Variation in mouse chemical signals is genetically controlled and environmentally modulated.

In most mammals and particularly in mice, chemical communication relies on the detection of ethologically relevant fitness-related cues from other individuals. In mice, urine is the primary source of these signals, so we employed proteomics and metabolomics to identify key components of chemical signalling. We show that there is a correspondence between urinary volatiles and proteins in the representation of genetic background, sex and environment in two house mouse subspecies Mus musculus musculus and M. m. domesticus. We found that environment has a strong influence upon proteomic and metabolomic variation and that volatile mixtures better represent males while females have surprisingly more sex-biased proteins. Using machine learning and combined-omics techniques, we identified mixtures of metabolites and proteins that are associated with biological features.

Perioral secretions enable complex social signaling in African mole-rats (genus Fukomys).

Subterranean common mole-rats of the genus Fukomys (family Bathyergidae) live in large, cooperatively-breeding families. Odor cues have been hypothesized to play an important role in mediating social behaviors in the underground ecotope, but only little is known about the role of olfactory signaling in burrowing mammals. Here we characterize the so far neglected perioral glands of Fukomys and other African mole-rats as an important source of olfactory social information. Histology demonstrates these structures to be derived sebaceous glands that are developed regardless of sex and reproductive status. However, gland activity is higher in Fukomys males, leading to sexually dimorphic patterns of stain and clotting of the facial pelage. Behavioral assays revealed that conspecifics prefer male but not female perioral swabs over scent samples from the back fur and that male sebum causes similar attraction as anogenital scent, a known source of social information in Fukomys. Finally, we assessed volatile compounds in the perioral sebum of the giant mole-rat (Fukomys mechowii) via GCxGC-MS-based metabolomic profiling. Volatiles display pronounced sex-specific signatures but also allow to differentiate between intrasexual reproductive status groups. These different lines of evidence suggest that mole-rat perioral glands provide complex odor signals which play a crucial role in social communication.

Loss of hexokinase 1 sensitizes ovarian cancer to high-dose metformin.

BACKGROUND: Hexokinases (HKs) are well-studied enzymes catalyzing the first step of glycolysis. However, non-canonical regulatory roles of HKs are still incompletely understood. Here, we hypothesized that HKs comprise one of the missing links between high-dose metformin and the inhibition of the respiratory chain in cancer. METHODS: We tested the isoenzyme-specific regulatory roles of HKs in ovarian cancer cells by examining the effects of the deletions of HK1 and HK2 in TOV-112D ovarian adenocarcinoma cells. We reverted these effects by re-introducing wild-type HK1 and HK2, and we compared the HK1 revertant with the knock-in of catalytically dead HK1 p.D656A. We subjected these cells to a battery of metabolic and proliferation assays and targeted GC×GC-MS metabolomics. RESULTS: We found that the HK1 depletion (but not the HK2 depletion) sensitized ovarian cancer cells to high-dose metformin during glucose starvation. We confirmed that this newly uncovered role of HK1 is glycolysis-independent by the introduction of the catalytically dead HK1. The expression of catalytically dead HK1 stimulated similar changes in levels of TCA intermediates, aspartate and cysteine, and in glutamate as were induced by the HK2 deletion. In contrast, HK1 deletion increased the levels of branched amino acids; this effect was completely eliminated by the expression of catalytically dead HK1. Furthermore, HK1 revertants but not HK2 revertants caused a strong increase of NADPH/NADP ratios independently on the presence of glucose or metformin. The HK1 deletion (but not HK2 deletion) suppressed the growth of xenotransplanted ovarian cancer cells and nearly abolished the tumor growth when the mice were fed the glucose-free diet. CONCLUSIONS: We provided the evidence that HK1 is involved in the so far unknown glycolysis-independent HK1-metformin axis and influences metabolism even in glucose-free conditions.

Krill Oil Supplementation Reduces Exacerbated Hepatic Steatosis Induced by Thermoneutral Housing in Mice with Diet-Induced Obesity.

