Biosynthesis of the defensive alkaloid cicindeloine in Stenus solutus beetles

To protect themselves from predation and microorganismic infestation, rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine, 3-(2-methyl-1-butenyl)pyridine, and cicindeloine in their pygidial glands. The biosynthesis of stenusine and 3-(2-methyl-1-butenyl)pyridine was previously investigated in Stenus bimaculatus and Stenus similis, respectively. Both molecules follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from l-lysine and the side chain from l-isoleucine. The different alkaloids are finally obtained by slight modifications of shared precursor molecules. The piperideine alkaloid cicindeloine occurs as a main compound additionally to (E)-3-(2-methyl-1-butenyl)pyridine and traces of stenusine in the pygidial gland secretion of Stenus cicindeloides and Stenus solutus. Feeding of S. solutus beetles with [D,¹⁵N]-labeled amino acids followed by GC/MS analysis techniques showed that cicindeloine is synthesized via the identical pathway and precursor molecules as the other two defensive alkaloids.     

Stenusine,an antimicrobial agent in the rove beetle genus <Emphasis Type="Italic">Stenus</Emphasis> (Coleoptera,Staphylinidae)

Stenusine is well known as the alkaloid, discharged by the rove beetle, genus Stenus Latreille (Coleoptera, Staphylinidae). The Stenus beetles employ the alkaloid as an escape mechanism when on water surfaces. In the case of danger, they lower their abdomen and emit stenusine from their pygidial glands. Stenusine shows a low surface tension and therefore a high spreading pressure; these properties propel the beetle quickly over the water. Many Steninae do not live in habitats with open waters, but in detritus, leaf litter, mosses, etc. This raises the possibility that stenusine might also have another function, e.g., as antibiotic or fungicide. Stenus beetles show an intense grooming behaviour. With gas chromatography-mass spectrometry analyses we could prove that they cover themselves with their secretion. To tests its antimicrobial properties we conducted agar diffusion tests with stenusine and norstenusine, another substance that is abundant in most Stenus species. Both compounds have an antimicrobial effect on entomopathogenic bacteria and fungi. Stenusine not only allows for an extraordinary method of locomotion on water surfaces, it also protects the Steninae from being infested with microorganisms.     

Intrageneric differences in the four stereoisomers of stenusine in the rove beetle genus, Stenus (Coleoptera, Staphylinidae)

Most species of the rove beetle genus Stenus employ the spreading alkaloid stenusine as an escape mechanism on water surfaces. In the case of danger, they emit stenusine from their pygidial glands, and it propels them over the water very quickly. Stenusine is a chiral molecule with four stereoisomers: (2′R,3R)-, (2′S,3R)-, (2′S,3S)-, and (2′R,3S)-stenusine. The percentile ratio of these four isomers is only known for the most common species of the genus: Stenus comma. With the intention of determining the stereoisomer ratios of five additional species from the two subgenera, Stenus and Hypostenus, we used GC/mass spectrometry measurements with a chiral phase. The results showed that the ratio differs among the genus. These findings can be a basis for chemotaxonomy. It is also possible that the biological function of stenusine, e.g., as antibiotic or fungicide, varies with changing stereoisomer composition.

Hoopoes color their eggs with antimicrobial uropygial secretions

Uropygial gland secretions are used as cosmetics by some species of birds to color and enhance properties of feathers and teguments, which may signal individual quality. Uropygial secretions also reach eggshells during incubation and, therefore, may influence the coloration of birds’ eggs, a trait that has attracted the attention of evolutionary biologists for more than one century. The color of hoopoe eggs typically changes along incubation, from bluish-gray to greenish-brown. Here, we test experimentally the hypothesis that dark uropygial secretion of females is responsible for such drastic color change. Moreover, since uropygial secretion of hoopoes has antimicrobial properties, we also explore the association between color and antimicrobial activity of the uropygial secretion of females. We found that eggs stayed bluish-gray in nests where female access to the uropygial secretion was experimentally blocked. Furthermore, experimental eggs that were maintained in incubators and manually smeared with uropygial secretion experienced similar color changes that naturally incubated eggs did, while control eggs that were not in contact with the secretions did not experience such color changes. All these results strongly support the hypothesis that female hoopoes use their uropygial gland secretion to color the eggs. Moreover, saturation of the uropygial secretion was associated with antimicrobial activity against Bacillus licheniformis. Given the known antimicrobial potential of uropygial secretions of birds, this finding opens the possibility that in scenarios of sexual selection, hoopoes in particular and birds in general signal antimicrobial properties of their uropygial secretion by mean of changes in egg coloration along incubation.     

