Sex Pheromone of the Click Beetle Agriotes pilosellus (SCHöNHERR, 1718).

Agriotes pilosellus is a fairly common click beetle species distributed in open deciduous and mixed forests throughout a large area in Europe. To identify its sex pheromone, gland extracts of female beetles were analyzed using gas chromatography-mass spectrometry (GC-MS). The only volatile compounds present in the extracts were geranyl butanoate and (E)-8-hydroxygeranyl dibutanoate in a 1:3 ratio, identified by comparison with synthetic samples. Field experiments revealed a clear attraction of A. pilosellus - males towards traps baited with geranyl butanoate, which could be synergistically enhanced by the factor of almost ten by addition of (E)-8-hydroxygeranyl dibutanoate. The latter compound alone did not show any attractive effect. Both compounds correspond well to the structures known from other Agriotes species and may serve as an effective monitoring tool for entomofaunistic research.

Sex Pheromone of the Rare Click Beetle Betarmon bisbimaculatus.

The click beetle Betarmon bisbimaculatus (Fabricius, 1803) (Coleoptera: Elateridae) has a scattered distribution throughout a large area in Europe and the near East. Due to its scarcity, little is known about the ecology, biology, and development of this peculiar species. Here, we studied the composition of the female-released sex pheromone of B. bisbimaculatus. Neryl hexanoate, neryl octanoate, and neryl decanoate, in a ratio of approximately 3:1:6, were the only volatile compounds present in the extracts of pheromone glands. A synthetic mixture of all three compounds in the natural ratio was highly attractive to males in field traps. When the compounds were tested individually, only traps baited with neryl hexanoate were attractive, but they caught only a sixth of the males compared to the mixture. Based on the similarity of their sex pheromones, we propose that the tribe Pomachiliini with B. bisbimaculatus is closely related to the tribe Agriotini. This study shows the potential of sex pheromone studies for monitoring of rare and threatened insects as well as for elucidating phylogenetic relationships.

Does early learning drive ecological divergence during speciation processes in parasitoid wasps?

Central to the concept of ecological speciation is the evolution of ecotypes, i.e. groups of individuals occupying different ecological niches. However, the mechanisms behind the first step of separation, the switch of individuals into new niches, are unclear. One long-standing hypothesis, which was proposed for insects but never tested, is that early learning causes new ecological preferences, leading to a switch into a new niche within one generation. Here, we show that a host switch occurred within a parasitoid wasp, which is associated with the ability for early learning and the splitting into separate lineages during speciation. Lariophagus distinguendus consists of two genetically distinct lineages, most likely representing different species. One attacks drugstore beetle larvae (Stegobium paniceum (L.)), which were probably the ancestral host of both lineages. The drugstore beetle lineage has an innate host preference that cannot be altered by experience. In contrast, the second lineage is found on Sitophilus weevils as hosts and changes its preference by early learning. We conclude that a host switch has occurred in the ancestor of the second lineage, which must have been enabled by early learning. Because early learning is widespread in insects, it might have facilitated ecological divergence and associated speciation in this hyperdiverse group.

Early memory in the parasitoid wasp Nasonia vitripennis.

We studied early memory in Nasonia vitripennis, a parasitoid of fly pupae and emerging model organism for ecological questions in the context of learning. After associative training consisting of one drilling experience in a host in the presence of cinnamon, females showed a preference to cinnamon for at least 24 h. To study sensitisation, wasps were trained by one drilling in a host without cinnamon. These wasps were not attracted to cinnamon immediately afterwards. Obviously, sensitisation is not involved in early memory. Wasps that were anaesthetised by CO2 directly after associative training did not react to cinnamon 30-45 min later, but after 1 and 24 h. CO2 treatment≥5 min after training did not erase the reaction to cinnamon. This indicates the existence of four early memory phases. (a) One phase<5 min after training, in which CO2 has to be applied to become effective. (b) One subsequent phase which is resistant to CO2. (c) One phase<1 h in which memory can be erased by CO2 treatment directly after training. (d) One phase 1-24 h not affected by CO2 treatment. Together with earlier data this enables us to establish a complete memory structure for N. vitripennis.