Preclinical evidence suggests that n-3 fatty acids EPA and DHA (Omega-3) supplemented as phospholipids (PLs) may be more effective than triacylglycerols (TAGs) in reducing hepatic steatosis. To further test the ability of Omega-3 PLs to alleviate liver steatosis, we used a model of exacerbated non-alcoholic fatty liver disease based on high-fat feeding at thermoneutral temperature. Male C57BL/6N mice were fed for 24 weeks a lard-based diet given either alone (LHF) or supplemented with Omega-3 (30 mg/g diet) as PLs (krill oil; ω3PL) or TAGs (Epax 3000TG concentrate; ω3TG), which had a similar total content of EPA and DHA and their ratio. Substantial levels of TAG accumulation (~250 mg/g) but relatively low inflammation/fibrosis levels were achieved in the livers of control LHF mice. Liver steatosis was reduced by >40% in the ω3PL but not ω3TG group, and plasma ALT levels were markedly reduced (by 68%) in ω3PL mice as well. Krill oil administration also improved hepatic insulin sensitivity, and its effects were associated with high plasma adiponectin levels (150% of LHF mice) along with superior bioavailability of EPA, increased content of alkaloids stachydrine and trigonelline, suppression of lipogenic gene expression, and decreased diacylglycerol levels in the liver. This study reveals that in addition to Omega-3 PLs, other constituents of krill oil, such as alkaloids, may contribute to its strong antisteatotic effects in the liver.

Omega-3 Phospholipids from Krill Oil Enhance Intestinal Fatty Acid Oxidation More Effectively than Omega-3 Triacylglycerols in High-Fat Diet-Fed Obese Mice.

Antisteatotic effects of omega-3 fatty acids (Omega-3) in obese rodents seem to vary depending on the lipid form of their administration. Whether these effects could reflect changes in intestinal metabolism is unknown. Here, we compare Omega-3-containing phospholipids (krill oil; ω3PL-H) and triacylglycerols (ω3TG) in terms of their effects on morphology, gene expression and fatty acid (FA) oxidation in the small intestine. Male C57BL/6N mice were fed for 8 weeks with a high-fat diet (HFD) alone or supplemented with 30 mg/g diet of ω3TG or ω3PL-H. Omega-3 index, reflecting the bioavailability of Omega-3, reached 12.5% and 7.5% in the ω3PL-H and ω3TG groups, respectively. Compared to HFD mice, ω3PL-H but not ω3TG animals had lower body weight gain (-40%), mesenteric adipose tissue (-43%), and hepatic lipid content (-64%). The highest number and expression level of regulated intestinal genes was observed in ω3PL-H mice. The expression of FA ω-oxidation genes was enhanced in both Omega-3-supplemented groups, but gene expression within the FA ß-oxidation pathway and functional palmitate oxidation in the proximal ileum was significantly increased only in ω3PL-H mice. In conclusion, enhanced intestinal FA oxidation could contribute to the strong antisteatotic effects of Omega-3 when administered as phospholipids to dietary obese mice.

Lipokine 5-PAHSA Is Regulated by Adipose Triglyceride Lipase and Primes Adipocytes for De Novo Lipogenesis in Mice.