Antifungal activity of the pygidial gland secretion of <Emphasis Type="Italic">Laemostenus punctatus</Emphasis> (Coleoptera: Carabidae) against cave-dwelling micromycetes

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.     

A salamander’s toxic arsenal: review of skin poison diversity and function in true salamanders,genus <Emphasis Type="Italic">Salamandra</Emphasis>

Terrestrial salamanders of the genus Salamandra represent one of the most prominent groups of amphibians. They are mainly distributed across Europe but also reach Northern Africa and the Near East. Members of the six currently accepted species have long been known to be poisonous; however, work on their toxins was mostly published in German language, and therefore, many nuances of these studies have remained hidden from the majority of herpetologists and toxinologists. Several Salamandra species are called fire salamanders due to their highly contrasted, black-yellow colouration which probably serves to deter predators, although thorough evidence for aposematism in Salamandra is still lacking. Salamandra skin toxins do not only represent a potent antipredator defence but may also have antimicrobial effects. A better understanding of this dual function of Salamandra skin secretions is of utmost importance in the face of the emergence of a fungal disease causing catastrophic declines of fire salamanders in Central Europe, caused by the fungus Batrachochytrium salamandrivorans. In this review, we summarize the knowledge on Salamandra toxins, providing a list of the compounds so far isolated from their secretion and focusing on the bioactivity of the major compounds in Salamandra secretions, the steroidal alkaloids. We identify priorities for future research, including a screening of co-occurrence of steroidal alkaloids and tetrodotoxins in salamandrids, chemical characterization of already identified novel steroidal compounds, elucidation of the presence and role of peptides and proteins in the secretion, and experimental in vitro and in vivo study of the interactions between bioactive compounds in Salamandra skin secretions and cutaneous fungal and bacterial pathogens.     

1-Tridecene—male-produced sex pheromone of the tenebrionid beetle <Emphasis Type="Italic">Parastizopus transgariepinus</Emphasis>

Males of the genus Parastizopus (Coleoptera: Tenebrionidae) exhibit a special pheromone-emitting behaviour. They do a headstand, expose the aedeagus and remain in this posture for a few seconds. The pheromone emitted by P. transgariepinus was collected by solid-phase micro-extraction (100 microm polydimethylsiloxane fibre) and identified as 1-tridecene by gas chromatography/mass spectrometry. Presumably, this compound originates from the aedeagal gland, a special feature in Parastizopus, as 1-tridecene is the main compound in the gland reservoirs (23.6+/-3.8%), accompanied by various less volatile fatty acid esters (25.2+/-2.0%) and hydrocarbons (51.2+/-5.7%). 1-Tridecene is also part of the pygidial defensive secretion of both sexes, together with other 1-alkenes, monoterpene hydrocarbons and 1,4-benzoquinones, but as none of these other compounds was detected during calling, the pygidial gland could be ruled out as pheromone source. Extracts of the aedeagal gland reservoirs and the pygidial defensive secretion contained comparable amounts of 1-tridecene, 1.24+/-0.41 and 1.88+/-0.54 microg/male, respectively. Chemo-orientation experiments using a servosphere showed that 1 microg of 1-tridecene was attractive to females but not to males.     