Age and aggregation trigger mating behaviour in the small hive beetle, Aethina tumida (Nitidulidae).

This study aimed to investigate the poorly documented reproductive behaviour of the small hive beetle, Aethina tumida (Nitidulidae), a honey bee (Apis mellifera) parasite. We described the mating behaviour in detail and tested the hypothesis that beetle aggregation plays a vital role in mating in this species. Gender preference was examined in the context of age-dependency and possible chemical communication. Beetles started mating at a high frequency 18 days after emergence from the soil but only if they were aggregated (p < 0.001); mating was infrequent when beetles were paired. Males in aggregation also tried to copulate with males and only copulated more frequently with females at 18 days after emergence from soil (p < 0.001) in contrast to newly emerged, 7-day-old and 60-day-old beetles. Males and females spent more time in social contact with the opposite sex (p < 0.01) when they were 18 days old in contrast to 7-day-old beetles. Filter papers which had been in contact with 21-day-old beetles were highly attractive to similar-aged beetles of the opposite sex (p < 0.01). This suggests that chemical substances produced by the beetles themselves play a role in mating. Mating behaviour was characterised by a short pre-copulation courtship and female aggression towards other females and copulating couples. Both behaviours may be indicative of cryptic female choice. Delayed onset of reproductive behaviour is typical of many polygamous species, whilst the indispensability of aggregation for onset of sexual behaviour seems to be a feature unique to A. tumida. Both strategies support mass reproduction in this parasitic species, enabling A. tumida to overcome its honey bee host colony, and are probably triggered by chemotactic cues..

Decrypting Cryptic Click Beetle Species by Analysis of Sex Pheromones.

Despite sex pheromones being highly species specific, their use as phylogenetic characters and a tool for the verification of species status are still relatively few compared to use of morphological and molecular characters. Earlier studies revealed that within the click beetle species Idolus picipennis, two types can be separated based on pheromone composition. Gas chromatography/mass spectrometry analysis of pheromone from a third type of Idolus revealed the presence of geranyl hexanoate and geranyl octanoate in a ratio of ca. 1:9. Neryl esters and farnesyl esters, present in the glands of the other two species, are absent in this type. In field experiments, males of all three types were attracted specifically to synthetic mixtures of pheromone resembling their own females. This suggests that cross attraction among different types is unlikely and indicates that they are likely distinct species. Using the large numbers of male beetles caught in pheromone traps, morphological differences between the species were studied and an identification key derived. This study highlights the role of sex pheromones as a powerful tool in integrative taxonomy and systematics to study the phylogenetic position and evolution of taxa and to determine the taxonomic status of cryptic species.

Identification and synthesis of macrolide pheromones of the grain beetle Oryzaephilus surinamensis and the frog Spinomantis aglavei.

Macrolide lactones, the so called cucujolides derived from unsaturated fatty acids, are aggregation pheromones of cucujid grain beetles. Thirty years ago, Oehlschlarger et al. showed that (3Z,6Z)-dodeca-3,6-dien-11-olide (4) and the respective 12-olide (7) attract the sawtoothed grain beetle Oryzaephilus surinamensis, whereas (5Z,8Z,13R)-tetradeca-5,8-dien-13-olide (5) increases the response synergistically. The frass of this beetle is attractive for its parasitoid Cephalonomia tarsalis, which potentially can be used for pest control. A GC/MS analysis of attractive frass showed the presence of 5, together with an unknown isomer. Cucujolide V was tentatively identified also in the femoral glands, pheromone-releasing structures, of the Madagascan mantelline frog Spinomantis aglavei. Therefore, a new route to synthesize doubly unsaturated macrolides allowing the flexible attachment of the side chain was developed. A straightforward method to obtain Z configured macrolides involves ring-closing alkyne metathesis (RCAM) followed by Lindlar-catalyzed hydrogenation. This methodology was extended to homoconjugated diene macrolides by using RCAM after introduction of one Z configured double bond in the precursor by Wittig reaction. A tungsten benzylidyne complex was used as the catalyst in the RCAM reaction, which afforded the products in high yield at room temperature. With the synthetic material at hand, the unknown isomer was identified as the new natural product (5Z,8Z,12R)-tetradeca-5,8-dien-12-olide, cucujolide X (8). Furthermore, the route also allowed the synthesis of cucujolide V in good yield. The natural products were identified by the synthesis of enantiomerically pure or enriched material and gas chromatography on chiral phases. The new macrolide (R)-8 proved to be biologically active, attracting female O. surinamensis, but no males. The synthetic material allowed the identification of (R)-5 in both the beetle and the frog.

