Discovery of novel trimethylalkanes in the internal hydrocarbons of developing pupae of Heliothis virescens and Helicoverpa zea

Novel trimethyl-branched alkanes which eluted with the monomethylalkanes were identified in the internal lipids of Helicoverpa zea but were not present in Heliothis virescens. Their structures were unique in that the first methyl branch occurred on carbon 2 and the 2nd and 3rd methyl branch points were separated by a single methylene. Novel trimethylalkanes identified from their chemical ionization and electron impact mass spectra were 2,18,20-trimethyltetratriacontane, 2,18,20-trimethylhexatriacontane, and 2,24,26-trimethyldotetracontane. Previous reports did not find these trimethylalkanes in the cuticular surface lipids of larvae, pupae or adults of either species. The internal pupal hydrocarbons of H. virescens and H. zea amounted to 123 μg and 304 μg per pupa, respectively. They consisted of n-alkanes (8 and 4%, respectively) and methyl-branched alkanes (88 and 94%, respectively). The n-alkanes ranged in chain length from approximately 21 to 35 carbons and the methyl-branched alkanes from approximately 26 to 55 carbons vs. methyl-branched alkanes from 28 to 37 carbons previously reported for hydrocarbons from the pupal cuticular surface. The major n-alkane was heptacosane (3.3 and 1.2%, respectively, in H. virescens and H. zea). The major methyl-branched alkanes in H. virescens were methylhentriacontane (15%), methyltritriacontane (12%) and dimethyltritriacontane (10%), and in H. zea were methylnonacosane (17%), dimethylnonacosane (9%) and methylhentriacontane (20%). Except for the novel trimethylalkanes, the methylalkane branch points were predominantly on odd-numbered carbons as has been reported for these and other species.     

Hydrocarbons of Rhodnius prolixus, a Chagas disease vector

The surface hydrocarbons of the blood-sucking insect, Rhodnius prolixus, a major Chagas disease vector in Venezuela, Colombia and Central America, were characterized by capillary gas chromatography coupled to mass spectrometry (CGC-MS). A total of 54 single or multicomponent peaks of saturated, straight-chain and methyl-branched hydrocarbons were identified. Major n-alkanes were n-C27, n-C29, n-C31 and n-C33 hydrocarbons. In the branched fraction, methyl groups were at positions 3, 5, 7, 11, 13, 15 and 17- for monomethyl isomers, and separated by three or five methylene groups for the trimethyl or tetramethyl derivatives. For the higher molecular weight components of 37, 39 and 41 atoms in the carbon skeleton, the di-, tri- and tetramethyl branches were usually separated by three or five, and sometimes 7, 11 or 13, methylene groups. The internal hydrocarbon pool contained larger amounts of the higher molecular weight methyl-branched components. Qualitative differences among epicuticular and internal hydrocarbon compositions were detected, both in adult and nymphal stages. No significant sexual dimorphism was detected, but a significant shift in the major n-alkane components was evident from the nymphal to the adult stage, differing also in the relative amounts of the higher molecular weight methyl-branched chains. Comparison of the hydrocarbon components to that of other Chagas disease vectors is discussed.     

Prevalence and distribution of parasites and pathogens of Triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV

Chagas’ disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n = 1646), Triatoma guasayana (n = 4), Triatoma platensis (n = 1) and Triatoma sordida (n = 5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina.     

Seasonal changes in cuticular hydrocarbons in response to polyphenism in the host-alternating aphid Prociphilus oriens

In most terrestrial arthropods, cuticular hydrocarbons (CHCs) function to assist in desiccation tolerance and chemical communications. However, few studies have clarified whether CHC profiles change among developmental stages or among different morphs in non-social insects. In the present study, we evaluated how CHC profiles change in accordance with polyphenism in the host-alternating aphid Prociphilus oriens, which exhibits a complex life cycle and five distinct morphs. These morphs are sexual or asexual and adapt to different host plants. We found that all generations of P. oriens shared high proportions of n-alkanes, but its composition varied among morphs. Three morphs that are attended by ants were characterized by relatively high proportions of n-C25 to n-C27, whereas two morphs that are not attended by ants had higher proportions of longer-chain n-alkanes, such as n-C27 and n-C29. The CHC profiles of sexual females were largely different from those of males. Considering that sexual females of Prociphilus spp. lack organs that secrete sex pheromones (scent plaques), the CHCs of sexual females are likely to function as a sex attractant. High proportions of methyl-branched alkanes were detected in the long and flocculent waxy substances of autumnal migrants. These methyl-branched alkanes are considered a cue to recognize conspecifics. We concluded that the functions and components of CHCs differ among morphs, and that those of sexual females differ from those of males and asexual generations because of their function in sexual communication.     