Fossil mesostigmatid mites (Mesostigmata: Gamasina, Microgyniina, Uropodina), associated with longhorn beetles (Coleoptera: Cerambycidae) in Baltic amber

Fossil mesostigmatid mites are extremely rare. Inclusions assignable to the tortoise mites (Mesostigmata, Uropodina) are described here for the first time from Eocene (ca. 44–49 Ma) Baltic amber. This is the oldest record of Uropodina and documents the first unequivocal amber examples potentially assignable to the extant genus Uroobovella Berlese, 1903 (Uropodoidea: Urodinychidae). Further mites in the same amber pieces are tentatively assigned to Microgynioidea (Microgyniina) and Ascidae (Gamasina), both potentially representing the oldest records of their respective superfamily and family groups. This new material also preserves behavioural ecology in the form of phoretic deutonymphs attached to their carriers via a characteristic anal pedicel. These deutonymphs in amber are intimately associated with longhorn beetles (Coleoptera: Cerambycidae), probably belonging to the extinct species Nothorhina granulicollis Zang, 1905. Modern uropodines have been recorded phoretic on species belonging to several beetle families, including records of living Uroobovella spp. occurring on longhorn beetles. Through these amber inclusions, a uropodine–cerambycid association can now be dated back to at least the Eocene.     

Prey-rolling behavior of coatis (Nasua spp.) is elicited by benzoquinones from millipedes

Coatis (Nasua spp.), gregarious, omnivorous carnivores that range in forests from the southwestern USA to south America, dispatch millipedes by rolling them on the ground using rapid, alternating movements of their forepaws. Prey rolling of millipedes is thought to stimulate the depletion of their defensive secretions and to wipe off secretions before millipedes are consumed. We report that prey-rolling behavior in Nasua spp. is elicited by 1,4-benzoquinone; 2-methyl-1,4-benzoquinone; and 2-methoxy-3-methyl-1,4-benzoquinone, the chief components of the defensive secretions of julidan, spirobolidan, and spirostreptidan millipedes. Chemicals elaborated for defense sometimes evolutionarily “backfire,” providing cues to predators on the presence or identity of prey. The elicitation of prey-rolling behavior in Nasua spp. by benzoquinones illustrates this effect for millipedes (and possibly other arthropods) that defensively discharge these compounds.     

Orange/lemon-scented beetles: opposite enantiomers of limonene as major constituents in the defensive secretion of related carabids

The major constituent in the pygidial gland defensive fluid of the carabid beetle Ardistomis schaumii is (R)-(+)-limonene, whereas that of Semiardistomis puncticollis is (S)-(−)-limonene. This was an unanticipated result, since it is not very common to find the opposite enantiomers of the same compound among the secondary metabolites of related species. Moreover, the glandular liquid of A. schaumii contains 1,8-cineole, and that of S. puncticollis has β-pinene, β-phellandrene, sabinene, and p-cymene. Of about 500 carabid species that have been chemically investigated, this is the first report of the presence of such complex mixtures of monoterpenes in their defensive secretions.     

Recycling plant wax constituents for chemical defense: hemi-biosynthesis of triterpene saponins from β-amyrin in a leaf beetle

Several species of Doryphorina leaf beetles from Central- and South America produce oleanane triterpene glycosides in their defensive glands. The presence of pentacyclic triterpenes in insects is intriguing since they lack the key enzymes necessary to synthesize these compounds. Since -amyrin is a common constituent of leaf waxes, we hypothesized that these leaf beetles use this compound as a precursor to their oleanane glycosides. To test this hypothesis we first confirmed the presence of -amyrin in Ipomoea batatas, the food plant of Platyphora kollari. Next, adults of P. kollari were fed for 10 days with I. batatas leaf disks painted with a solution of [2,2,3-²H₃]-amyrin ([2,2,3-²H₃]-1). The secretion from their defensive glands was collected and analyzed by HPLC-ESIMS. The results demonstrated that the secretion of beetles fed with an amount of [2,2,3-²H₃]-amyrin corresponding to the quantity of unlabeled (natural) -amyrin present in the leaf disks contained on average 5.1% of [2,2,3-²H₃]-3-O--d-glucopyranosyl-(1-->4)--d-glucuronopyranosyl-hederagenin ([2,2,3-²H₃]-2), whereas the secretions of beetles fed with 10 times this amount of [2,2,3-²H₃]-amyrin contained on average 23.9% of [2,2,3-²H₃]-2. In both series of experiments, the percentage of labeled versus unlabeled triterpene glycoside in the secretion was positively correlated with the amount of deuterated -amyrin ingested. These results demonstrate for the first time that some leaf beetles are able to metabolize a widespread triterpenic constituent of leaf wax into more complex glycosides that are stored in their defensive glands.     