Identification of the sex pheromone of Idolus picipennis (Bach, 1852) revealed the presence of a cryptic sibling species.

The click beetle Idolus picipennis represents the only species of its genus in Europe, where it is widely distributed but is rare and only occurs locally. In order to identify its sex pheromone we investigated gland extracts of females from populations in southern Germany. GC/MS analyses revealed two distinct types of gland compositions that correspond to slight but consistent morphological differences in the respective beetles. Extracts of one type contain four compounds, geranyl hexanoate (~ 40 %), (Z,E)-farnesyl hexanoate (~ 10 %), (E,E)-farnesyl hexanoate (~ 40 %), and (E,E)-farnesyl octanoate (~ 10 %), and this type belongs to the authentic I. picipennis (Bach 1852). Extracts of a second type contain neryl hexanoate (~10 %) and neryl octanoate (~ 90 %), and this type belongs to an Idolus species that apparently has been overlooked to date, presumably due to similarity with the authentic I. picipennis and insufficient material in collections. Synthetic blends of the identified compounds in their naturally-occurring ratios, as well as the main compounds alone, proved to be highly attractive to swarming males of the respective species in the field. A strong species-specific attraction also was observed in a locality where both species co-occur, thus confirming effective reproductive isolation. This study shows the potential of sex pheromones for monitoring rare and threatened insects as well as for detecting hitherto unknown cryptic species.

Highly transmissible cytoplasmic incompatibility by the extracellular insect symbiont Spiroplasma.

Cytoplasmic incompatibility (CI) is a form of reproductive manipulation caused by maternally inherited endosymbionts infecting arthropods, like Wolbachia, whereby matings between infected males and uninfected females produce few or no offspring. We report the discovery of a new CI symbiont, a strain of Spiroplasma causing CI in the parasitoid wasp Lariophagus distinguendus. Its extracellular occurrence enabled us to establish CI in uninfected adult insects by transferring Spiroplasma-infected hemolymph. We sequenced the CI-Spiroplasma genome and did not find any homologues of any of the cif genes discovered to cause CI in Wolbachia, suggesting independent evolution of CI. Instead, the genome contains other potential CI-causing candidate genes, such as homologues of high-mobility group (HMG) box proteins that are crucial in eukaryotic development but rare in bacterial genomes. Spiroplasma's extracellular nature and broad host range encompassing medically and agriculturally important arthropods make it a promising tool to study CI and its applications.

The emergence of ecotypes in a parasitoid wasp: a case of incipient sympatric speciation in Hymenoptera?

BACKGROUND: To understand which reproductive barriers initiate speciation is a major question in evolutionary research. Despite their high species numbers and specific biology, there are only few studies on speciation in Hymenoptera. This study aims to identify very early reproductive barriers in a local, sympatric population of Nasonia vitripennis (Walker 1836), a hymenopterous parasitoid of fly pupae. We studied ecological barriers, sexual barriers, and the reduction in F1-female offspring as a postmating barrier, as well as the population structure using microsatellites. RESULTS: We found considerable inbreeding within female strains and a population structure with either three or five subpopulation clusters defined by microsatellites. In addition, there are two ecotypes, one parasitizing fly pupae in bird nests and the other on carrion. The nest ecotype is mainly formed from one of the microsatellite clusters, the two or four remaining microsatellite clusters form the carrion ecotype. There was slight sexual isolation and a reduction in F1-female offspring between inbreeding strains from the same microsatellite clusters and the same ecotypes. Strains from different microsatellite clusters are separated by a reduction in F1-female offspring. Ecotypes are separated only by ecological barriers. CONCLUSIONS: This is the first demonstration of very early reproductive barriers within a sympatric population of Hymenoptera. It demonstrates that sexual and premating barriers can precede ecological separation. This indicates the complexity of ecotype formation and highlights the general need for more studies within homogenous populations for the identification of the earliest barriers in the speciation process.

