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.

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.

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.

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.

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.

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.

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.

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.

Biological control of Ixodes ricinus larvae and nymphs with Metarhizium anisopliae blastospores.

The entomopathogenic fungus Metarhizium anisopliae is used as a biological pest control agent against various arthropod species, including ticks. However, the efficacy depends on tick species, tick stage and fungus strain. We studied the effect of M. anisopliae on engorged larvae and nymphs of Ixodes ricinus, the most abundant tick species in Europe, under laboratory and semi-field conditions. A significant reduction of engorged larvae and nymphs could be shown under laboratory as well as under semi-field conditions. Only 3.5% of the larvae treated in the lab and only 18.5% kept under semi-field conditions were able to develop into nymphs compared to the recovered nymphs of the control groups, which were regarded as 100%. Only 7.1% of nymphs were recovered as adult ticks after fungal treatment under semi-field conditions compared to the control (100%). The efficacy of blastospores of M. anisopliae against engorged larvae and nymphs of I. ricinus under semi-field conditions was demonstrated in this study, showing their high potential as a biological control agent of ticks. Further studies will have to investigate the effect of this agent against other stages of I. ricinus as well as other tick species before its value as a biological control agent against ticks can be fully assessed.

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.

Sexy Mouth Odour? Male Oral Gland Pheromone in the Grain Beetle Parasitoid Lariophagus distinguendus (Förster) (Hymenoptera: Pteromalidae).

Throughout the animal kingdom, sexual pheromones are used for the attraction of mates and as courtship signals but also enable sexual isolation between species. In the parasitic wasp Lariophagus distinguendus, male courtship behaviour consisting of wing fanning, antennal stroking of the female antenna, and head nodding stimulates female receptivity leading to copulation. Recently L. distinguendus was reported to consist of two different lineages, which are sexually isolated because males fail to elicit receptivity in foreign females. It is unclear, however, which part of the courtship behaviour triggers female receptivity and therefore could be a mechanism causing sexual isolation. Here we show that in L. distinguendus a nonvolatile male oral pheromone is essential to release the female receptivity signal. In contrast, male wing fanning and antennal contact play a minor role. Additionally, the composition of the oral pheromone depends on the developmental host and females learn the composition upon emergence from the host substrate. These results will enable more detailed work on oral sexual pheromones to answer the question of how they are involved in the speciation process of L. distinguendus and other parasitoid species, for a better understanding of the huge biodiversity in this group.

Identification of a Grain Beetle Macrolide Pheromone and Its Synthesis by Ring-Closing Metathesis Using a Terminal Alkyne.

A major C18-macrolide was found during analysis of the frass of the storage beetle Oryzaephilus surinamensis to be (9Z,12Z,15R)-octadeca-9,12-dien-15-olide (10, cucujolide XI). The synthesis used ring-closing alkyne metathesis as a key step. The highly active 2,4,6-trimethylbenzylidyne molybdenum complex [MesCMo{OC(CF3)2Me}3] (12) allowed the use of a terminal alkyne and afforded the product in excellent yield. Bioassays proved the activity of the R-enantiomer 10 in the aggregation of the beetle. Cucujolide XI is the first macrolide pheromone oxidized at the ω-4 position.

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.

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..

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.

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.

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.

Pheromonal Communication in the European House Dust Mite, Dermatophagoides pteronyssinus.

Despite the sanitary importance of the European house dust mite Dermatophagoides pteronyssinus (Trouessart, 1897), the pheromonal communication in this species has not been sufficiently studied. Headspace analysis using solid phase micro extraction (SPME) revealed that nerol, neryl formate, pentadecane, (6Z,9Z)-6,9-heptadecadiene, and (Z)-8-heptadecene are released by both sexes whereas neryl propionate was released by males only. Tritonymphs did not produce any detectable volatiles. In olfactometer experiments, pentadecane and neryl propionate were attractive to both sexes as well as to tritonymphs. (Z)-8-heptadecene was only attractive to male mites. Therefore it is discussed that pentadecane and neryl propionate are aggregation pheromones and (Z)-8-heptadecene is a sexual pheromone of the European house dust mite D. pteronyssinus. To study the potential use of pheromones in dust mite control, long-range olfactometer experiments were conducted showing that mites can be attracted to neryl propionate over distances of at least 50 cm. This indicates that mite pheromones might be useable to monitor the presence or absence of mites in the context of control strategies.

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.

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.