Cuticular hydrocarbons of triatomines

Triatomine insects (Hemiptera) are the vectors of Chagas disease. Their cuticular surface is covered by a thin layer of lipids, mainly hydrocarbons, wax esters, fatty alcohols, and free or esterified fatty acids. These lipids play a major role in preventing a lethal desiccation, altering the absorption of chemicals and microorganism penetration, they also participate in chemical communication events. Lipid components are biosynthetically related, the synthesis of long chain and very long chain fatty acids was first shown in the integument of Triatoma infestans through the concerted action of fatty acid synthases (FAS's) and fatty acyl-CoA elongases. A final decarboxylation step produces the corresponding hydrocarbon. Capillary gas chromatography coupled to mass spectrometry analyses showed that cuticular hydrocarbons of Triatominae comprise saturated straight and methyl-branched chains, from 18 to more than 43 carbon atoms. Odd-chain hydrocarbons, mostly from 27 to 33 carbons, are the major straight chains. Different isomers of mono, di, tri, and tetramethylcomponents, mostly from 29 to 39 atoms in the carbon skeleton, account for the major methyl-branched hydrocarbons. The presence, absence, and relative quantities of these hydrocarbons represent characters for their chemical phenotype, and are useful for differentiating genera, species and populations. In this review, we will discuss the metabolic pathways involved in hydrocarbon formation, and their structure, together with their role in insect survival. We will also review the utility of cuticular hydrocarbon fingerprints in chemotaxonomy.     

Cuticular hydrocarbons corroborate the distinction between lowland and highland Natal fruit fly (Tephritidae,Ceratitis rosa) populations

The cuticular hydrocarbons (CHs) and morphology of two Ceratitis rosa Karsch (Diptera: Tephritidae) populations, putatively belonging to two cryptic taxa, were analysed. The chemical profiles were characterised by two-dimensional gas chromatography with mass spectrometric detection. CHs of Ceratitis rosa that originated from the lowlands and highlands of Kenya comprised of n-alkanes, monomethylalkanes, dimethylalkanes and unsaturated hydrocarbons in the range of the carbon backbone from C₁₄ to C₃₇. Hydrocarbons containing C₂₉, C₃₁, C₃₃ and C₃₅ carbon atoms predominated in these two populations. 2-Methyltriacontane was the predominant compound in both populations. Quantitative differences in the distribution of hydrocarbons of different chain lengths, mainly the C₂₂, C₃₂, C₃₃ and C₃₄ compounds of these two populations, were observed despite indistinct qualitative differences in these hydrocarbons. Morphological analyses of male legs confirmed that the flies belong to different morphotypes of Ceratitis rosa previously labelled as R1 and R2 for lowland and highland populations, respectively. A statistical analysis of the CH compositions of the putative R1 and R2 species showed distinct interspecific identities, with several CHs specific for each of the lowland and highland populations. This study supports a hypothesis that the taxon Ceratitis rosa consists of at least two biological species.     

Methyl-branched hydrocarbons, major components of the waxy material coating the embryos of the viviparous cockroach Diploptera punctata

The viviparous cockroach Diploptera punctata carries a wax-coated batch of embryos in a brood sac. When the embryos are expelled into saline, flakes of wax from the surface of the embryos float to the surface. In contrast, embryos of the ovoviviparous species such as Rhyparobia maderae are not nourished by the mother during embryogenesis and do not have a copious waxy coating. As a first step in determining the function of this copious wax layer on the batch of embryos of D. punctata, its composition was compared to that of the waxy material on the outer cuticular surface of the mother (female cuticle) by thin-layer chromatography (TLC) and gas chromatography-mass spectrometry. The major lipid class on the embryos was hydrocarbons with lesser amounts of wax esters and long-chain alcohols. Hydrocarbons from both sources had similar elution times and chemical composition, but were markedly different in the amounts of the major methyl-branched hydrocarbon components. A mixture of 3,X-dimethyl alkanes were 44% of the hydrocarbons on the embryos and were only 29% on the female cuticle. However, trimethylalkanes were only 22% of the hydrocarbons on the embryos and were 34% of the hydrocarbons on the female cuticle. The major hydrocarbons from both sources were mixtures of methyl-branched alkanes with backbones of 33 and 35 carbon atoms. Methyl-branched tritriacontanes were 59% of embryo and 35% of female cuticular hydrocarbons; methyl-branched pentatriacontanes were 19% of embryo and 42% of female hydrocarbons. The difference in proportions of the similar hydrocarbons on the outer cuticular surface of the female and those covering the embryos may suggest that the evolution of copious nutrient secretion for the embryos was accompanied by selection for a mixture of hydrocarbons that prevents water loss by the embryos and protects them against invasion by microorganisms without preventing the movement of nutrient fluid into the embryos.     

Characterization and discrimination of three Rhopalosiphum species (Homoptera: aphididae) using cuticular hydrocarbons and cuticular surface patterns

• 1.1. Cuticular hydrocarbons of two clones of Rhopalosiphum maidis, two of R. padi, one of R. insertum, and one of Schizaphis graminum were identified as n-alkanes, monomethylalkanes and dimethylalkanes.
• 2.2. No qualitative differences in hydrocarbon content were apparent among the six aphid populations studied; however, hydrocarbon profiles were discriminatory.
• 3.3. Discriminant analysis of the proportions of the cuticular hydrocarbons selected 29 hydrocarbon components that provided discrimination among populations except for the two R. padi clones which were indistinguishable.
• 4.4. Scanning electron micrographs showed very clear differences in the cuticular surface patterns among the three Rhopalosiphum species.

     

Chemical basis for inter-colonial aggression in the stingless bee Scaptotrigona bipunctata (Hymenoptera: Apidae)

Inter-colonial aggression was tested using three colonies of Scaptotrigona bipunctata in a natural setting when their nests were moved and by artificial contact between individuals. Examination of the cuticular lipids of individuals from two colonies kept under identical conditions showed clear differences in their cuticular hydrocarbon profiles. The cuticular lipids were a mixture of hydrocarbons (saturated and unsaturated alkanes and alkenes) within the range of C23-C29. The use of multivariate analysis (PCA and discriminant analysis) showed that seven of the identified surface compounds are enough to separate workers from colonies A and B from each other.     

Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain

A novel bacterial strain, DQ12-45-1b, was isolated from the production water of a deep subterranean oil-reservoir. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia with both alkB (coding for alkane monooxygenase) and CYP153 (coding for P450 alkane hydroxylase of the cytochrome CYP153 family) genes and their induction detected. It was capable of utilizing a wide range of n-alkanes (C6-C40), aromatic compounds and crude oil as the sole carbon sources for growth. In addition, it preferentially degraded short-chain hydrocarbons (?C25) in the early cultivation phase and accumulated hydrocarbons with chain-lengths from C23 to C27 during later cultivation stage with crude oil as the sole carbon source. This is the first study to report the different behaviors of a bacterial species toward crude oil degradation as well as a species of Dietzia degrading a wide range of hydrocarbons.     

