the internal lipids. Internal fatty acids were present in large amounts—7.4 mg/g (female) and 10.1 mg/g (male). Only traces of internal alcohols (from C14 to C26 in males, from C14 to C22 in females) were found in L. sericata. Large amounts of internal cholesterol were identified in L. sericata males and females (0.49 and 0.97 mg/g of the insect body, respectively)

Trichothecenes Production by Entomopathogenic Fungus Conidiobolus Coronatus

Entomo pathogenic fungi are of particular interest now as the likely source of a new class of insecticides. This interest stems from the fact that these organisms naturally present in the environment, in most cases selectively affect specific organisms, and metabolites produced by them do not pass into the food chain. Using naturally or artificially introduced organisms to reduce populations of arthropod pests, provide you more than ever the security of the consumers as well as crops and the environment. This is particularly important from the point of view of organic farming, in which there is a need to extend the scope of alternatives against harmful insects. Careful study of the selection of organisms used, as well as the methods of their application are necessary for the efficient and safe prevent losses caused by the pests. Much attention is paid to these issues, with a view to both human health and reducing exposure to unintended infections other living organisms, non-target conservation measures. The aim of this study was to identify toxic metabolites from trichothecene group produced by the parasitic fungus Conidiobolus coronatus. The results of our study allow conclude that the T-2 toxin is present in C. coronatus extract. It was also shown that C. coronatus produced higher amount of trichothecenes when is grown on LB medium. The optimal temperature for production of both T-2 and HT-2 by C. coronatus was 20°C. However, the pH value is the same for grown of C. coronatus mycelium and trichothecenes production. In any of the tested extracts DON and NIV was not detected. Toxicity of C. coronatus homogenates and post-incubation filtrates to G. mellonela larvae and Sf9 cells was also determined.</p

Quantitative summary of the experiment: Numbers of used <i>Galleria mellonella</i> larvae, pupae and adults; masses of extracts.

<p>Quantitative summary of the experiment: Numbers of used <i>Galleria mellonella</i> larvae, pupae and adults; masses of extracts.</p

Cuticular fatty acids of <i>Galleria mellonella</i> (Lepidoptera) inhibit fungal enzymatic activities of pathogenic <i>Conidiobolus coronatus</i>

<div><p>The entomopathogenic fungus <i>Conidiobolus coronatus</i> produces enzymes that may hydrolyze the cuticle of <i>Galleria mellonella</i>. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by <i>C</i>. <i>coronatus</i> enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free <i>N</i>-glucosamine was in the hydrolysis of <i>G</i>. <i>mellonella</i> thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of <i>G</i>. <i>mellonella</i> confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from <i>G</i>. <i>mellonella</i> imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of <i>G</i>. <i>mellonella</i> cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles.</p></div

<i>G</i>. <i>mellonella</i> larvae (A), pupae (B) and adults (C) infected with the entomopathogenic fungus <i>C</i>. <i>coronatus</i> and control groups.

<p>Circles indicate changes occurring during fungal infection.</p

Hydrolysis of cuticular proteins (A), chitin (B) and lipids (C) by <i>C</i>. <i>coronatus</i> enzymes.

<p>Products released during 8 h of incubation are presented as mean±standard deviation μM/mg of cuticle dissected from larvae, pupae or adults (thoraces and wings) of <i>G</i>. <i>mellonella</i>.</p

Correlation coefficients (r) the concentration of fatty acids identified in the cuticle of <i>G</i>. <i>mellonella</i> and the efficiency <i>of C</i>. <i>coronatus</i> proteases, chitinases and lipases in degrading the cuticle.

<p>Correlation coefficients (r) the concentration of fatty acids identified in the cuticle of <i>G</i>. <i>mellonella</i> and the efficiency <i>of C</i>. <i>coronatus</i> proteases, chitinases and lipases in degrading the cuticle.</p

Fatty acid contents in the epicuticular lipids of the <i>Galleria mellonella</i> larvae, pupae and adults (μg/g of insect body).

<p>Fatty acid contents in the epicuticular lipids of the <i>Galleria mellonella</i> larvae, pupae and adults (μg/g of insect body).</p

Relative content (%) of fatty acids in the epicuticular lipids of the <i>Galleria mellonella</i> larvae, pupae and imago.

<p>Relative content (%) of fatty acids in the epicuticular lipids of the <i>Galleria mellonella</i> larvae, pupae and imago.</p

The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus.

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence