Uptake and turn-over of glucosinolates sequestered in the sawfly Athalia rosae

Larvae of the sawfly Athalia rosae sequester glucosinolates from their various host plants of the Brassicaceae into their hemolymph for defensive purposes. We found that the glucosinolate concentration in the insect varies in a fluctuating manner during larval development. Analyses of larvae which had been offered diets with different glucosinolate profiles showed that there is an equilibrium between a rapid uptake of glucosinolates into the hemolymph and a continuous turn-over. Injection of glucotropaeolin into the hemolymph and ingestion of the same amount resulted in similar levels of intact glucosinolates recovered from larvae after different periods of time. This indicates that hemolymph glucosinolates are the principal source for glucosinolate degradation. Feeding experiments with [14C]-labeled glucotropaeolin revealed that the majority of the ingested glucosinolate is excreted as one or more unidentified metabolite(s) within 14 h. We found no indication for the presence of an insect myrosinase, or sulfatase in A. rosae, which have been shown to be involved in glucosinolate metabolism in other specialists feeding on Brassicaceae. Furthermore, the metabolism of sinalbin in A. rosae seems to result in different products than its metabolism in the caterpillar Pieris rapae. Obviously, A. rosae has yet another way of coping with the glucosinolates.     

Interactions between glucosinolate- and myrosinase-containing plants and the sawfly Athalia rosae

Several insects have specialised on using Brassicaceae as host plants. Therefore, they evolved metabolic pathways to cope with the defensive glucosinolate–myrosinase system of their diet. Larvae of the turnip sawfly, Athalia rosae L. (Hymenoptera: Tenthredinidae), incorporate various glucosinolates from their hosts into their haemolymph. The ability to sequester these metabolites makes A. rosae a useful model system to study mechanisms of glucosinolate metabolism in this species compared to other specialists, and to study effects of sawfly feeding on levels of glucosinolates and their hydrolysing enzymes in plants. The levels of plant metabolites might in turn directly affect the performance of the insect. On the one hand, costs for glucosinolate uptake and avoidance of myrosinase activity were postulated. On the other hand, sequestration of glucosinolates can be part of the insect’s defence against several predators. Here, the findings on glucosinolate metabolic pathways are compared between different herbivores and the sawfly. The impact of different glucosinolate levels and myrosinase activities on the performance of A. rosae is discussed. Furthermore, effects of feeding by A. rosae larvae on the chemical composition and enzyme activities of various Brassicaceae species are summarised. Induction patterns vary not only between different plant species and cultivars but also due to the inducing agent. Finally, the plant–herbivore interactions are discussed with regard to the sawflies’ defence abilities against different carnivore guilds.     

Genetics and biology of the sawfly,Athalia rosae (Hymenoptera)

Hymenopteran insects are a unique group of animals in which arrhenotokous reproduction (haploid males develop from unfertilized eggs) is a rule. Males produce sperm through a non-reductional maturation division. A sawfly species,Athalia rosae ruficornis Jakovlev (Tenthredinidae, Symphyta, Hymenoptera), has been introduced as a new experimental material for studies on genetics and developmental biology. Basic features relating to the potential usefulness of the species in elucidating some of the important genetic and developmental biological problems are described.     

Production of haploid-haploid chimeras by sperm injection in the sawfly,Athalia rosae (Hymenoptera)

Mature unfertilized eggs (oocytes) dissected from the ovary of the sawfly Athalia rosae (Hymenoptera) begin parthenogenetic development if exposed to distilled water and produce haploid males. Injection of sperm into mature oocytes through the anterior pole resulted in karyogamy in a fraction of cases which developed as diploid females. No haploid-haploid chimeras due to independent participation of the injected sperm in development were produced. When sperm were injected through the posterior pole, however, fertilization never occurred but haploid-haploid chimeras were produced in a smaller fraction of cases. Both egg nucleus-derived and injected sperm-derived nuclei contributed in forming the germ cells of the chimeric males.     

Effectiveness of the defence mechanism of the turnip sawfly, Athalia rosae (Hymenoptera: Tenthredinidae), against predation by lizards