A new class of mealybug pheromones: a hemiterpene ester in the sex pheromone of Crisicoccus matsumotoi

Mealybugs, which include several agricultural pests, are small sap feeders covered with a powdery wax. They exhibit clear sexual dimorphism; males are winged but fragile and short lived, whereas females are windless and less mobile. Thus, sex pheromones emitted by females facilitate copulation and reproduction by serving as a key navigation tool for males. Although the structures of the hitherto known mealybug pheromones vary among species, they have a common structural motif; they are carboxylic esters of monoterpene alcohols with irregular non-head-to-tail linkages. However, in the present study, we isolated from the Matsumoto mealybug, Crisicoccus matsumotoi (Siraiwa), a pheromone with a completely different structure. Using gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy, we identified the pheromone as 3-methyl-3-butenyl 5-methylhexanoate. Its attractiveness to males was confirmed in a series of field trapping experiments involving comparison between the isolated natural product and a synthetic sample. This is the first report of a hemiterpene mealybug pheromone. In addition, the acid moiety (5-methylhexanoate) appears to be rare in insect pheromones.     

Identification and electrophysiological studies of (4S,5S)-5-hydroxy-4-methyl-3-heptanone and 4-methyl-3,5-heptanedione in male lucerne weevils

An investigation to identify a sex or aggregation pheromone of Sitona discoideus Gyllenhål (Coleoptera: Curculionidae) is presented. Antenna flicking and attraction behaviors evoked by conspecifics of both sexes were recorded in arena bioassays, where attraction of females to males was observed. Air entrainment of both males and females was conducted in separate chambers. Gas chromatographic–mass spectrometric analysis of headspace volatiles revealed that two male-specific compounds, 4-methyl-3,5-heptanedione (major) and (4S,5S)-5-hydroxy-4-methyl-3-heptanone (minor), were emitted during the autumnal post-aestivatory flight period. The stereoisomers of the minor component were separated by enantioselective gas chromatography and their absolute configurations assigned by NMR (diastereomers) and the known preference of enantioselective transesterification reactions catalyzed by Candida antarctica lipase B. Electroantennogram and single sensillum recording studies indicate that 4-methyl-3,5-heptanedione as well as all individual stereoisomers of 5-hydroxy-4-methyl-3-heptanone are detected by the antennae of male and female S. discoideus. Further, single sensillum recordings suggest that both sexes of S. discoideus have specialized olfactory receptor neurons (ORNs) for detecting 4-methyl-3,5-heptanedione and different populations of stereoselective ORNs for detecting the stereoisomers of 5-hydroxy-4-methyl-3-heptanone. Some of these stereoselective ORNs appear to be sex-specific in S. discoideus.     

Chirality determines pheromone activity for flour beetles

Olfactory perception and orientation behaviour of female and male flour beetles (Tribolium castaneum, T. confusum) to single stereoisomers of their aggregation pheromone revealed maximal receptor potentials and optimal attraction in response to 4R,8R-(?)-dimethyldecanal, whereas its optical antipode 4S,8S-(+)-dimethyldecanal was found to be inactive in this respect. Female flour beetles of both species were ≈ 10³ times less attracted to 4R,8S-(+)- and 4S,8R-(?)-dimethyldecanal than to 4R,8R-(?)-dimethyldecanal, while male flour beetles failed to respond to the R,S-(+)- and S,R-(?)-stereoisomers. Pheromone extracts of prothoracic femora from unmated male flour beetles elicited higher receptor potentials in the antennae of females than in those of males. The results suggest that the aggregation pheromone emitted by maleT. castaneum as well as maleT. confusum has the stereochemical structure of 4R,8R-(?)-dimethyl-decanal, which acts as sex attractant for the females and as aggregant for the males of both species.     