Biosynthetic and environmental effects on the stable carbon isotopic compositions of anteiso- (3-methyl) and iso- (2-methyl) alkanes in tobacco leaves

Nicotiana tabacum is the only plant known to synthesise large quantities of anteiso- (3-methyl) alkanes and iso- (2-methyl) alkanes. We investigated the carbon isotope ratios of individual long-chain n-alkanes, anteiso- and iso-alkanes (in the C₂₉-C₃₃ carbon number range) extracted from tobacco grown in chambers under controlled conditions to confirm the pathway used by the tobacco plant to synthesise these particular lipids and to examine whether environmental data are recorded in these compounds. Tobacco was grown under differing temperatures, water availabilities and light intensities in order to control its stable carbon isotope ratios and evaluate isotopic fractionations associated with the synthesis of these particular lipids. The anteiso-alkanes were found to have a predominant even-carbon number distribution (maximising at C₃₂), whereas the iso-alkanes exhibit an odd-carbon number distribution (maximising at C₃₁). Iso-alkanes were relatively more abundant than the anteiso-alkanes and only two anteiso-alkanes (C₃₀ and C₃₂) were observed.The anteiso-alkanes and iso-alkanes were found to be enriched in ¹³C by 2.8-4.3‰ and 0-1.8‰ compared to the n-alkanes, respectively, consistent with different biosynthetic precursors. The assumed precursor for the odd-carbon-numbered iso-alkanes is iso-butyryl-CoA (a C₄ unit derived from valine) followed by subsequent elongation of C₂ units and then decarboxylation. The assumed precursor for even-carbon-numbered anteiso-alkanes is α-methylbutyryl-CoA (a C₅ unit derived from isoleucine) and subsequent elongation by C₂ units followed by decarboxylation. The ratio of carbon atoms derived from α-methylbutyryl-CoA and subsequent C₂ units (from malonyl-CoA) is 1:5 for the biosynthesis of a C₃₀anteiso-alkane. The ratio of carbon atoms derived from iso-butyryl-CoA and subsequent C₂ units (from malonyl-CoA) is 4:25 for the synthesis of a C₂₉iso-alkane. An order of ¹³C depletion n-alkanes > iso-alkanes > anteiso-alkanes is evident from compound specific isotope data. This trend can probably be attributed to the ratio of the two different sources of carbon atoms in the final wax components.Higher water availability generally results in more depleted stable carbon isotope ratios due to maximised discrimination during carboxylation, associated with less diffusional limitation. This was confirmed in the present study by compound specific isotope analyses of iso-alkanes, anteiso-alkanes and n-alkane lipids extracted from the tobacco leaves. Likewise, light intensity has been shown to influence plant bulk δ¹³C in previous studies. The carbon isotope ratios of n-alkanes in tobacco grown under low-light conditions were about 2‰ more depleted in ¹³C than those of lipids extracted from tobacco grown under elevated light conditions. A similar order of difference is observed for the iso-alkanes and anteiso-alkanes (1.8‰ and 1.9‰, respectively). A negligible depletion in carbon isotope ratios was observed for the iso-alkanes and anteiso-alkanes extracted from tobacco grown under elevated temperatures. These results are consistent with the work of Farquhar [Farquhar, G.D., 1980. Carbon isotope discrimination by plants: effects of carbon dioxide concentration and temperature via the ratio of intercellular and atmospheric CO₂ concentrations. In: Pearman, G.I. (Ed.), Carbon Dioxide and Climate: Australian Research. Springer, Berlin, pp. 105-110] where temperature appears to have only a minor effect on plant bulk δ¹³C.     

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.     

Qualitative and quantitative differences in cuticular hydrocarbons between laboratory and field colonies of Pogonomyrmex barbatus

Ants held in the laboratory and field ants of the species Pogonomyrmex barbatus have quantitative differences in their cuticular hydrocarbons and a qualitative difference in their methyl-branched hydrocarbons. Laboratory-held workers showed twice the hydrocarbon content as field ants. This difference was mainly due to higher amounts of straight-chain alkanes and methyl-branched alkanes in laboratory ants, whereas the proportion of the alkenes remained the same for both groups. In addition to the absence of some hydrocarbons in the field colonies, one of the methyl-branched hydrocarbons differed in amount and branching pattern between the two groups of ants. Whereas, notable peaks of 2-methylalkanes were identified in ants kept in the laboratory, these compounds could not be identified in ants living in their natural habitat. However, a trace amount of 4-methyltriacontane was found in lieu of the 2-methyltriacontane counterpart in field ants. Possible explanations for both qualitative and quantitative differences are discussed.     

