Emilia Włóka

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C(16:0), C(16:1), C(18:0), C(18:1), C(18:2), C(18:3), C(20:0) and C(20:1). At high concentrations spore germination was inhibited by C(7:0), C(8:0), C(9:0), C(10:0), C(12:0), C(18:2) and C(18:3) and hyphal growth was merely retarded by C(5:0), C(6:0), C(6:2), C(14:0), C(16:0), C(16:1), C(18:0,) C(18:1), C(20:0) and C(20:1). The presence of C(15:0) at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C(16:0) and C(18-20) fatty acids. Low concentrations of C(5:0), C(6:0), C(6:2) and C(7:0) enhanced sporulation. Fatty acids C(5-12) as well as C(18:3), C(20:0) and C(20:1) decreased the ability of fungal colonies to infect G. mellonella while C(16:1) elevated it thus suggesting that C(16:1) may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C(6:0), C(6:2,) C(7:0), C(9:0), C(12:0), C(16:1), C(18:2), C(18:3), C(20:0) and C(20:1); other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.

Free fatty acids in the cuticular and internal lipids of Calliphora vomitoria and their antimicrobial activity

The cuticular and internal lipid composition in Calliphora vomitoria larvae, pupae, and male and female adults was studied. The free fatty acid (FA) compositions of the lipids were chemically characterized using gas chromatography (GC) and gas chromatography-electron impact mass spectrometry (GC-MS). Analyses of cuticular extracts from larvae, pupae, and male and female adults revealed that the carbon numbers of the acids ranged from C7:0 to C22:0, from C8:0 to C24:0, from C7:0 to C24:0 and from C7:0 to C22:0 respectively. The internal lipids of C. vomitoria larvae, pupae, male and female adults contained FAs ranging from C8:0 to C20:0, from C9:0 to C22:0, from C8:0 to C24:0 and from C9:0 to C22:0 respectively. Nine FAs with odd-numbered carbon chains from C7:0 to C21:0 were identified in the cuticular lipids of the larvae. The internal lipids of C. vomitoria larvae contained 8 odd-numbered FAs ranging from C9:0 to C19:0. Eight odd-numbered FAs from C9:0 to C21:0 were identified in the cuticular and internal lipids of pupae, while nine such FAs were found in the cuticular lipids of male and female adults. The internal lipids of adult males and females respectively contained nine and seven odd-numbered FAs, while both larvae and pupae contained eight such compounds. Eight unsaturated FAs were identified in the cuticular lipids of larvae, adult males and females and also in the internal lipids of females. Seven unsaturated FAs were identified in the cuticular lipids of pupae. The internal lipids of larvae, pupae and males contained 10, 11 and 12 unsaturated FAs respectively. Developmental changes were found both in the amounts of extracted cuticular and internal FAs and in their profiles. Four cuticular FAs (C7:0, C9:0, C10:0 and C15:1), identified as being male-specific, were either absent in the female cuticle or present there only in trace amounts. Cuticular and internal extracts obtained from larvae, pupae, adult males and females were tested for their potential antimicrobial activity. The minimal inhibitory concentrations of extracts against reference strains of bacteria and fungi were determined. Antimicrobial activity was the strongest against Gram-positive bacteria; Gram-negative bacteria, on the other hand, turned out to be resistant to all the lipids tested. Overall, the activities of the internal lipids were stronger. All the lipid extracts were equally effective against all the fungal strains examined. In contrast, crude extracts containing both cuticular and internal lipids displayed no antifungal activity against the entomopathogenic fungus Conidiobolus coronatus, which efficiently killed adult flies, but not larvae or pupae.

