Reproductive biology of viviparous and oviparous species of the leaf beetle genus Oreina

In five species of the genus Oreina Chevrolat (Coleoptera, Chrysomelidae) we compared the size of offspring, the fecundity of the females, the timing of offspring production and female investment over the season. Two of the species, O. elongata and O. luctuosa, laid eggs, while O. cacaliae, O. gloriosa and O. variabilis gave birth to larvae. Offspring size corrected for female size was similar in the two oviparous species and in the viviparous O. cacaliae. In the two other viviparous species the larvae were two to three times bigger in relation to the female. The greater size of the offspring was not traded off for lower fecundity in these latter two species, yet the production of bigger larvae was associated with a longer laying period and thereby a spreading of reproductive investment over the season. The prediction of life history theory that higher investment in individual offspring should be traded off for lower fecundity could not be confirmed. The investigation of egg and larval development showed that in one of the oviparous species, O. luctuosa, the length of the egg stage was more variable. This corroborates the view that in this species the eggs can be retained for varying times before being laid. Greater size at birth does not necessarily lead to shortened developmental times: the larval periods of O. cacaliae, O. elongata, O. gloriosa and O. variabilis were all comparable although the larvae of the first two species were relatively smaller when laid; only the small larvae of O. luctuosa needed significantly longer for their development. For all growth parameters examined the differences between species were larger than the differences between populations. A comparison of larval growth of the oligophagous species O. cacaliae on three plant genera showed that larval growth rate is influenced by the food plant. However, the plant on which the larvae grew worst is apparently not chosen for oviposition in the field. A comparison with a phylogeny of the species based on allozymes suggests that species with similar reproductive parameters are closely related, yet that viviparity evolved independently in O. cacaliae on one hand and O. variabilis and O. gloriosa on the other.     

Performance of leaf beetle larvae on sympatric host and non-host plants

Studies asking the ability of insects to utilize novel host plants often use novel hosts that are allopatric with the insect population under investigation. However, since the outcomes of species interactions are often site-specific, such studies cannot tell us whether a plant would actually be used by a given insect population if the plant grew sympatrically with it. We therefore performed a quantitative genetics experiment to analyse the performance of larvae of the leaf beetle Oreina elongata Suffrian (Coleoptera: Chrysomelidae, Chrysomelinae) on two host and three non-host plants, collected from a site where insects and plants co-occur in the Western Alps. When raised on the non-host Petasites albus (L.), larvae were able to survive equally well as on the two hosts, Adenostyles alliariae (Gouan) and Cirsium spinosissimum (L.), whereas they did not survive on the two other non-hosts, Peucedanum ostruthium (L.) and Rumex alpinus L. On P. albus, growth rate was slightly lower and development time slightly longer than on the two hosts. We found a genotype by environment interaction only for growth rate but not for development time and survival. However, the shape of the reaction norms of growth rates suggests that it is unlikely that selection could favour the inclusion of P. albus into the host range of the study population.     

Pyrrolizidine alkaloids in and on the leaf surface of Senecio jacobaea L

This is the first study showing that alkaloids are present on the leaf surface of plants. A concentration of 30-230 pmol/cm2 pyrrolizidine alkaloids (PA's) was detected in 8 different samples taken from Senecio jacobaea. PA concentration on the leaves was marginally correlated with PA concentration of the total leaf tissues. The PA spectrum on the leaf differed from the PA spectrum of the total leaf.     

An alternative hibernation strategy involving sun-exposed 'hotspots', dispersal by flight, and host plant finding by olfaction in an alpine leaf beetle

Oreina cacaliae (Schrank) (Coleoptera: Chrysomelidae) has a 2‐year life cycle that it has to complete within the short warm seasons of the harsh alpine environment. Three years of field observations and experiments revealed that not all beetles overwintered in the soil next to their principal host Adenostyles alliariae (Asteraceae), as was previously assumed, but that many O. cacaliae left their host in autumn and flew to overwintering sites that were extensively sun‐exposed. In spring, these individuals became active 2 months earlier than their conspecifics that had remained in the soil close to the host plant. These early beetles flew from their hibernation sites against the direction of the prevailing wind. After a random landing in snow, they walked to the spring host Petasites paradoxus (Asteraceae) and fed on its floral stalks, the only plant parts present at that time. A few weeks later, they took flight again to locate newly emerging A. alliariae on which they would feed and deposit larvae as did individuals that had overwintered close to A. alliariae. Leaves of A. alliariae contain pyrrolizidine alkaloids (PAs), which the beetles sequester for their own defence. The dominating PA (seneciphylline) was also found to be present in the floral stalks of P. paradoxus. With additional behavioural assays in the field and laboratory, we demonstrated the importance of plant odours in the short‐range host location process. This study reveals a unique hibernation behaviour in which part of the beetle population uses exceptionally warm locations from which they emerge in spring, long before all the snow has melted. This early, but risky emergence allows them to exploit a second, highly suitable host plant, which they locate first by wind‐guided flight and then by odour‐guided walking. The well‐fed beetles then use odour again to move to their principal host plant, on which they reproduce.     

Effects of leaf pubescence in Salix borealis on host- plant choice and feeding behaviour of the leaf beetle, Melasoma lapponica

Density of leaf trichomes in Salix borealis affected both the choice of individual host plants and feeding behaviour of adults and last instar larvae of the willow feeding leaf beetle, Melasoma lapponica. Beetles clearly preferred shaved disks to unshaved ones taken from the same leaf; this preference was highest in leaves of the most pubescent plants. High leaf pubescence explained the low preference for willow clones from the high density site in among-site preference trials; shaving significantly increased the consumption of these pubescent willow clones. In no-choice experiments, the food consumption by both adults and last instar larvae decreased with an increase in leaf pubescence. The time budget of adults did not depend on leaf pubescence of the host plants, however adults compelled to feed on highly pubescent plants changed their feeding sites twice as often as on less pubescent willow clones. Larvae feeding on highly pubescent plants spend moving three times as much time as larvae feeding on less pubescent plants. Combined with our earlier observations on the increase in leaf pubescence in the year(s) following defoliation, these data suggest that leaf hairiness may have contributed to the delayed induced resistance in S. borealis by disturbing the feeding behaviour of M. lapponica.     

Host race formation in the meadowsweet and strawberry feeding leaf beetle Galerucella tenella

We investigated host race formation in Galerucella tenella, a leaf beetle which feeds preferentially on meadowsweet (Rosaceae) in its natural habitats, but has become an important pest on strawberry (Rosaceae) in agricultural areas. Beetles from two isolated sites (Skeppsvik in Sweden and Solf in Finland) were compared with respect to preference and performance. At Skeppsvik the beetles were found feeding on meadowsweet, while at Solf large populations only develop in strawberry plantations, despite the presence of meadowsweet.In reciprocal field transplantations and laboratory bioassays, beetles from both sites discriminated against their foreign host, using their natal plant to a significantly higher degree for both egg laying and feeding, but with one interesting exception, namely that beetles from Solf increased their use of and even preferred meadowsweet for feeding in the laboratory. This increased use of meadowsweet by adult Solf beetles in the laboratory (without conspecific competitors) may be attributed to a density-dependent strategy, whereby mothers avoid pre-emptying the nutritional resources of the plants that will host their offspring in cases where no competitors are present.Larval fitness did not differ significantly between host plants, although larval survival of Solf beetles was halved when reared on their non-natal host plant (meadowsweet). Although beetles from both sites preferred to oviposit on their local host plant, our results provide little evidence that this presumed adaptation has to do with the nutritional quality of the plants involved. Instead, we suggest that other factors associated with the plants, such as enemy-free space and resistance to drought may be more important selective agents, shaping host preference in the field.     

Changes in flight track angles of cabbage root fly, Delia radicum, in diffuse clouds and discrete plumes of the host-plant volatile allylisothiocyanate

Flight tracks of female cabbage root fly, Delia radicum (L.), in odourless air, and in difuse clouds and discrete plumes of the host-plant volatile allylisothiocyanate (ANCS), were investigated. In diffuse ANCS D. radicum flew upwind with a smaller mean straight leg track angle with respect to the wind than in odourless air. In discrete ANCS, a larger mean straight leg track angle and a larger range of track angles occurred than in diffuse odour. The frequency distribution of these angles may have been bi-modal. It is suggested that perceived changes in odour concentration may alter the flight track angle. This behaviour is compared to the odour-modulated anemotaxis of male moths locating pheromone sources.

---

Résumé Changements des angles de vol de Delia radicum en présence de nuages diffus ou de panaches discontinus d'allylisothiocyanate, substance volatile émise par la plante-hôte.Nous avons étudié les chemins de vol des D. radicum femelles, en air pur, avect des émissions diffuses ou dicontinues de l'allylisothiocyanate (ANCS), substance volatilede la plante-hôte. En présence d'ANCS diffus, D. radicum vole contre le vent. Ce vol présente des portions droites faisant un angle avec la direction du vent; l'angle moyen de ce crochets est plus petit que celui observé en air pur. En présence d'ANCS discontinu, l'angle moyen est plus grand qu'en atmosphère diffuse et surtout l'histogramme de valeur de ces angles est bimodal.On peut envisager que la perception de changements dans la concentration de l'odeur peut modifier l'angle de vol. Un tel comportement est à comparer à l'anémotaxie contre le vent, modulée par l'odeur, observée chez les papillons mâles décelant une source de phéromones.

