Changes in the Composition of Triacylglycerols in the Fat Bodies of Bumblebee Males During Their Lifetime

The age-dependent changes in the composition of triacylglycerols (TAG) in the fat bodies of bumblebee males were studied using HPLC/MS. Two related species (Bombus terrestris and B. lucorum) were compared, with the age of the males being 0–30 days. The total amount of TAG in B. lucorum was about 2.7 times higher than that in B. terrestris for all of the ages studied. One to three-day-old males had the highest content of TAG in their fat bodies (1.6–2.3 mg/individual in B. terrestris and 3.8–4.2 mg/individual in B. lucorum). The analytical data show different patterns in both species. The qualitative composition of fatty acids in TAG was similar, but the mean relative abundance between B. terrestris and B. lucorum differed: 14:0, 7 and 14%; 16:0, 20 and 44%; 18:3, 62 and 23%; 18:1, 3 and 8%, respectively (the data is based on a GC/MS integration). A statistical evaluation of the dynamic changes in the TAG composition revealed that in B. terrestris different age classes were well separated according to their TAG composition while in B. lucorum the TAG did not change substantially during the male’s life. The TAG analyses provide more precise information on the differences between the classes studied than the FA composition alone.     

Characterisation of Acetyl‐CoA Thiolase: The First Enzyme in the Biosynthesis of Terpenic Sex Pheromone Components in the Labial Gland of Bombus terrestris

Buff‐tailed bumblebees, Bombus terrestris, use a male sex pheromone for premating communication. Its main component is a sesquiterpene, 2,3‐dihydrofarnesol. This paper reports the isolation of a thiolase (acetyl‐CoA thiolase, AACT_BT), the first enzyme involved in the biosynthetic pathway leading to formation of isoprenoids in the B. terrestris male sex pheromone. Characterisation of AACT_BT might contribute to a better understanding of pheromonogenesis in the labial gland of B. terrestris males. The protein was purified to apparent homogeneity by column chromatography with subsequent stepwise treatment. AACT_BT showed optimum acetyltransferase activity at pH 7.1 and was strongly inhibited by iodoacetamide. The enzyme migrated as a band with an apparent mass of 42.9 kDa on SDS‐PAGE. MS analysis of an AACT_BT tryptic digest revealed high homology to representatives of the thiolase family. AACT_BT has 96 % amino acid sequence identity with the previously reported Bombus impatiens thiolase.     

Comparison of Age-dependent Quantitative Changes in the Male Labial Gland Secretion of <Emphasis Type="Italic">Bombus Terrestris</Emphasis> and <Emphasis Type="Italic">Bombus Lucorum</Emphasis>

Age-related changes of antennal-active components of male labial gland extracts were studied in two closely related bumblebee species, Bombus terrestris and B. lucorum. In B. terrestris, compounds eliciting electroantennogram (EAG) responses of virgin queens were ethyl dodecanoate, 2,3-dihydrofarnesal, 2,3-dihydrofarnesol, hexadecan-1-ol, octadeca-9,12,15-trien-1-ol, and geranylcitronellol. Compounds that elicited EAG responses from queens of B. lucorum were ethyl dodecanoate, ethyl tetradec-7-enoate, ethyl tetradec-9-enoate, ethyl hexadec-9-enoate, hexadecan-1-ol, hexadec-7-enal, octadeca-9,12-dien-1-ol, octadeca-9,12,15-trien-1-ol, and octadecan-1-ol. Quantities of these compounds in the labial glands changed significantly over the lifetime of the respective males of the two species. In both species, concentrations of the respective compounds reached their maximum within seven days after eclosion. Subsequently, a rapid decrease in the amount of EAG-active compounds occurred in B. terrestris, whereas in B. lucorum the amount of active compounds stayed approximately constant or decreased at a slow rate. Microscopy showed that in B. terrestris secretory cells of the labial glands undergo apoptosis from the fifth to the tenth day of life, whilst in B. lucorum labial gland cells remain unchanged throughout the life of the males.     

Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron‐withdrawing aryl‐alkyl side chains which inhibited the growth of Gram‐negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ～1–4 μg mL^?1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially “rescued” by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (～2–6 μg mL^?1) against Gram‐positive but not Gram‐negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ～1–2 μg mL^?1.     

Analysis of insect triacylglycerols using liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry

An HPLC non‐aqueous reversed‐phase separation system was adapted for analyzing insect triacylglycerols (TAG). The method uses two conventional Nova‐Pak C18 columns connected in series, for a total length of 45 cm. The mobile phase gradient is mixed from acetonitrile and 2‐propanol, and the flow rate is programmed from 1.0 to 0.7 mL/min. TAG are detected by atmospheric pressure chemical ionization‐mass spectrometry. The method ensures efficient separation of isomers and analysis of high‐molecular‐weight TAG with equivalent chain lengths up to 72. The method performance is demonstrated on analysis of TAG isolated from the fat body of the bumblebee Bombus lucorum.     