Cuticular and internal n-alkane composition of Lucilia sericata larvae, pupae, male and female imagines: application of HPLC-LLSD and GC/MS-SIM

The composition of cuticular and internal n-alkanes in Lucilia sericata larvae, pupae, and male and female imagines were studied. The cuticular and internal lipid extracts were separated by HPLC-LLSD, after which the hydrocarbon fraction was identified by GC/MS in selected ion monitoring (SIM) and total ion current (TIC) modes. The cuticular lipids of the larvae contained seven n-alkanes from C23 to C31. The major n-alkane in L. sericata larvae was C29 (42.1%). The total cuticular n-alkane content in the cuticular lipids was 31.46 μg g-1 of the insect body. The internal lipids of L. sericata larvae contained five n-alkanes ranged from C25 to C31. The most abundant compound was C27 (61.71 μg g-1 of the insect body). Eighteen n-alkanes from C14 to C31 were identified in the cuticular lipids of the pupae. The most abundant n-alkanes ranged from C25 to C31; those with odd-numbered carbon chains were particularly abundant, the major one being C29:0 (59.5%). Traces of eight cuticular n-alkanes were present. The internal lipids of L. sericata pupae contained five n-alkanes, ranging from C25 to C31. The cuticular lipids of female imagines contained 17 n-alkanes from C12 to C30. Among the cuticular n-alkanes of females, C27 (47.5%) was the most abundant compound. Four n-alkanes, with only odd-numbered carbon chains, were identified in the internal lipids of females. The lipids from both sexes of L. sericata had similar n-alkane profiles. The cuticular lipids of adult males contained 16 n-alkanes ranging from C13 to C31. C27 (47.9%) was the most abundant cuticular n-alkanes in males. The same n-alkanes only with odd-numbered carbon chains and in smaller quantities of C27 (0.1%) were also identified in the internal lipids of males. The highest amounts of total cuticular n-alkanes were detected in males and females of L. sericata (330.4 and 158.93 μg g-1 of the insect body, respectively). The quantities of total cuticular alcohols in larvae and pupae were smaller (31.46 μg g-1 and 42.08 μg g-1, respectively). The internal n-alkane contents of larvae, pupae, and male and female imagines were significantly higher than the cuticular n-alkane contents (153.53, 99.60, 360.06 and 838.76 μg g-1 of the insect body, respectively).

Antimicrobial activity of alcohols from Musca domestica

SUMMARY Information on the stimulatory and inhibitory effects of cuticular alcohols on growth and virulence of insecticidal fungi is unavailable. Therefore, we set out to describe the content of cuticular and internal alcohols in the body of housefly larvae, pupae, males and females. The total cuticular alcohols in larvae, males and females of Musca domestica were detected in comparable amounts (4.59, 3.95 and 4.03 μg g−1 insect body, respectively), but occurred in smaller quantities in pupae (2.16 μg g−1). The major free alcohol in M. domestica larvae was C12:0 (70.4%). Internal alcohols of M. domestica larvae were not found. Among cuticular pupae alcohols, C12:0 (31.0%) was the most abundant. In the internal lipids of pupae, only five alcohols were identified in trace amounts. The most abundant alcohol in males was C24:0 (57.5%). The percentage content of cuticular C24:0 in males and females (57.5 and 36.5%, respectively) was significantly higher than that of cuticular lipids in larvae and pupae (0.9 and 5.6%, respectively). Only two alcohols were present in the internal lipids of males in trace amounts (C18:0 and C20:0). The most abundant cuticular alcohols in females were C24:0 (36.5%) and C12:0 (26.8%); only two alcohols (C18:0 and C20:0) were detected in comparable amounts in internal lipids (3.61±0.32 and 5.01±0.42 μg g−1, respectively). For isolated alcohols, antimicrobial activity against 10 reference strains of bacteria and fungi was determined. Individual alcohols showed approximately equal activity against fungal strains. C14:0 was effective against gram-positive bacteria, whereas gram-negative bacteria were resistant to all tested alcohols. Mixtures of alcohols found in cuticular lipids of larvae, pupae, males and females of M. domestica generally presented higher antimicrobial activity than individual alcohols. In contrast, crude extracts containing both cuticular and internal lipids showed no antifungal activity against the entomopathogenic fungus Conidiobolus coronatus, which efficiently kills adult house flies.