Branched esters of palmitic acid and hydroxystearic acid (PAHSA) are anti-inflammatory and antidiabetic lipokines that connect glucose and lipid metabolism. We aimed to characterize involvement of the 5-PAHSA regioisomer in the adaptive metabolic response of white adipose tissue (WAT) to cold exposure (CE) in mice, exploring the cross talk between glucose utilization and lipid metabolism. CE promoted local production of 5- and 9-PAHSAs in WAT. Metabolic labeling of de novo lipogenesis (DNL) using 2H2O revealed that 5-PAHSA potentiated the effects of CE and stimulated triacylglycerol (TAG)/fatty acid (FA) cycling in WAT through impacting lipogenesis and lipolysis. Adipocyte lipolytic products were altered by 5-PAHSA through selective FA re-esterification. The impaired lipolysis in global adipose triglyceride lipase (ATGL) knockout mice reduced free PAHSA levels and uncovered a metabolite reservoir of TAG-bound PAHSAs (TAG estolides) in WAT. Utilization of 13C isotope tracers and dynamic metabolomics documented that 5-PAHSA primes adipocytes for glucose metabolism in a different way from insulin, promoting DNL and impeding TAG synthesis. In summary, our data reveal new cellular and physiological mechanisms underlying the beneficial effects of 5-PAHSA and its relation to insulin action in adipocytes and independently confirm a PAHSA metabolite reservoir linked to ATGL-mediated lipolysis.

Omega-3 index in the Czech Republic: No difference between urban and rural populations.

Naturally occurring long-chain omega-3 PUFA such as eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) exert multiple effects on health, which are related to the intake of these lipids in the diet and correlate with the levels of omega-3 PUFA in the body. These levels are reflected by the omega-3 PUFA index, i.e. the EPA and DHA content as % of all fatty acids in red blood cells. The aim of this study was to evaluate omega-3 index in the Czech Republic, using blood samples collected from the capital city (n = 476) and the rural region (n = 388). The mean omega-3 index was 3.56 mol % with a maximal value of 8.10% and a minimal value of 1.12%. There was no difference in the index value between rural and urban / industrial regions, but this value was higher in subjects who reported eating fish or omega-3 PUFA supplements. In conclusion, the results indicated suboptimal values of the omega-3 index in the Czech population independent of the sampling region.

Dietary saturated fatty acid type impacts obesity-induced metabolic dysfunction and plasma lipidomic signatures in mice.

Saturated fatty acid (SFA) intake is associated with obesity, insulin resistance, and hepatic steatosis, but scant work examines the impact of SFA type upon these outcomes. We tested the hypothesis that an obesogenic diet prepared with medium chain SFA (MCSFA), mostly as lauric acid-derived from coconut oil, reduces obesity-induced outcomes compared to obesogenic diets prepared with increasing amounts long chain SFA (LCSFA), primarily palmitic acid. Mice were fed (16 weeks) a control, low fat diet or obesogenic diets prepared with differing content of MCSFA or LCSFA in which polyunsaturated and monounsaturated fatty acids (PUFA; MUFA) were kept constant. Inclusion of MCSFA in an obesogenic diet prevented hepatic lipid accumulation and lowered indices of insulin resistance. Obesogenic diets reduced hepatic levels of de novo lipogenesis proteins (SCD1 and FASN) but elevated the adipose levels of mRNA for the pro-inflammatory markers Mcp-1 and Tnfα. Lipidomic analysis of plasma indicated that MCSFA intake resulted in a different lipidomic signature than LCSFA intake, prevented elevation of pro-inflammatory ceramides, but elevated concentrations of some lipids associated with elevated cardiovascular disease risk. Intake of the obesogenic diets in an SFA-type dependent manner elevated plasma concentrations of several phosphatidylcholine (PC) lipids having the long chain PUFA (LCPUFA) arachidonic acid (ARA) and docosahexaenoic acid (DHA), altered phospholipid ethers, and changed the triacylglyceryl environments of these LCPUFA. Our data indicate that (1) MCSFA reduce the severity of some obesogenic co-morbidities, (2) SFA-type modulates lipidomic signatures associated with cardiovascular disease and diabetes, and (3) dietary SFA type impacts LCPUFA metabolism.

Lipidomic Impacts of an Obesogenic Diet Upon Lewis Lung Carcinoma in Mice.