Mimicry of queen Dufour's gland secretions by workers of Apis mellifera scutellata and A. m. capensis

The development of the Dufour's gland of workers of the two honey bee races Apis mellifera scutellata and A. m. capensis was measured. The Dufour's glands of A. m. capensis workers were longer and increased in length more rapidly than the glands of workers of A. m. scutellata at comparable ages. Analysis of the Dufour's gland secretions of workers and queens of both races revealed that there were caste and racial differences. Secretions of queenright A. m. scutellata workers were dominated by a series of long-chain hydrocarbons. In contrast the secretions of the A. m. capensis workers both under queenright and queenless conditions were a mixture of hydrocarbons and wax-type esters, as were those of queens. Multivariate analysis of the secretion profiles indicated that laying workers of both races mimic queens. The secretions of the A. m. capensis laying workers mimicked queen secretions most closely, enabling them to act as successful social parasites.     

Influence of hedgerow and grassy field borders on ground beetle (Coleoptera: Carabidae) activity in fields of corn

Agricultural landscapes may be manipulated in ways that benefit predatory invertebrates by providing alternate food sources, overwintering sites, and refuge from farming activities. Ecological theory predicts that complex plant communities should support a richer community of natural enemies of pest insects than a simple plant community. A study was conducted in Iowa, USA to investigate the influence of the vegetative diversity of field boarders on the activity, species richness, and community similarities of predatory beetles occurring in corn fields.Ground beetle (Coleoptera: Carabidae) populations were compared among corn fields bounded by either complex hedges or simple grass edges. Directional pitfall traps were used to investigate activity patterns of beetles between border types and their adjacent corn fields. Beetles were trapped during four seasonal periods, based on the growth stage of corn. During corn emergence (May–June) when fields are barren, carabids were more active and species richness was higher in the corn fields bordered by woody hedges. The carabid species Scarites quadriceps, Scarites subterraneus, and Harpalus pensylvanicus, were more dominant in hedge sites as compared to grass sites at this time. Following corn–canopy closure, carabids were now more active in fields bordered by grassy edges, but beetle activity also remained high in the fields adjacent to woody hedges. Further analysis of the carabid communities by Bray–Curtis Similarity Index showed no difference among field edge types at any time of the season. Results indicate that both complex and simple field border habitats support abundant and diverse populations of carabids during most of the growing season. However, during the early growing season hedges appear to be more important than grass edges in supplying carabid beetles to corn fields. Woody hedges may serve as very important overwintering sites and as an early season refuge for predatory beetles in corn.     

A review of myrmecophily in ant nest beetles (Coleoptera: Carabidae: Paussinae): linking early observations with recent findings

Myrmecophily provides various examples of how social structures can be overcome to exploit vast and well-protected resources. Ant nest beetles (Paussinae) are particularly well suited for ecological and evolutionary considerations in the context of association with ants because life habits within the subfamily range from free-living and predatory in basal taxa to obligatory myrmecophily in derived Paussini. Adult Paussini are accepted in the ant society, although parasitising the colony by preying on ant brood. Host species mainly belong to the ant families Myrmicinae and Formicinae, but at least several paussine genera are not host-specific. Morphological adaptations, such as special glands and associated tufts of hair (trichomes), characterise Paussini as typical myrmecophiles and lead to two different strategical types of body shape: while certain Paussini rely on the protective type with less exposed extremities, other genera access ant colonies using glandular secretions and trichomes (symphile type). We compare these adaptations with other taxonomic groups of insects by joining contemporary research and early sources and discuss the possibility of an attracting or appeasing effect of the secretion. Species that are ignored by their host ants might use chemical mimicry instead. Furthermore, vibrational signals may contribute to ant–beetle communication, and chemical signals have proven to play a role in host finding. The powerful defense chemistry of paussines as “bombardier beetles” is not used in contact with host ants. We attempt to trace the evolution of myrmecophily in paussines by reviewing important aspects of the association between paussine beetles and ants, i.e. morphological and potential chemical adaptations, life cycle, host specificity, alimentation, parasitism and sound production.