Reevaluation of the Role of Mandibular Glands in Regulation of Reproduction in Bumblebee Colonies

Possible pheromonal control of worker reproduction was tested in Bombus terrestris. The mode of assay included exposure of callow workers to extracts originating from different queen parts and measuring the effect on the in vitro biosynthesis of juvenile hormone (JH), the apparent gonadotropin in this species. Both queen total body extracts applied to dummies consisting of oven-dried or Soxhlet-washed virgin queen bodies and cuticular washes applied to living virgin queens effectively inhibited the biosynthesis of JH in callow workers. None of the five exocrine glands (mandibular, hypopharyngeal, salivary, Dufour's, and tarsal) demonstrated inhibitory activity. Likewise, the use of synthetic 3-hydroxy acids, found in queen mandibular glands, were ineffective in blocking JH biosynthesis in queenless workers. The results suggest that the queen may use a primer pheromone spread on the epicuticle as a means to inhibit worker reproduction. However, our results are not consistent with the prevailing hypothesis that in B. terrestris the main source of the pheromone that inhibits worker reproduction is in the queen's mandibular glands.     

Biosynthesis of Trehangelin in Polymorphospora rubra K07–0510: Identification of Metabolic Pathway to Angelyl‐CoA

Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07‐0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase‐like protein (thgC), α‐amylase (thgD), 3‐ketoacyl‐ACP synthase III (thgI), 3‐ketoacyl‐ACP reductase (thgK), enoyl‐CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl‐CoA and methylmalonyl‐CoA to 2‐methylacetoacetyl‐CoA (MAA‐CoA), ThgK catalyses NADPH‐dependent reduction of MAA‐CoA to 3‐hydroxy‐2‐methylbutyryl‐CoA (HMB‐CoA) and ThgH catalyses the dehydration of HMB‐CoA to angelyl‐CoA (AN‐CoA). This is the first report on the elucidation of the enzymatic formation of AN‐CoA.     

Host recognition and the study of a chemical basis for attraction by cuckoo bumble bees (Hymenoptera: Apidae)

Species ofPsithyrus (Hymenoptera; Apidae) are obligate bumble bee social parasites. In this study, females ofP. vestalis andP. ashtoni were presented with pentane extracts prepared from different body parts of queens of their respective host species,Bombus terrestris andB. terricola. Parasites of both species were capable of distinguishing host bees from other bumble bee species using chemical cues contained within extracts. Among extracts of several body parts presented to parasites, the abdomen produced the greatest behavioral response, with Dufour's gland and terminal tergal segments eliciting the greatest response among abdominal regions. Extracts of these two body parts obtained fromB. terrestris queens shared a number of compounds, identified by GC-MS. Among the identified compounds are a number that have been reported to be of importance in bee sociochemistry.     

The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones

Many moth pheromones are composed of mixtures of acetates of long‐chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl‐CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non‐insect acetyltransferase alternative, we expressed a plant‐derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with EaDAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27‐ and 10‐fold higher in vivo and in vitro efficiency, respectively, compared to EaDAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.     

Biosynthetic Code for Divergolide Assembly in a Bacterial Mangrove Endophyte

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3‐amino‐5‐hydroxybenzoate and the rare extender units ethylmalonyl‐CoA and isobutylmalonyl‐CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer–Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.     

Isolation and characterization of insect attractant lipids

Isolation and characterization of the naturally occurring sex attractants of the silkworm moth (Bombyx mori), gypsy moth (Porthetria dispar), cotton leafworm (Prodenia litura), American cockroach (Periplaneta americana), an introduced pine sawfly (Diprion similis), and mating attractants of the honey bee (Apis mellifera) and bumblebee (Bombus terrestris) are discussed.     

Plant Cytosolic Acyl-CoA-Binding Proteins

A gene family encoding six members of acyl‐CoA‐binding proteins (ACBP) exists in Arabidopsis and they are designated as AtACBP1–AtACBP6. They have been observed to play pivotal roles in plant lipid metabolism, consistent to the abilities of recombinant AtACBP in binding different medium‐ and long‐chain acyl‐CoA esters in vitro. While AtACBP1 and AtACBP2 are membrane‐associated proteins with ankyrin repeats and AtACBP3 contains a signaling peptide for targeting to the apoplast, AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. They were verified to be subcellularly localized in the cytosol using diverse experimental methods, including cell fractionation followed by western blot analysis, immunoelectron microscopy and confocal laser‐scanning microscopy using autofluorescence‐tagged fusions. AtACBP4 (73.2 kDa) and AtACBP5 (70.1 kDa) are the largest, while AtACBP6 (10.4 kDa) is the smallest. Their binding affinities to oleoyl‐CoA esters suggested that they can potentially transfer oleoyl‐CoA esters from the plastids to the endoplasmic reticulum, facilitating the subsequent biosynthesis of non‐plastidial membrane lipids in Arabidopsis. Recent studies on ACBP, extended from a dicot (Arabidopsis) to a monocot, revealed that six ACBP are also encoded in rice (Oryza sativa). Interestingly, three small rice ACBP (OsACBP1, OsACBP2 and OsACBP3) are present in the cytosol in comparison to one (AtACBP6) in Arabidopsis. In this review, the combinatory and distinct roles of the cytosolic AtACBP are discussed, including their functions in pollen and seed development, light‐dependent regulation and substrate affinities to acyl‐CoA esters.     