Metabolic reprogramming of lipid metabolism is a hallmark of cancer. Consumption of a high-fat obesogenic diet enhances spontaneous metastasis using a Lewis lung carcinoma (LLC) model. In order to gain further insights into the mechanisms by which dietary fats impact cancer progression, we conducted a lipidomic analysis of primary tumors originated from LLC from mice fed with a standard AIN93G diet or a soybean oil-based high-fat diet (HFD). Hierarchical clustering heatmap analysis of phosphatidylcholine (PC) lipids and phosphatidylethanolamine (PE) lipids demonstrated an increase in polyunsaturated fatty acids (PUFA)-containing phospholipids and a decrease in monounsaturated fatty acids (MUFA)-containing lipids in tumors from mice fed the HFD. The quantities of 51 PC and 24 PE lipids differed in primary tumors of LLC from mice fed the control diet and the HFD. Analysis of triacylglycerol (TAG) lipids identified differences in 32 TAG (by brutto structure) between the two groups; TAG analysis by neutral loss identified 46 PUFA-containing TAG species that were higher in mice fed with the HFD than in the controls. Intake of the HFD did not alter the expression of the de novo lipogenesis enzymes (fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase-1). Our results demonstrate that the dietary fatty acid composition of the HFD is reflected in the higher order lipidomic composition of primary tumors. Subsequent studies are needed to investigate how these lipidomic changes may be used for targeted dietary intervention to reduce tumor growth and malignant progression.

Differential modulation of white adipose tissue endocannabinoid levels by n-3 fatty acids in obese mice and type 2 diabetic patients.

n-3 polyunsaturated fatty acids (n-3 PUFA) might regulate metabolism by lowering endocannabinoid levels. We examined time-dependent changes in adipose tissue levels of endocannabinoids as well as in parameters of glucose homeostasis induced by n-3 PUFA in dietary-obese mice, and compared these results with the effect of n-3 PUFA intervention in type 2 diabetic (T2DM) subjects. Male C57BL/6J mice were fed for 8, 16 or 24 weeks a high-fat diet alone (cHF) or supplemented with n-3 PUFA (cHF + F). Overweight/obese, T2DM patients on metformin therapy were given for 24 weeks corn oil (Placebo; 5 g/day) or n-3 PUFA concentrate as above (Omega-3; 5 g/day). Endocannabinoids were measured by liquid chromatography-tandem mass-spectrometry. Compared to cHF-fed controls, the cHF + F mice consistently reduced 2-arachidonoylglycerol (up to ~2-fold at week 24) and anandamide (~2-fold) in adipose tissue, while the levels of endocannabinoid-related anti-inflammatory molecules N-eicosapentaenoyl ethanolamine (EPEA) and N-docosahexaenoyl ethanolamine (DHEA) increased more than ~10-fold and ~8-fold, respectively. At week 24, the cHF + F mice improved glucose tolerance and fasting blood glucose, the latter being positively correlated with adipose 2-arachidonoylglycerol levels only in obese cHF-fed controls, like fasting insulin and HOMA-IR. In the patients, n-3 PUFA failed to reduce 2-arachidonoylglycerol and anandamide levels in adipose tissue and serum, but they increased both adipose tissue and serum levels of EPEA and DHEA. In conclusion, the inability of n-3 PUFA to reduce adipose tissue and serum levels of classical endocannabinoids might contribute to a lack of beneficial effects of these lipids on glucose homeostasis in T2DM patients.

Selective enrichment of n-3 fatty acids in human plasma lipid motifs following intake of marine fish.