Biosynthetic origin of the saw-toothed profile in δC and δΗ of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (¹³C) and hydrogen (²H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in ¹³C but relatively depleted in ²H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in ¹³C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (²H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of ²H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly ²H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.     

Cuticular hydrocarbons of Chagas disease vectors in Mexico

Capillary gas-liquid chromatography was used to analyse the cuticular hydrocarbons of three triatomine species, Triatoma dimidiata, T. barberi and Dipetalogaster maxima, domestic vectors of Chagas disease in Mexico. Mixtures of saturated hydrocarbons of straight and methyl-branched chains were characteristic of the three species, but quantitatively different. Major methylbranched components mostly corresponded to different saturated isomers of monomethyl, dimethyl and trimethyl branched hydrocarbons ranging from 29 to 39 carbon backbones. Sex-dependent, quantitative differences in certain hydrocarbons were apparent in T. dimidiata.     

Varroa destructor changes its cuticular hydrocarbons to mimic new hosts

Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host''s cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.     

Regional variation in cuticular hydrocarbon composition in the common house cricket,Acheta domesticus

• 1.1. The hydrocarbon composition of different cuticular regions (pronotum, legs, abdominal tergites and sternites, pleural membrane) was determined for adult female crickets (Acheta domesticus).
• 2.2. Hydrocarbon groups included n-alkanes, 2-methylalkanes, long-chain internally branched monomethyl- and dimethyalkanes, n-alkenes, 2-methylalkenes and alkadienes.
• 3.3. Saturated hydrocarbons were more abundant than unsaturated hydrocarbons and branched saturates more abundant than n-alkanes in all regions of the cuticle examined.
• 4.4. Except for a higher percentage of n-alkanes in the pleural membrane (soft cuticle), little difference was noted in compositional patterns or relative amounts of individual molecules from the different cuticular regions.

     

Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus

The cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus, were found to contain minor amounts of novel wax esters, in addition to the major components, hydrocarbons. The wax esters ranged in carbon number from C19 to C31 and consisted of esters of both odd- and even-numbered alcohols and acids. Each wax ester with a given carbon number eluted at several different retention times indicating possible methyl branching in either the fatty acid or alcohol moiety, or in both moieties. Each eluting peak of wax esters consisted of a mixture of wax esters of the same carbon number in which the fatty acid moiety ranged from C8 to C18, and the alcohol moiety ranged from C8 to C17. Some wax esters were largely found on the head indicating they may be of a glandular origin. The hydrocarbons consisted of: n-alkanes, C23 to C33; odd-numbered n-alkenes, C27 to C35; and the major components, methyl-branched alkanes, C26 to over C49. Notable components of the methyl-branched alkanes were 2-methyltriacontane, and the novel trimethylalkanes with a single methylene between the first and second branch points, 13,15,19-trimethylhentriacontane and 13,15,21-trimethyltritriacontane.     

Anaerobic oxidation of long-chain n-alkanes by the hyperthermophilic sulfate-reducing archaeon,Archaeoglobus fulgidus

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C₁₀–C₂₁) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C₁₆) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.     

Cuticular hydrocarbon composition and phenotypic variability in sympatric populations of Ixodes ricinus ticks from Poland

Gas chromatography of cuticular hydrocarbons was used to assess genetic similarity and heterozygosity between 18 sympatric populations of adult Ixodes ricinus ticks collected from Poland. Sixteen n-alkanes, 45 monomethylalkanes, and 36 dimethylalkanes were identified. Forty-one compounds were detected in 90% of the specimens and nine hydrocarbons were present in all the specimens studied. Visual inspection of chromatograms revealed major differences in the relative abundance of hydrocarbons between populations of I. ricinus. The average proportion of heterozygotic hydrocarbons was 50.68%, while the average genetic similarity among all populations was 0.967. Two other methods were used to measure the intrapopulation variability: the number of isomeric forms for each hydrocarbon and the range of hydrocarbon abundance. It was found that some hydrocarbons manifested an unexpectedly high number of isomeric patterns, such as nonacosane (21 patterns), triacontane (23), pentacosane (26), and hexacosane (28). Our data suggest that I. ricinus ticks from Poland consist of several interbreeding, non-isolated populations, with a high degree of genetic flow between them.