A Study of Cuticular Hydrocarbons of All Life Stages in Sarcophaga peregrina (Diptera: Sarcophagidae).

Sarcophaga peregrina (Robineau-Desvoidy, 1830), a synanthropic flesh fly species found in different parts of the world, is of medical and forensic importance. Traditional methods of inferring developmental age rely on the life stage of insects and morphological changes. However, once the larvae reach the pupal and adult stage, morphological changes would become barely visible, so that the classic method would be invalid. Here, we studied the cuticular hydrocarbon profile of S. peregrina of the whole life cycle from larval stage to adult stage by GC-MS. Sixty-three compounds with carbon chain length ranging from 8 to 36 were detected, which could be categorized into four classes: n-alkanes, branched alkanes, alkenes, and unknowns. As developmental increased, branched alkanes dominant, and the content of high-molecular-weight hydrocarbons is variable, especially for 2-methyl C19, DiMethyl C21, docosane (C22), and tricosane (C23). This study shows that the composition of CHC could be used to determine the developmental age of S. peregrina and aid in postmortem interval estimations in forensic science.

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial ß-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata.

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of ß-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of ß-Ala-Tyr and its admixtures have been converted into preparative purification of ß-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the ß-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Unraveling complex molecular transformations of N-ß-alanyldopamine that account for brown coloration of insect cuticle.

RATIONALE: N-ß-Alanyldopamine (NBAD) and N-acetyldopamine (NADA) are catecholamines that are used by insects as sclerotizing precursors to harden their cuticle. They share a common pathway utilizing the same set of sclerotizing enzymes. Yet, cuticles using NBAD are brown, while cuticles using NADA are colorless. To identify the cause of this major unresolved color difference, molecular transformations of NBAD with cuticular enzymes were investigated. METHODS: Reactions of NBAD and NADA with native cuticle isolated from the wandering stages of Sarcophaga bullata larvae as well as the reactions of NBAD with cuticular sclerotization enzymes - phenoloxidase, quinone isomerase and quinone methide isomerase - were investigated using UV-Vis spectroscopy, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). In addition, the reactivity of enzymatically generated NBAD quinone was investigated by MS. RESULTS: Reactions of NBAD with sclerotizing enzymes isolated from Sarcophaga bullata larvae generate colorless products such as N-ß-alanylnorepinephrine, N-ß-alanylarterenone, dehydro NBAD, the benzodioxan dimers of dehydro NBAD and other minor adducts, the same kind of compounds generated by NADA reaction with cuticular enzymes. However, oxidation of NBAD produces colored quinone adducts, in addition. NADA, which lacks the amino group, did not produce these quinone adducts. CONCLUSIONS: LC/MS analysis of the reaction mixture of NBAD-cuticular enzyme reactions reveals the novel production of colored quinone adducts that are not possible for NADA. Therefore, our results suggest that the brown coloration of cuticle formed through NBAD crosslinking is likely due to the formation and accumulation of NBAD quinone and its adducts, while NADA quinone adducts tend not to form during NADA crosslinking, producing a nearly colorless cuticle.

Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via resazurin-based turbidometric assay.