Developmental Changes in the Sterol Composition and the Glycerol Content of Cuticular and Internal Lipids of Three Species of Flies

The glycerol concentration and the composition of cuticular and internal sterols in three medically and forensically important fly species, viz., Musca domestica, Sarcophaga carnaria, and Calliphora vicina, were analyzed. The cuticular and internal lipid extracts were separated by HPLC‐LLSD, after which the sterol fraction was characterized by GC/MS in total ion current (TIC) mode. The cuticular lipids of M. domestica larvae contained seven sterols, while in pupae and females, six sterols were identified. Five sterols were found in the cuticular lipids of M. domestica males. The internal lipids of M. domestica larvae and pupae contained six and seven sterols, respectively, while those of male and female flies contained only five sterols. Sitosterol, cholesterol, and campesterol were the dominant sterols in M. domestica, while campestanol, stigmasterol, sitostanol, and fucosterol were identified in low concentrations or in traces. In contrast, cuticular and internal lipids of S. carnaria and C. vicina contained only cholesterol. Glycerol was identified in all stages of M. domestica, S. carnaria, and C. vicina. For all the three examined fly species, the present study clearly showed species‐specific developmental changes in the composition of cuticular and internal sterols as well as in the glycerol concentration.

Application of Two-Way Hierarchical Cluster Analysis for the Identification of Similarities between the Individual Lipid Fractions of Lucilia sericata

The composition of the cuticular and internal lipids of larvae and pupae of Lucilia sericata was studied using chromatographic techniques. The lipids from both stages of L. sericata had similar free fatty acid (FFA) profiles and also contained alcohols and cholesterol. The range of the number of C‐atoms detected for these classes of compounds was to some extent similar in larvae and pupae, but the relative amounts of each class differed between stages. Saturated as well as unsaturated FFAs with even and odd numbered C‐atom chains were present in both cuticular and internal lipids. The alcohol fractions of L. sericata were represented by free, straight‐chain primary alcohols containing an even number of C‐atoms. The lipid composition of male and female L. sericata adults and the hydrocarbon composition of all stages of L. sericata had previously been analyzed. To have a full overview of the lipid composition and to identify similarities or dissimilarities between the individual lipid fractions in this insect species, two‐way hierarchical cluster analysis (HCA) was performed using also the data from these previous publications. The content of FFA 18 : 1 (n‐9) was noticed to be very high in the cuticular fractions of larvae and pupae as well as in all internal fractions (male, female, larvae, and pupae) and low in the cuticular fractions of male and female imago. The contents of FFAs 16 : 0 and 16 : 1 (n‐9), cholesterol, and the n‐alkanes n‐C31, n‐C29, n‐C27, n‐C25, and n‐C23 varied between particular fractions, whereas the amounts of other compounds were similar in all fractions.

Effect of exposure to chlorpyrifos on the cuticular and internal lipid composition of Blattella germanica males.

The results of our research on the cuticular and internal lipids of Blattella germanica males provide new information on variation in the composition of the cuticular and internal lipids of B. germanica males after exposure to the presence of the insecticide. gas chromatography and gas chromatography‐mass spectrometry analyses were used to identify and quantify the cuticular and internal lipid composition in males and males exposed to insecticide. There were significantly more acids having an even number of carbon atoms in the molecule, and these were also generally in higher concentrations. The following acids were in a higher concentration: C16:0 and C18:1, C18:2, C18:0. In both males and males exposed to insecticide, 24 fatty acids ranging from C6 to C22 were determined. However, there was a significantly higher content of fatty acids in the surface lipids of B. germanica males after exposure to insecticide. Our results indicate a higher content of n‐alkanes, sterols, particularly cholesterol, fatty acids, and fatty acid methyl esters in the B. germanica surface after exposure to chlorpyrifos than in males that were not exposed.