柘林湾表层沉积物中甲藻孢囊的分布与浮游植物休眠体萌发研究

于2008年11月采集了广东柘林湾表层沉积物样品,对沉积物中的甲藻孢囊进行分类鉴定,同时对沉积物进行了直接萌发,并对萌发的浮游植物进行了定性定量分析,以了解柘林湾海域甲藻孢囊的分布状况,揭示赤潮发生潜势.研究结果显示,柘林湾海域甲藻孢囊种类多样性较低,仅分析鉴定出甲藻孢囊16种,有毒有害赤潮藻类亚历山大藻和链状裸甲藻的孢囊分布广泛.孢囊组成以异养型甲藻的孢囊占优势,其中,原多甲藻孢囊种类和密度最为丰富,显示了该海域较高的富营养化程度.孢囊密度较低,在16.59~83.37 cysts·g-1(以干重计,下同)之间,平均为39.52 cysts·g-1.空孢囊百分比均较高,平均百分比达到56.3%,说明大部分孢囊已经萌发.萌发的浮游植物种类数较少,共萌发出浮游植物26属33种,硅藻优势种类主要有拟菱形藻(Pseudonitzschia spp.)、角毛藻(Chaetoceros spp.)、中肋骨条藻(Skeletonema costatum)、圆筛藻(Coscinodiscus spp.)等.甲藻种类数较少,其中以woloszynskioid类中的Biecheleriopsis adriatica为优势甲藻.萌发的浮游植物中还出现了大量金色藻(Chrysochromulina spp.)及球形棕囊藻(Phaeocystis globaosa)等其他类别的潜在有毒种类.本研究结果表明,柘林湾海域沉积物中有毒有害赤潮藻类孢囊分布广泛,并萌发出大量的潜在有毒藻类,说明柘林湾海域具有较高的有害赤潮发生风险.     

利用T-RFLP解析生物强化去除吡啶过程中微生物种群动态变化

在接种活性污泥处理含吡啶废水的序批式反应器中,引入吡啶降解菌Paracoccus sp.KT-5构成生物强化反应器,研究了对吡啶的生物强化去除特性及效果,利用末端限制性片段长度多态性(T-RFLP)手段解析了微生物种群结构的动态变化.结果表明,投加高效降解菌株KT-5可以加速反应器的启动,但随着反应器的运行,当吡啶初始浓度增加至195.6 mg·L^-1以后,生物强化反应器对吡啶降解的促进作用已不再明显;当吡啶初始浓度在293.4~586.8 mg·L^-1变化时,起初强化反应器对吡啶的去除速率出现了波动,尽管随后逐渐恢复,但仍然没有表现出明显的强化作用.T-RFLP的分析结果表明,当吡啶初始浓度达到978 mg·L^-1以后,生物强化反应器中已检测不到KT-5,表明生物强化作用的消失可能是因为引入的高效降解菌株KT-5的流失造成的.     

生物沸石对吡啶、喹啉的降解与吸附作用

探讨了通过生物沸石的降解及吸附作用,解决吡啶、喹啉及转化产物NH 4+-N的污染问题.结果表明,生物沸石中的吡啶降解菌Shinella zoogloeoides BC026及喹啉降解菌Pseudomonas sp.BW003能有效去除吡啶、喹啉,同时转化后的NH 4+-N也能被天然沸石或改性沸石所吸附.尽管改性沸石吸附能力不如天然沸石,但其表面能更有效附着微生物,在实际工程应用上更具前景.