Plasma levels of n-3 long chain polyunsaturated fatty acids (LCPUFA) are associated with a reduction in risk of cardiovascular disease and other chronic, age-related diseases like Alzheimer's disease. In this work, we tested the hypothesis that n-3 LCPUFA fatty acids in human plasma are incorporated into selective lipid species following intake of n-3 LCPUFA rich marine fish. To test this hypothesis, we performed lipidomic analysis on plasma samples from a clinical trial in which participants consumed increasing amounts of farmed Atlantic salmon (Salmo salar). Under basal conditions, n-3 and n-6 LCPUFA were selectively incorporated into plasma phosphatidylcholine (PC) species containing saturated fatty acids (SFA) versus unsaturated fatty acids as the complementary fatty acids. LCPUFA were incorporated into selective triacylglycerol (TAG) species with complementary diacylglyceryl environments of 34:1 or 34:2 (for 20:5 and 22:5) and 36:2>36:3>36:4 and 36:1 (for 20:4 and 22:6). High n-3 LCPUFA marine fish intake resulted in selective increases of PC SFA_n-3 LCPUFA species and LCPUFA-containing TAG species. Changes in cholesteryl esters and phosphatidylethanolamines also occurred following fish intake. Our results highlight the importance of discriminating phospholipid and TAG species and dietary background when evaluating lipidomic outcomes and disease associations.

Species Specificity of Aldehyde and Fatty Acid Profiles of Four Family Group Representatives within the Insect Infraorder Pentatomomorpha (Hemiptera: Heteroptera).

Reactive α,ß-unsaturated aldehydes, including 4-oxoalk-2-enals, are known to be present in volatile secretions of numerous heteropteran insect species. Because the aldehydes are likely to originate from metabolism of fatty acids (FAs), the present study aimed to examine and compare the aldehyde and FA profiles of four model heteropteran species. The model species consisted of adult family group representatives within the infraorder Pentatomomorpha (Hemiptera: Heteroptera): seed bug (Lygaeus equestris (Lygaeoidea)), dock leaf bug (Coreus marginatus (Coreoidea)), red firebug (Pyrrhocoris apterus (Pyrrhocoroidea)), and European stink bug (Graphosoma lineatum (Pentatomoidea)). Solid-phase microextraction combined with two-dimensional gas-chromatography/time-of-flight mass spectrometry was used to establish the profiles of volatile secretions in stressed living insects. The FA profiles of acylglyceride and phospholipid fractions deposited in fat body and/or hemolymph were obtained by liquid chromatography/mass spectrometry and gas chromatography with flame ionization detection techniques. Our results based on multivariate statistical analyses of the data imply that volatile secretion blends as well as fat body and/or hemolymph lipid profiles are species specific but the differences in volatile blends between different species do not mirror the changes in corresponding fat body and/or hemolymph lipid profiles of stressed and non-stressed individuals.

Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion-trap mass spectrometer.

Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PCs. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PCs. This approach is comprised of two MS methods: a precursor ion scanning (PIS) of mass m/z 184 in positive mode (PIS m/z +184) and MS3 fragmentation in negative mode, both performed on the same instrument, a hybrid triple quadrupole ion-trap mass spectrometer. The MS3 experiment identified the FA composition and the relative abundance of isobaric and sn-1, sn-2 positional isomeric PC species, which were subsequently combined with absolute quantitative data obtained by PIS m/z +184 scan. This approach was applied to the analysis of a National Institute of Standards and Technology human blood plasma standard reference material (SRM 1950). We quantified more than 70 PCs and confirmed that a majority are present in isobaric and isomeric mixtures. The FA content determined by this method was comparable to that obtained using GC with flame ionization detection, supporting the quantitative nature of this MS method. This methodology will provide more in-depth biomarker information for clinical and mechanistic studies.

Regulation of Isoprenoid Pheromone Biosynthesis in Bumblebee Males.

Males of the closely related species Bombus terrestris and Bombus lucorum attract conspecific females by completely different marking pheromones. MP of B. terrestris and B. lucorum pheromones contain mainly isoprenoid (ISP) compounds and fatty acid derivatives, respectively. Here, we studied the regulation of ISP biosynthesis in both bumblebees. RNA-seq and qRT-PCR analyses indicated that acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and farnesyl diphosphate synthase (FPPS) transcripts are abundant in the B. terrestris labial gland. Maximal abundance of these transcripts correlated well with AACT enzymatic activity detected in the LG extracts. In contrast, transcript abundances of AACT, HMGR, and FPPS in B. lucorum were low, and AACT activity was not detected in LGs. These results suggest that transcriptional regulation plays a key role in the control of ISP biosynthetic gene expression and ISP pheromone biosynthesis in bumblebee males.