     

Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae)

This paper is the first record of the satellite DNA of the specialized phytophagous genus Chrysolina. The satellite DNA of Chrysolina americana is organized in a tandem repeat of monomers 189 bp long, has a A + T content of 59.6 % and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation sensitive enzymes suggests that this repetitive DNA is undermethylated. In situ hybridization with a biotinylated probe of the satellite DNA showed the pericentromeric localization of these sequences in all meiotic bivalents. The presence of this repetitive DNA in other species of the genus was also tested by Southern analysis. The results showed that this satellite DNA sequence is specific to the C. americana genome and has not been found in three other species of Chrysolina with a different choice of host plants than in the former. This revised version was published online in July 2006 with corrections to the Cover Date.     

Feeding damage by larvae of the mustard leaf beetle deters conspecific females from oviposition and feeding

Herbivorous insects may be informed about the presence of competitors on the same host plant by a variety of cues. These cues can derive from either the competitor itself or the damaged plant. In the mustard leaf beetle Phaedon cochleariae (Coleoptera, Chrysomelidae), adults are known to be deterred from feeding and oviposition by the exocrine glandular secretion of conspecific co-occurring larvae. We hypothesised that the exocrine larval secretion released by feeding larvae may adsorb to the surface of Chinese cabbage leaves, and thus, convey the information about their former or actual presence. Further experiments tested the influence of leaves damaged by conspecific larvae, mechanically damaged leaves, larval frass and regurgitant on the oviposition and feeding behaviour of P. cochleariae. Finally, the effect of previous conspecific herbivory on larval development and larval host selection was assessed. Our results show that (epi)chrysomelidial, the major component of the exocrine secretion from P. cochleariae larvae, was detectable by GC-MS in surface extracts from leaves upon which larvae had fed. However, leaves exposed to volatiles of the larval secretion were not avoided by female P. cochleariae for feeding or oviposition. Thus, we conclude that secretion volatiles did not adsorb in sufficient amounts on the leaf surface to display deterrent activity towards adults. By contrast, gravid females avoided to feed and lay their eggs on leaves damaged by second-instar larvae for three days when compared to undamaged leaves. Mechanical damage of leaves and treatment of artificially damaged leaves with larval frass or regurgitant did not affect oviposition and feeding of P. cochleariae. Since no adverse effects of previous herbivory on larval development were detected, we suggest that female P. cochleariae avoid Chinese cabbage leaves damaged by feeding larvae for other reasons than escape from competition or avoidance of direct negative effects that result from consuming induced plant material.     

Oviposition experience on a host-infested plant affects flight and antennal searching behavior of Cotesia kariyai toward the host-plant complex

The flight response of Cotesia kariyaiWatanabe (Hymenoptera: Braconidae), a parasitoid of the polyphagous herbivore, Mythimna separataWalker (Lepidoptera: Noctuidae), to pairs of different plant species infested by M. separatalarvae was tested under a dual choice condition in the laboratory. The oviposition-inexperienced (naive) wasps showed preference in the order: corn > kidney bean > Japanese radish. Wasps that had previously oviposited on the less preferred plant in a pair were found to have shifted their preference to this plant at 2 h after oviposition. However, this shift became indistinct at 17 h after oviposition. Prior oviposition on a plant species other than those being compared also affected the preference. These data suggest that learning is involved in the wasp's flight response. Prior oviposition was also observed to have an effect on the antennal searching behavior of the wasp on corn leaves. Such behavioral plasticity may enhance the efficiency by which C. kariyaisearches for polyphagous hosts.     