Sex Pheromone of Agriotes acuminatus (Stephens, 1830) (Coleoptera: Elateridae).

The click beetle species Agriotes acuminatus is distributed in open deciduous forests throughout a large area in Europe. In order to identify its sex pheromone, gland extracts of female beetles were investigated by using GC/MS. Neryl butanoate and 2,6-dimethyl-(Z,E)-2,6-octadien-1,8-diol dihexanoate, in a ratio of approximately 1:5, were the only volatile compounds present in the extracts. Structures of both esters were confirmed by synthesis. Field experiments revealed a strong attraction of A. acuminatus males towards neryl butanoate, which could be synergistically enhanced by addition of 2,6-dimethyl-(Z,E)-2,6-octadien-1,8-diol dihexanoate. The latter compound alone did not show any attractive effect. While all Agriotes spp. investigated to date use geranyl and/or (E,E)-farnesyl esters as sex pheromones, the nerol derivatives of A. acuminatus are the first (Z)-2-configurated pheromones within this genus.

Olfactory host finding, intermediate memory and its potential ecological adaptation in Nasonia vitripennis.

Associative learning of host-associated chemical cues was studied in Nasonia vitripennis, a parasitoid of fly pupae in nests of hole-nesting birds. When females encountered a fly pupa and performed one sequence of host recognition behaviour including drilling the ovipositor into the host in the presence of the artificial odour furfurylheptanoate (FFH), they were afterwards arrested by FFH in olfactometer experiments. The response vanished after 4 days and could be blocked after 3 days by feeding wasps with ethacrynic acid prior and after the training. This indicates the formation of an intermediate form of memory by one host experience in N. vitripennis. Interestingly, the trained wasps avoided odours that were not present during the host encounter, although naive wasps did not react to these odours. This unique behaviour probably causes wasps to focus during host searching on those chemical cues they have experienced in the host environment. Studies in nests of hole-nesting birds revealed that about 30% of all nests contained only one fly pupa, and laboratory studies showed that N. vitripennis females are able to parasitise around 100 fly pupae in their life. It is discussed that under these conditions, the formation of a non-permanent intermediate memory for host-associated odours after one host encounter is adaptive to avoid costs involved with formation and maintenance of memory for misleading cues. The demonstration of associative olfactory learning in N. vitripennis, the first parasitoid species with sequenced genome, opens the gate to study molecular mechanisms of memory formation and its ecological adaptation in parasitoids.

Mate finding in the parasitic wasp Cephalonomia tarsalis (Ashmead): more than one way to a female's heart.

The parasitic wasp, Cephalonomia tarsalis (Hymenoptera: Bethylidae), parasitizes larvae of the sawtoothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae), and is used for biological control of this worldwide pest of stored grain. To study the hypothesis that C. tarsalis not only mates at its natal patch but also uses olfactory cues to find mating partners elsewhere, we investigated semiochemical use by male C. tarsalis. Olfactometer experiments revealed that male C. tarsalis are arrested by odors emanating from the cocoons of conspecifics, from young unmated females, and from feces of the host. Dodecanal, which was identified from extracts of filter paper contaminated by young females, had an arresting effect on males but not on females and was, therefore, considered as a sex pheromone. These findings indicate that C. tarsalis is a species with partial local mate competition. Males mate with females: 1) at the emergence site following location of females by sex pheromones from their cocoons, and 2) after dispersal from the natal patch following location of females directly by dodecanal and indirectly by unidentified sexual kairomones from host feces.

Reproductive isolation due to prezygotic isolation and postzygotic cytoplasmic incompatibility in parasitoid wasps.