BACKGROUND: Antimicrobial resistance is currently a major global issue. As the rate of emergence of antimicrobial resistance has superseded the rate of discovery and introduction of new effective drugs, the medical arsenal now is experiencing shortage of effective drugs to combat diseases, particularly against diseases caused by the dreadful multidrug-resistant strains, such as the methicillin-resistant Staphylococcus aureus (MRSA). The ability of fly larvae to thrive in septic habitats has prompted us to determine the antibacterial activity and minimum inhibitory concentrations (MICs) of larval extract of flies, namely Lucilia cuprina, Sarcophaga peregrina and Musca domestica against 4 pathogenic bacteria [Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli] via a simple and sensitive antibacterial assay, resazurin-based turbidometric (TB) assay as well as to demonstrate the preliminary chemical profile of larval extracts using gas chromatography-mass spectrophotometry (GC-MS). RESULTS: The resazurin-based TB assay demonstrated that the L. cuprina larval extract was inhibitory against all tested bacteria, whilst the larval extract of S. peregrina and M. domestica were only inhibitory against the MRSA, with a MIC of 100 mg ml-1. Subsequent sub-culture of aliquots revealed that the larval extract of L. cuprina was bactericidal against MRSA whilst the larval extracts of S. peregrina and M. domestica were bacteriostatic against MRSA. The GC-MS analysis had quantitatively identified 20 organic compounds (fatty acids or their derivatives, aromatic acid esters, glycosides and phenol) from the larval extract of L. cuprina; and 5 fatty acid derivatives with known antimicrobial activities from S. peregrina and M. domestica. CONCLUSION: The resazurin-based turbidometric assay is a simple, reliable and feasible screening assay which evidently demonstrated the antibacterial activity of all fly larval extracts, primarily against the MRSA. The larval extract of L. cuprina exerted a broad spectrum antibacterial activity against all tested bacteria. The present study revealed probable development and use of novel and effective natural disinfectant(s) and antibacterial agent(s) from flies and efforts to screen more fly species for antibacterial activity using resazurin-based TB assay should be undertaken for initial screening for subsequent discovery and isolation of potential novel antimicrobial substances, particularly against the multi-drug resistant strains.

New MALDI matrices based on lithium salts for the analysis of hydrocarbons and wax esters.

Lithium salts of organic aromatic acids (lithium benzoate, lithium salicylate, lithium vanillate, lithium 2,5-dimethoxybenzoate, lithium 2,5-dihydroxyterephthalate, lithium α-cyano-4-hydroxycinnamate and lithium sinapate) were synthesized and tested as potential matrices for the matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry analysis of hydrocarbons and wax esters. The analytes were desorbed using nitrogen laser (337.1 nm) and ionized via the attachment of a lithium cation, yielding [M + Li](+) adducts. The sample preparation and the experimental conditions were optimized for each matrix using stearyl behenate and n-triacontane standards. The performance of the new matrices in terms of signal intensity and reproducibility, the mass range occupied by matrix ions and the laser power threshold were studied and compared with a previously recommended lithium 2,5-dihydroxybenzoate matrix (LiDHB) (Cvacka and Svatos, Rapid Commun. Mass Spectrom. 2003, 17, 2203). Several of the new matrices performed better than LiDHB. Lithium vanillate offered a 2-3 times and 7-9 times higher signal for wax esters and hydrocarbons, respectively. Also, the signal reproducibility improved substantially, making this matrix a suitable candidate for imaging applications. In addition, the diffuse reflectance spectra and solubility of the synthesized compounds were investigated and discussed with respect to the compound's ability to serve as MALDI matrices. The applicability of selected matrices was tested on natural samples of wax esters and hydrocarbons.

The antifungal activity of fatty acids of all stages of Sarcophaga carnaria L. (Diptera: Sarcophagidae).

Fatty acids as components of cuticular lipids of insects play a significant role in antifungal in protection against fungal infection. The chemical composition of cuticular and internal extracts obtained from all developmental stages of flesh flies Sarcophaga carnaria was identified. The fatty acids were detected using gas chromatography coupled with mass spectrometry and the most abundant for all examined stages were: 18:1 > 16:0 > 16:1 > 18:0 > 18:2. Polyunsaturated fatty acids (PUFA) C20 were found in both, cuticular and internal extracts. GC-MS analysis showed higher relative content of PUFA in adults than in preimaginal stages. Fatty acids alone as well as their cuticular and internal extracts obtained from larvae, pupae male and female of S. carnaria were tested according to their potential antimicrobial activity against entomopathogenic fungi: Paecilomyces lilacinus, Paecilomyces fumosoroseus, Lecanicillium lecanii, Metarhizium anisopliae, Beauveria bassiana (Tve-N39) and B. bassiana (Dv-1/07). FA presented diverse antimicrobial activity depending on the length of the chain and the presence of unsaturated bonds. Short chain and unsaturated FA (6:0, 11:0, 13:0) have shown significantly stronger activity against fungi but they were detected in lower concentrations. PUFA inhibit fungal growth more effectively than unsaturated long chain fatty acids. Cuticular and internal extracts of all living forms of S. carnaria exhibited approximately equal activity against tested entomopathogenic fungi. We presumed that the most abundant saturated long chain FA and additionally PUFA founded in our analysis are involved in protecting the flies against fungal infection.