Colour polymorphism in relation to population dynamics of the leaf beetle, Chrysomela lapponica

We studied colour morph diversity and frequencies of light and dark morphs in non-fluctuating and fluctuating populations of willow feeding leaf beetle Chrysomela lapponica in the Kola Peninsula, NW Russia. Population-specific Shannon–Weaver diversity index positively correlated with dark morph frequencies, indicating that the larger part of colour polymorphism is related with numbers and diversity of dark morphs. Among-population variation in studied characters was not explained by pollution load or predation rates, but depended on the type of the population and the stage of density change in the fluctuating populations: both colour morph diversity and frequency of dark morphs were low in declining post-outbreak populations but equally high in non-fluctuating populations and in fluctuating populations at peak densities. In time-series, both diversity index and frequency of dark morphs decreased with post-outbreak density decline in the fluctuating population, but did not change in the non-fluctuating population. In the experiment, when adults received low quality food (plants from post-outbreak site), mortality of dark morphs during the hibernation was almost doubled relative to the mortality of light morphs, whereas on high quality food the colour morphs demonstrated similar mortality. This may indicate, that decrease in colour polymorphism extent and dark morph frequencies in the declining populations is due to selective mortality of dark morphs imposed by density dependent (induced by heavy herbivore damage during an outbreak) decrease in host-plant quality (delayed inducible resistance, DIR). DIR is known as one of the factors driving herbivore populations, but our result is the first evidence that DIR may act as a factor of natural selection. Dark morphs are not only susceptible to low food quality, but also have smaller size compared to light morphs, and therefore the dark females are presumably less fecund. Thus, decrease in frequency of low-fitness (dark) individuals in post-outbreak populations and accumulation of low-fitness phenotypes at the popu-lation peak may create feedbacks contributing to regulation of density fluctuations in Ch. lapponica.     

Site of synthesis,metabolism and translocation of senecionine N-oxide in cultured roots of Senecio erucifolius

Root cultures of Senecio erucifolius (Asteraceae) efficiently took up and incorporated [¹⁴C]putrescine and [¹⁴C]arginine into the pyrrolizidine alkaloid (PA) senecionine N-oxide. Pulse-chase experiments covering a growth period of 10 to 19 days revealed the absence of any significant alkaloid turnover. The only metabolic activity was a slow but progressive transformation of senecionine N-oxide into its dehydrogenation product, seneciphylline N-oxide. Tracer experiments with single roots showed that the sites of enhanced PA synthesis coincided with the sites of preferred protein synthesis, i.e. root apices, indicating a close correlation between growth activity and alkaloid synthesis. Long-term pulse-chase experiments (10 to 12 days) with ¹⁴C-labelled arginine, putrescine and senecionine fed to single roots indicated that in spite of its metabolic inertia, senecionine N-oxide is a mobile compound which is translocated into tissues newly grown during the chase.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Effect of variation in photoperiodic response on diapause induction and developmental time in the willow leaf beetle, Plagiodera versicolora

The willow leaf beetle, Plagiodera versicolora (Coleoptera: Chrysomelidae) overwinters in adult diapause. In this study, the photoperiodic responses for diapause induction and developmental time were examined in the Ishikari (Hokkaido, Japan) population of P. versicolora. All females entered reproductive diapause under short daylength (L10:D14), but 31.7% of females did not enter diapause under long daylength (L16:D8). The developmental time from oviposition to adult emergence was significantly longer at L10:D14 than that at L16:D8. Norm of reaction curves illustrated variation among families in the photoperiodic responses for diapause induction and for developmental time. ANOVA indicated significant family × photoperiod interactions in the developmental time. At L16:D8, developmental time was positively correlated with the incidence of diapause in females. This means that a female having a longer developmental time tends to have a longer critical photoperiod. Such variation may be maintained by differences in selection pressures on the growth rate and the critical photoperiod for diapause induction between univoltine and bivoltine genotypes because Ishikari is located in a transitional area between populations with univoltine and bivoltine life cycles.     

Nematodes of the genus Pristionchus are closely associated with scarab beetles and the Colorado potato beetle in Western Europe

Evolutionary developmental biology examines how changes in developmental programmes give rise to developmental and, ultimately, morphological novelty. To this end, comparisons of related but distinct organisms have to be performed. The diplogastrid nematode Pristionchus pacificus has been developed as a satellite system for a detailed comparison of various developmental processes to the model organism Caenorhabditis elegans, a rhabditid nematode. In addition to developmental and genetic studies, a genomic platform has been established to analyse the biology of this organism. However, only little is known about where and how Pristionchus pacificus and its relatives live in the wild. Here we show that nematodes of the genus Pristionchus live in close association with scarabaeoid beetles and the Colorado potato beetle. In total, we generated 371 isogenic female lines from 4242 beetles collected at 25 sampling sites all over Europe. Isogenic female lines were subjected to sequence analysis and mating experiments for species determination. The 371 isolates fell into six species. Two hermaphroditic species account for about 60% of the collected nematodes. We found Pristionchus maupasi almost exclusively on cockchafers and Pristionchus entomophagus predominantly on dung beetles. Colorado potato beetles carried the gonochoristic species Pristionchus uniformis, which was only rarely observed on scarabaeoid beetles. We describe the initial evidence for the association of Pristionchus nematodes with beetles and provide a phylogeny based on sequence analysis of the small subunit ribosomal RNA gene.     