Biosynthetic Studies of the Male Marking Pheromone in Bumblebees by Using Labelled Fatty Acids and Two-Dimensional Gas Chromatography with Mass Detection.

Two-dimensional comprehensive gas chromatography (GC×GC) coupled with mass detection was used as a tool for biosynthetic studies of bumblebee pheromones. Prior to biosynthetic experiments, the chromatographic behaviour of isotopically modified esters in the GC×GC system as well as their behaviour in mass detection was studied. The male marking pheromones of Bombus lucorum, Bombus lapidarius and Bombus terrestris were investigated. Main pheromonal components are ethyl tetradec-9-enoate (53 %) and ethyl dodecanoate (6 %) in B. lucorum, hexadec-9-en-1-ol (52 %) and hexadecan-1-ol (31 %) in B. lapidarius, and 2,3-dihydrofarnesol (58 %) and ethyl dodecanoate (15 %) in B. terrestris. The research strategy was based on 1) in vivo incubation of isotopically (2 H, 13 C) modified fatty acids (FAs) and analysis of their metabolites and 2) feeding experiments with 2 H- and 13 C-labelled FAs mixed with food. It was observed that labelled FAs were modified into the most abundant aliphatic compounds present in labial gland secretions. In feeding experiments, the labelled FAs were transformed into pheromone components. Transport of the FA precursors from the fat body through haemolymph was confirmed. The results show that FAs, stored in the form of triacylglycerols in the fat body, are likely to participate in the biosynthesis of some aliphatic pheromone components.

Antifungal effect of essential oil components against Aspergillus niger when loaded into silica mesoporous supports.

BACKGROUND: Essential oil components (EOCs) are known for their antifungal properties; however, their high volatility limits their application as antimicrobial agents. Strategies used for controlling the volatility of EOCs include encapsulation or loading into porous materials. This study evaluated the in vitro antifungal activity of selected EOCs (carvacrol, cinnamaldehyde, eugenol and thymol) against the fungus Aspergillus niger when loaded into MCM-41 and ß-cyclodextrin (ß-CD). RESULTS: Carvacrol and thymol in Mobil Composition of Matter No. 41 (MCM-41) displayed remarkable enhanced antifungal properties in comparison to the pure or ß-CD-encapsulated EOCs. In fact, carvacrol and thymol were able to maintain antifungal activity and inhibit fungal growth for 30 days, suggesting better applicability of these EOCs as natural preservatives. CONCLUSIONS: The sustained antifungal effect of EOCs encapsulated into silica mesoporous supports was described.

The role of desaturases in the biosynthesis of marking pheromones in bumblebee males.

Bumblebee males (Hymenoptera) produce species-specific labial gland secretions called marking pheromones (MPs). MPs generally consist of terpenoids and fatty-acid-derived aliphatic compounds with various chain lengths predominantly containing one or no double bonds. The unsaturated fatty-acid-derived MP components were hypothesized to be produced by fatty acid desaturases (FADs) that exhibit diverse substrate specificities. To address this hypothesis, we isolated and functionally characterized FADs from three bumblebee species: Bombus lucorum, Bombus terrestris, and Bombus lapidarius. By employing RNA sequencing of the male labial glands and fat bodies of B. lucorum and B. terrestris, we identified five paralogous FAD-like sequences but only two FAD lineages were abundant and differentially expressed in the labial glands. We found that abundant FAD lineages were also expressed in the labial gland and fat body of Bombus lapidarius. Functional characterization of FADs in a yeast expression system confirmed that Δ4-FADs exhibited a unique Δ4-desaturase activity exclusively on 14-carbon fatty acyls and Δ9-FADs displayed Δ9-desaturase activity on 14- to 18-carbon fatty acyls. These results indicate that Δ9-FADs are involved in the biosynthesis of major unsaturated components of MPs in B. lucorum and B. lapidarius despite the diverse MP composition of these bumblebee species. The contribution of lipases, acyltransferases, esterases, and fatty acid reductases to production of the species-specific MP composition is also discussed in light of the transcriptomic data obtained in this study.