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty‐acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl‐CoA‐dependent or acyl‐CoA‐independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl‐CoA‐dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane‐bound and soluble forms of DGAT have been identified with very different amino‐acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl‐CoA‐independent pathways via the catalytic action of membrane‐bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty‐acid composition of TAG is also discussed.     

Short-Term changes in hepatic HMG-CoA reductase in rats fed diets containing cholesterol or oat bran

In vivo regulation of hepatic HMG-CoA reductase (HMGR) (mevalonate: NADP⁺ oxidoreductase [acylating CoA]; EC 1.1.1.34] by phosphorylation/dephosphorylation has not been demonstrated. Rats were meal-fed semipurified diets; effects of inclusion of cholesterol (2%) or oat bran (15%) in a single meal on expressed (phosphorylated) and total (dephosphorylated) activities of HMGR were measured from 15 min to 4 hr after presentation of the meal. Expressed activity was not significantly altered in response to the control diet during the time periods examined, while total HMGR activity declined by 15 min and increased through 4 hr to an activity about 1.5 times control levels. Addition of cholesterol resulted in little change in expressed activity but a greater and more sustained reduction in total activity. Oat bran caused reductions in both total and expressed activities, which were maintained through 4 hr. Total HMGR activity was best correlated with apparent demand for cholesterol synthesis.     

Biosynthesis of the Insecticidal Xenocyloins in Xenorhabdus bovienii

The biosynthesis gene cluster for the production of xenocyloins was identified in the entomopathogenic bacterium Xenorhabdus bovienii SS‐2004, and their biosynthesis was elucidated by heterologous expression and in vitro characterization of the enzymes. XclA is an S‐selective ThDP‐dependent acyloin‐like condensation enzyme, and XclB and XclC are examples of the still‐rare acylating ketosynthases that catalyze the acylation of the XclA‐derived initial xenocyloins with acetyl‐, propionyl‐, or malonyl‐CoA, thereby resulting in the formation of further xenocyloin derivatives. All xenocyloins were produced mainly by the more virulent primary variant of X. bovienii and showed activity against insect hemocytes thus contributing to the overall virulence of X. bovienii against insects.     

Identity and Function of Scent Marks Deposited by Foraging Bumblebees

Foraging bumblebees can detect scents left on flowers by previous bumblebee visitors and hence avoid flowers that have been depleted of nectar. Tarsal secretions are probably responsible for this repellent effect. The chemical components of the tarsal glands were analyzed by combined gas chromatography–mass spectrometry for three species of bumblebee, Bombus terrestris, B. lapidarius, and B. pascuorum. The hydrocarbons identified were similar for each species, although there were interspecific differences in the relative amounts of each compound present. The tarsal extracts of all three species comprised complex mixtures of long-chain alkanes and alkenes with between 21 and 29 carbon atoms. When B. terrestris tarsal extracts were applied to flowers and offered to foraging bumblebees of the three species, each exhibited a similar response; concentrated solutions produced a repellent effect, which decreased as the concentration declined. We bioassayed synthetic tricosane (one of the compounds found in the tarsal extracts) at a range of doses to determine whether it gave a similar response. Doses 10^–12 ng/flower resulted in rejection by foraging B. lapidarius. Only when 10^–14 ng was applied did the repellent effect fade. We bioassayed four other synthetic compounds found in tarsal extracts and a mixture of all five compounds to determine which were important in inducing a repellent effect in B. lapidarius workers. All induced repellency but the strength of the response varied; heneicosane was most repellent while tricosene was least repellent. These findings are discussed in relation to previous studies that found that tarsal scent marks were attractive rather than repellent.     

Production of Dehydrogingerdione Derivatives in Escherichia coli by Exploiting a Curcuminoid Synthase from Oryza sativa and a β‐Oxidation Pathway from Saccharomyces cerevisiae

Gingerol derivatives are bioactive compounds isolated from the rhizome of ginger. They possess various beneficial activities, such as anticancer and hepatoprotective activities, and are therefore attractive targets of bioengineering. However, the microbial production of gingerol derivatives has not yet been established, primarily because the biosynthetic pathway of gingerol is unknown. Here, we report the production of several dehydrogingerdione (a gingerol derivative) analogues from a recombinant Escherichia coli strain that has an “artificial” biosynthesis pathway for dehydrogingerdione that was not based on the original biosynthesis pathway of gingerol derivatives in plants. The system consists of a 4‐coumarate:CoA ligase from Lithospermum erythrorhizon, a fatty acid CoA ligase from Oryza sativa, a β‐oxidation system from Saccharomyces cerevisiae, and a curcuminoid synthase from O. sativa. To our knowledge, this is the first report of the microbial production of a plant metabolite the biosynthetic pathway of which has not yet been identified.