Application of headspace solid‐phase microextraction followed by gas chromatography coupled with mass spectrometry to determine esters of carboxylic acids and other volatile compounds in Dermestes maculatus and Dermestes ater lipids

A constant problem in veterinary medicine, human healthcare, agriculture, forestry and horticulture is the large number of pests, and the lack of effective methods to combat them which cause no harm to the rest of the environment. It is recommended and desired to reduce the use of chemicals and increase the use of agents based on knowledge acquired in the fields of biology, chemistry and agrochemicals. To learn the defense mechanisms of insects we should consider not only the site of their physiological ability to protect against external factors (cuticle), but also the possibility of chemical protection, formed by all compounds on the surface and in the body of insects. In this study, a procedure was developed to determine the esters of carboxylic acids in insect lipids. Headspace solid-phase microextraction was followed by gas chromatography coupled with gas spectrometry. First, the best conditions were selected for the analysis to obtain the best chromatographic separation. An RTx-5 column was used for this purpose. Polydimethylsiloxane/divinylbenzene (PDMS/DVB) and polyacrylate fibers were used to isolate acid esters. PDMS/DVB fiber achieved the best conditions for the extraction; the extraction time was 50 min, the extraction temperature was 105°C and the desorption time was 10 min at 230°C. These solid-phase microextraction conditions were used to analyze volatile compounds extracted from insects belonging to the Dermestidae family.

Erratum to: Fatty acids and amino acids of entomopathogenic fungus Conidiobolus coronatus grown on minimal and rich media

Entomopathogenic fungi are referred to as potential candidates as insect pest control agents. The objective of the study was to identify fatty acids and amino acids from Conidiobolus coronatus cultured on two different media. Each medium was extracted with ethyl acetate and its mixtures with isopropanol, acetonitrile and methanol. Analyses of fatty acids and amino acids of entomopathogenic fungus C. coronatus were performed by means of gas chromatography coupled with mass spectrometry. The analysis showed that the fungus C. coronatus produces the following groups of compounds: fatty acids and amino acids; α- and β-glucopyranose were also identified. The identified fatty acids included 12–20, 22 and 24 carbon atoms per chain. The highest content of fatty acids was detected in a mycelium sample cultured in a liquid minimal medium extracted with ethyl acetate. The lowest content of these organic compounds was identified in mycelium cultured in a liquid nutrient-rich medium extracted with ethyl acetate–methanol mixture. Fatty acids were found to account for 62.0 mass % to 94.4 mass % of all organic compounds in the analyzed mycelia. C18:1 acids were detected in the highest amounts when ethyl acetate was used as the extracting agent. The identified amino acids accounted for 4 mass % to 21 mass % of all organic compounds. Upon extraction of C. coronatus mycelium samples with the ethyl acetate—methanol mixture, two anomeric forms of glucose were also identified. An analysis of the studied material confirmed, that the entomopathogenic fungus C. coronatus is a very rich source of organic compounds, which might encourage its further research so as to identify an even larger number of compounds being produced by this species.

The effect of the entomopathogenic fungus Conidiobolus coronatus on the composition of cuticular and internal lipids of Blatta orientalis females

The composition of cuticular and internal lipids in females of the cockroach Blatta orientalis L. exposed to the entomopathogenic fungus Conidiobolus coronatus is investigated. The compositions of the fatty acids, n‐alkanes, alcohol, sterols and methyl esters in the lipids are chemically characterized. Although contact with virulent colonies of the fungus does not induce insect mortality, significant changes in the lipid profiles, both cuticular and internal, are found. The cuticular extracts of a control group of B. orientalis females contain 24 compounds varying in carbon chain length from C6 to C22. The main cuticular fatty acids identified are: C16:1, C16:0, C18:1 and C18:0. The cuticular lipids of B. orientalis females after exposure to C. coronatus contain only 14 free fatty acids from C8 to C20. The highest concentrations identified are C16:0, C18:2 and C18:1. Analysis by gas chromatography‐mass spectrometry identifies the presence of a homologous series of n‐alkanes containing from 25 to 31 carbon atoms. In the case of the insects after fungal exposure, the content of the n‐alkanes in the cuticular lipid is two‐fold higher compared with the controls. Of the cuticular lipids, 11 alcohols are found, ranging from C12:0 to C20:0. There is no presence of alcohols in the internal lipids of the control B. orientalis females and in all of the extracts from the B. orientalis females after fungal exposure. In the samples analyzed, the most common sterol is cholesterol. This is present in the cuticular lipids and the internal lipids of all of the insects sampled. The cuticular and internal lipids of females contain five fatty acid methyl esters, ranging in size from C15 to C19.