De novo biosynthesis of sexual pheromone in the labial gland of bumblebee males.

De novo biosynthesis of male sex pheromone from two bumblebee species (Bombus terrestris and Bombus lucorum) was studied by using in vitro incubations of labial glands (LGs) with radioactive [1,2-(14)C]acetate and deuterated [D(3)]acetate. The labeled substrate was incorporated into several types of compounds, such as terpenic alcohols, fatty acids, esters, and hydrocarbons. A similar incubation of [1,2-(14)C]acetate with fat bodies (FB) led to the formation of fatty acids, triacylglycerols (TAG), and hydrocarbons. To support the results from in vitro incubations, PCR analysis of fatty acid synthase (FAS) transcripts in LG and FB was performed. Relative quantification of FAS transcription levels revealed that the abundance of mRNA from the FAS gene is a function of the age of B. terrestris males. A comparison of the relative FAS mRNA gene transcription level in FB and LGs of B. terrestris and B. lucorum males proved that high biosynthetic activity takes place in the LGs of both species. Together, these results indicate that pheromone components are synthesized de novo in the LG.

Leg tendon glands in male bumblebees (Bombus terrestris): structure, secretion chemistry, and possible functions.

Among the large number of exocrine glands described in bees, the tarsal glands were thought to be the source of footprint scent marks. However, recent studies showed that the compounds used for marking by stingless bees are secreted by leg tendon instead of tarsal glands. Here, we report on the structure of leg tendon glands in males of Bombus terrestris, together with a description of the chemical composition of their secretions and respective changes of both during the males' lives. The ultrastructure of leg tendon glands shows that the secretory cells are located in three independent regions, separated from each other by unmodified epidermal cells: in the femur, tibia, and basitarsus. Due to the common site of secretion release, the organ is considered a single secretory gland. The secretion of the leg tendon glands of B. terrestris males differs in its composition from those of workers and queens, in particular by (1) having larger proportions of compounds with longer chain lengths, which we identified as wax esters; and (2) by the lack of certain hydrocarbons (especially long chain dienes). Other differences consist in the distribution of double bond positions in the unsaturated hydrocarbons that are predominantly located at position 9 in males but distributed at seven to nine different positions in the female castes. Double bond positions may change chemical and physical properties of a molecule, which can be recognized by the insects and, thus, may serve to convey specific information. The function of male-specific compounds identified from their tendon glands remains elusive, but several possibilities are discussed.

Females of the bumblebee parasite, Aphomia sociella, excite males using a courtship pheromone.

Aphomia sociella (Lepidoptera: Pyralidae: Galleriinae) is a parasitic moth of bumblebees. Behavioral experiments show that A. sociella females emit semiochemicals that influence male pre-mating behavior and serve as a courtship pheromone. GC/EAD and two-dimensional GC/MS (GCxGC-TOFMS) analyses of extracts of females revealed three antennally active compounds. Comparative GC and GCxGC-TOFMS analyses of extracts and synthetic standards confirmed the identity of the antennally active compounds as hexan-1-ol (1), 6,10,14-trimethylpentadecan-2-one (2), and 6,10,14-trimethylpentadecan-2-ol (3). In laboratory bioassays, alcohol 3 and, at higher doses, ketone 2 initiated male courtship behavior associated with ultrasonic production. Hexan-1-ol (1) and ketone 2 enhanced the activity of alcohol 3. These data suggest that hexan-1-ol, 6,10,14-trimethylpentadecan-2-ol, and 6,10,14-trimethylpentadecan-2-one constitute the female-produced courtship pheromone of A. sociella.