The reproductive barriers that prevent gene flow between closely related species are a major topic in evolutionary research. Insect clades with parasitoid lifestyle are among the most species-rich insects and new species are constantly described, indicating that speciation occurs frequently in this group. However, there are only very few studies on speciation in parasitoids. We studied reproductive barriers in two lineages of Lariophagus distinguendus (Chalcidoidea: Hymenoptera), a parasitoid wasp of pest beetle larvae that occur in human environments. One of the two lineages occurs in households preferably attacking larvae of the drugstore beetle Stegobium paniceum ("DB-lineage"), the other in grain stores with larvae of the granary weevil Sitophilus granarius as main host ("GW-lineage"). Between two populations of the DB-lineage, we identified slight sexual isolation as intraspecific barrier. Between populations from both lineages, we found almost complete sexual isolation caused by female mate choice, and postzygotic isolation, which is partially caused by cytoplasmic incompatibility induced by so far undescribed endosymbionts which are not Wolbachia or Cardinium. Because separation between the two lineages is almost complete, they should be considered as separate species according to the biological species concept. This demonstrates that cryptic species within parasitoid Hymenoptera also occur in Central Europe in close contact to humans.

The Asian bush mosquito Aedes japonicus japonicus (Diptera: Culicidae) in Europe, 17 years after its first detection, with a focus on monitoring methods.

After the first detection of the Asian bush mosquito Aedes japonicus japonicus in the year 2000 in France, its invasive nature was revealed in 2008 in Switzerland and Germany. In the following years, accumulating reports have shown that Ae. j. japonicus succeeded in establishing in several European countries. Surveillance efforts suggest that there are currently four populations in Europe, with the largest one, formed by the recent fusion of several smaller populations, ranging from West Germany, with extensions to Luxembourg and French Alsace, southwards to Switzerland and continuing westwards through Liechtenstein to western Austria. This paper summarises the present distribution of Ae. j. japonicus in Europe, based on published literature and hitherto unpublished findings by the authors, and critically reviews the monitoring strategies applied. A proposal for a more standardised monitoring approach is provided, aiming at the harmonisation of future data collections for improving the comparability between studies and the suitability of collected data for further research purposes, e.g. predictive modelling approaches.

Microdissection and whole chromosome painting confirm karyotype transformation in cryptic species of the Lariophagus distinguendus (Förster, 1841) complex (Hymenoptera: Pteromalidae).

Karyotypes of two cryptic species of parasitoid Hymenoptera with n = 5 and 6 belonging to the Lariophagus distinguendus (Förster, 1841) complex, which includes cosmopolitan parasitoids of coleopteran stored-product pests, were studied using glass-needle based microdissection, reverse and cross-species fluorescence in situ hybridisation (FISH). This experiment strongly indicates that the largest metacentric chromosome in the karyotype with n = 5 originated from a particular fusion between the only acrocentric and a smaller metacentric chromosome of the set with n = 6, therefore confirming our previous hypothesis based on the karyotypic analysis using chromosome morphometrics. This study represents the first successful application of both microdissection and whole chromosome painting for the reconstruction of karyotypic rearrangements in closely related species of parasitoids, as well as in the order Hymenoptera in general.

Molecular and cytogenetic differentiation within the Lariophagusdistinguendus (Förster, 1841) species complex (Hymenoptera, Pteromalidae).

Several strains of the apparently well-known cosmopolitan synanthropic parasitoid of coleopteran stored-product pests, Lariophagusdistinguendus (Förster, 1841) from Western Europe, were studied using DNA sequencing and chromosomal analysis. The presence of at least two cryptic species with different COI sequences and chromosome numbers (n = 5 and 6) was supported. The species with n = 6 is associated with the drugstore beetle Stegobiumpaniceum (Linnaeus, 1758), whereas the other one with n = 5 mostly develops on the granary weevil Sitophilusgranarius (Linnaeus, 1758). A phylogenetic study revealed that the karyotype with n = 6 represents an ancestral character state in this complex. Consequently, the chromosome set with n = 5 which is characteristic of a particular internal clade, apparently originated via chromosomal fusion which was probably preceded by a pericentric inversion. If this is true, inverted chromosome segments could accumulate a number of genetic loci responsible for certain interspecific differences.