Implication of tyramine in the biosynthesis of morphinan alkaloids in Papaver

Doubly-labeled [³H, ¹⁴C]tyrosines, [1-¹³C-]tyramine or [2-¹⁴C]tyramine, administered to the stems of intact Papaver somniferum L. plants, were found to be incorporated into the morphinan alkaloids of the plant with comparable efficiency. ³H/¹⁴C ratios of alkaloids from plants fed the tyrosines were consistent with an almost equal conversion of this amino acid into the tetrahydroisoquinoline (TIQ) and benzyl-derived segments. Nuclear magnetic resonance (NMR) analyses of morphine isolated after administration of [1-¹³C]tyramine demonstrated selective labeling of C-16 of the alkaloid, indicating the conversion of this amine primarily into the TIQ-derived moiety. Morphine and thebaine labeled by [2-¹⁴C]tyramine were degraded to phenanthridines and N,N-dimethyl ethylamines. Of the total radioactivity in the alkaloids 97% was found to be associated with the ethylamines, a distribution consistent with the NMR data. This preferential utilization of tyramine in the biosynthesis of morphinan alkaloids can be explained by the compartmentalization of intermediates and enzymes of the pathway.Abbreviations L-dopa L-3,4-dihydroxyphenylalanine - HPLC high-pressure liquid chromatography - NMR nuelear magnetic resonance - TIQ tetrahydroisoquinoline     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

Breakdown of indole alkaloids in suspension cultures of Tabernaemontana divaricata and Catharanthus roseus

The relative importance of breakdown on the accumulation of indole alkaloids has been determined in suspension cultures of Tabernaemontana divaricata and Catharanthus roseus by the feeding of stable isotope labelled alkaloids. In all cultures a considerable amount of the alkaloid biosynthesized was broken down. The breakdown was found to be dependent on the culture period and the half-life was in the order of several days. The breakdown could not explain the difference between producing and non-producing cultures. Further it was determined that in both cultures the breakdown was due to both biotic and abiotic factors.     

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t_1/2)<one day), were stable in solutions of acetonitrile, tetrahydrofuran and diluted hydrochloric acid (t_1/2>five months) and were unstable in solutions of methanol and ethanol (t_1/2<one month). These alkaloids were separated on an octadecylsilica column with isocratic elution using a solvent mixture of tetrahydrofuran and 0.2% trifluoroacetic acid (14:86, v/v), which was found to be the optimal solvent of the elution systems examined. Calibration curves with UV detection were linear on injection of amounts ranging from 2.5 to 500 ng, and the limit of detection was 1 ng (S/N = 3). These four alkaloids in aqueous solution were recovered almost totally by solid-phase extraction using the styrene polymer resin, Sep-Pak Plus PS-1, and were eluted using a mixture of acetonitrile and hydrochloric acid. These Aconitum alkaloids were confirmed by HPLC coupled with fast atom bombardment MS, giving their protonated molecular ions as base peaks. These alkaloids were detected by HPLC with UV detection from blood samples spiked with more than 50 ng ml⁻¹ of alkaloids, but were not detectable from urine samples spiked with 5 μg ml⁻¹ of alkaloids because of severe sample interference.     

Influence of endophyte infection, plant age and harvest interval on Rhopalosiphum padi survival and its relation to quantity of N-formyl and N-acetyl loline in tall fescue

Rhopalosiphum padi L. (Homoptera: Aphididae) is sensitive to loline alkaloids present in tall fescue, Festuca arundinacea Shreb., infected with the endophytic fungus, Acremonium coenophialum Morgan-Jones & Gams. Aphid survival was higher on endophyte-free plants regardless of plant age after germination or age of regrowth tissue after clipping. Survival of aphids on endophyte-infected grass was lower on young tissue but increased as plants aged, although it never reached the same level on endophyte-free plants. Both N-formyl and N-acetyl loline increased as uncut or regrowth tissue aged; however, this was influenced by the age of the plant at the initial cut and the clipping frequency. Although even small amounts of loline cause high aphid mortality, the aphids are able to survive on endophyte-infected plants if the tillers have senescing leaves which contain lower amounts of loline. Preference for senescing leaves may help R. padi avoid plant parts containing high amounts of toxic allelochemicals, thus contributing to higher numbers of aphids on older, endophyte-infected plants.