Cuticular hydrocarbons as a tool for the identification of insect species: puparial cases from Sarcophagidae.

The external surface of all insects is covered by a species-specific complex mixture of highly stable, very long chain cuticular hydrocarbons (CHCs). Gas chromatography coupled to mass spectrometry was used to identify CHCs from four species of Sarcophagidae, Peckia (Peckia) chrysostoma, Peckia (Pattonella) intermutans, Sarcophaga (Liopygia) ruficornis and Sarcodexia lambens. The identified CHCs were mostly a mixture of n-alkanes, monomethylalkanes and dimethylalkanes with linear chain lengths varying from 23 to 33 carbons. Only two alkenes were found in all four species. S. lambens had a composition of CHCs with linear chain lengths varying from C23 to C33, while the other three species linear chain lengths from 24 to 31 carbons. n-Heptacosane, n-nonacosane and 3-methylnonacosane, n-triacontane and n-hentriacontane occurred in all four species. The results show that these hydrocarbon profiles may be used for the taxonomic differentiation of insect species and are a useful additional tool for taxonomic classification, especially when only parts of the insect specimen are available.

Determining the age of adult flesh flies, Boettcherisca peregrina, using pteridine fluorescence.

To assess the potential application of pteridine fluorescence in determining the age of adult Boettcherisca peregrina (Diptera: Sarcophagidae) Robineau-Desvoidy and further for the postmortem interval, the age-dependent changes of pteridine fluorescence were investigated for the adults maintained at five constant temperatures. From the results, significant linear relationships were found between pteridine fluorescence and the age of the adults maintained at 16, 20, 24, 28 or 32 °C (P < 0.001, r(2) > 0.85). In addition, the relationships between the rate of pteridine accumulation and temperature were well described using linear equations for adult females and males. Then for each cohort of the flies at the ambient temperature, a calendar was constructed and used to determine the ages of females and males, respectively, in which was recorded in reverse time order the amount of pteridine accumulated per hour by the flies and their expected pteridine level when they emerged at the specified time. A significant linear relationship between estimated ages and chronological ages was observed for female or male adults, with the mean errors of the estimated ages of ±1.82 days for females and ±1.58 days for males. It is suggested that pteridine fluorescence analysis has a potential value in determining the age of adult B. peregrina.

Oostatic peptides containing D-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study.

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Reexamination of the mechanisms of oxidative transformation of the insect cuticular sclerotizing precursor, 1,2-dehydro-N-acetyldopamine.

1,2-dehydro-N-acetyldopamine (dehydro NADA) is an important catecholamine derivative formed during the sclerotization of insect cuticle. Earlier we have reported that tyrosinase-catalyzed oxidation of dehydro NADA produces a reactive quinone methide imine amide that forms adducts and cross-links through its side chain, thereby accounting for sclerotization reactions. Recently, laccase has also been identified as a key enzyme associated with sclerotization. Hence, we re-examined oxidation of dehydro NADA by tyrosinase and laccase using high performance liquid chromatography - tandem mass spectrometry. Tyrosinase-catalyzed oxidation of dehydro NADA not only generated dimers as reported earlier, but also generated significant amounts of oligomers. The course of laccase-catalyzed oxidation of dehydro NADA significantly differed from the tyrosinase reaction kinetically and mechanistically. Laccase failed to produce any detectable quinone or quinone methide as the primary two-electron oxidation product. Since laccases are known to generate primarily semiquinones as the initial products, lack of accumulation of two-electron oxidation products indicated that laccase reaction is primarily occurring via free radical coupling mechanism. Consistent with this proposal, laccase-catalyzed oxidation of dehydro NADA, resulted in the production of largely dimeric products and failed to produce any significant amount of oligomeric materials. These studies call for radical coupling as yet another major mechanism for sclerotization of insect cuticle.