Species-specific acquisition and consolidation of long-term memory in parasitic wasps.

Long-term memory (LTM) formation usually requires repeated, spaced learning events and is achieved by the synthesis of specific proteins. Other memory forms require a single learning experience and are independent of protein synthesis. We investigated in two closely related parasitic wasp species, Cotesia glomerata and Cotesia rubecula, whether natural differences in foraging behaviour are correlated with differences in LTM acquisition and formation. These parasitic wasp species lay their eggs in young caterpillars of pierid butterflies and can learn to associate plant odours with a successful egg laying experience on caterpillars on the odour-producing plant. We used a classical conditioning set-up, while interfering with LTM formation through translation or transcription inhibitors. We show here that C. rubecula formed LTM after three spaced learning trials, whereas C. glomerata required only a single trial for LTM formation. After three spaced learning trials, LTM formation was complete within 4 h in C. glomerata, whereas in C. rubecula, LTM formation took 3 days. Linking neurobiology with ecology, we argue that this species-specific difference in LTM acquisition and formation is adaptive given the extreme differences in both the number of foraging decisions of the two wasp species and in the spatial distributions of their respective hosts in nature.

Host habitat assessment by a parasitoid using fungal volatiles.

BACKGROUND: The preference-performance hypothesis predicts that oviposition preference of insects should correlate with host suitability for offspring development. Therefore, insect females have to be able to assess not only the quality of a given host but also the environmental conditions of the respective host habitat. Chemical cues are a major source of information used by insects for this purpose. Primary infestation of stored grain by stored product pests often favors the intense growth of mold. This can lead to distinct sites of extreme environmental conditions (hot-spots) with increased insect mortality. We studied the influence of mold on chemical orientation, host recognition, and fitness of Lariophagus distinguendus, a parasitoid of beetle larvae developing in stored grain. RESULTS: Volatiles of wheat infested by Aspergillus sydowii and A. versicolor repelled female parasitoids in an olfactometer. Foraging L. distinguendus females are known to be strongly attracted to the odor of larval host feces from the granary weevil Sitophilus granarius, which may adhere in remarkable amounts to the surface of the grains. Feces from moldy weevil cultures elicited neutral responses but parasitoids clearly avoided moldy feces when non-moldy feces were offered simultaneously. The common fungal volatile 1-octen-3-ol was the major component of the odor of larval feces from moldy weevil cultures and repelled female parasitoids at naturally occurring doses. In bioassays investigating host recognition behavior of L. distinguendus, females spent less time on grains containing hosts from moldy weevil cultures and showed less drumming and drilling behavior than on non-moldy controls. L. distinguendus had a clearly reduced fitness on hosts from moldy weevil cultures. CONCLUSION: We conclude that L. distinguendus females use 1-octen-3-ol for host habitat assessment to avoid negative fitness consequences due to secondary mold infestation of host patches. The female response to fungal volatiles is innate, suggesting that host-associated fungi played a crucial role in the evolution of host finding strategies of L. distinguendus. Research on the role of host-associated microorganisms in the chemically mediated orientation of parasitoids is still at the beginning. We expect an increasing recognition of this issue in the future.

Lariophagus distinguendus (Hymenoptera: Pteromalidae) (Förster)-Past, Present, and Future: The History of a Biological Control Method Using L. distinguendus against Different Storage Pests.

Legal requirements and consumer demands for residue-free products pose a big challenge for pest control in grain stores. One possible alternative to chemical insecticides is biological pest control with the pteromalid wasp Lariophagus distinguendus against the weevils Sitophilus granarius, S. oryzae (Coleoptera: Dryophtoridae), and many other storage pest beetles. The use of this wasp as a biocontrol agent was already suggested in 1919 by Prof. Dr. Hase [1]. Despite many studies on host-finding and behavioral biology, the applied aspect was neglected until 1994. Nowadays the wasps are commercially available and can now even be reared on-site, facilitating their use tremendously. This review highlights the milestones in L. distinguendus research, gives insights in current studies, and ventures a glimpse into the future.