The turnip sawfly, Athalia rosae Linnaeus, is a pest on cruciferous crops. Larvae sequester secondary plant compounds, namely glucosinolates, in their haemolymph. When attacked, their integument is easily disrupted and a droplet of haemolymph is exuded ('easy bleeding'). This has been shown to be an effective, chemical-based, defence against invertebrate predators. The efficiency of this proposed defence was tested against a vertebrate predator, using groups of the iguanid lizard Anolis carolinensis Voigt as a model predator. Caterpillars of Pieris rapae Linnaeus and Pieris brassicae Linnaeus served as control prey species that do not sequester glucosinolates. Lizards attacked far fewer sawfly larvae than pierid caterpillars. Several of the sawfly larvae were rejected after an initial attack, demonstrating unpalatability to the lizards, while the Pieris larvae were not rejected. However, P. rapae larvae topically treated with extracts of haemolymph of A. rosae had no deterrent effect on the lizards and no avoidance learning occurred over a period of two weeks. Adult sawflies do not easy bleed but have glucosinolates carried over from the larval stage. Lizards attacked them at a higher rate than larvae and they were never rejected. The results suggest that for the defensive effectiveness of the pest sawfly species against vertebrates the chemical cue is not necessarily sufficient. Movement and colour may be important additional factors triggering the behaviour of vertebrate predators.     

Ovarian development and vitellogenesis in the sawfly,Athalia rosae ruficornis Jakovlev (Hymenoptera,Tenthredinidae)

Summary

Ovarian development in Athalia rosae ruficornis Jakovlev (Hymenoptera: Tenthredinidae) is described. Number of nurse cells per egg chamber is most often around 60 (close to 63 according to the 2ⁿ–1 rule), but in many cases it deviates from this number significantly. Two major yolk proteins [vitellins: large (apparent molecular weight 160–170 kD) and small (48–50 kD] were identified by SDS-PAGE. Western blotting and immunochemical detection using polyclonal antibodies prepared against each of the vitellins revealed that adult female but not male (both haploid and diploid) hemolymph contains vitellogenins corresponding to these vitellins. Vitellogenins become detectable in the hemolymph of late pupae, and vitellins one day later in the oocytes of adults. Transplantation of immature ovaries into the adult male abdomen caused not only significant accumulation of vitellins in the oocyte but also appearance of small amounts of hemolymph vitellogenins in host males. Injection of homogenate of immature ovaries also caused appearance of small amounts of hemolymph vitellogenins in host males.     

Experiments inducing prospective polar body nuclei to participate in embryogenesis of the sawfly Athalia rosae (Hymenoptera)

Summary Mature eggs dissected from ovaries of unmated females of Athalia rosae (Hymenoptera: Tenthredinidae), if placed on a filter-paper soaked with distilled water, are activated and develop to haploid males. Occasionally, however, diploid females develop from these artificially activated eggs. Treatment of mature unfertilized eggs dissected from diploid females with ice-cold temperatures immediately before activation and with a high temperature (36° C) upon and immediately after activation resulted in the production of diploid males, diploid females, triploid females and gynandromorphs at high frequency. The same treatment of mature unfertilized eggs dissected from triploid females resulted in the production of only triploid survivors. These results, together with the results on the segregation of a marker mutation, yellow fatbody (yfb), appear to indicate that meiotic divisions were complete in the treated eggs, and that all four nuclei became potentially capable of participating in development with or without automictic fusion.Studies on the sawfly, Athalia rosae (Insecta, Hymenoptera, Tenthredinidae), part V     

Characterization of abdominal appendages in the sawfly, Athalia rosae (Hymenoptera), by morphological and gene expression analyses

Larvae of the sawfly, Athalia rosae, have remarkable abdominal prolegs. We analyzed the morphogenesis of appendages and the expression of decapentaplegic and Distal-less genes during embryonic development to characterize the origin of prolegs. Proleg primordia in abdominal segments A1–A9 appeared shortly after the inner lobes (endites) of gnathal appendages were formed. These were located on the ventral plates, medioventral to the appendages of the other segments in light of serial homology. Nothing was seen where the main axis of the appendage should develop in abdominal segments. The primordia in A1 and A9 disappeared before larval hatching. Anal prolegs appeared separate from cerci, the main axes of appendages, which were formed temporarily in A11. The expression of decapentaplegic, which reflects the primary determination of appendages, was detected in the lateral juxtaposition with the prolegs. Distal-less was expressed in the main axes of appendages, protruding endites and the cerci, but not in prolegs and anal prolegs or the gnathal endites which do not protrude. These findings suggest a possibility that the abdominal and anal prolegs of A. rosae are outgrowths of ventral plates which derived from coxopodal elements, but not main axes of appendages.     

Germline transformation of the sawfly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector

A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous helper plasmid containing an active piggyBac transposase gene into the posterior end of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera: Symphyta). These injected eggs, which developed as haploid male embryos upon artificial activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid adult males. These G(0) haploid males were individually mated to diploid females. The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1) diploid females were activated artificially, and the resultant embryos were examined for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2) haploid embryos segregated to GFP-positive and -negative individuals. By mating the G(2) adult haploid males individually to diploid females, stocks were established in which the piggyBac construct was stably integrated into the genome, as evidenced by GFP expression and Southern blot hybridization. The piggyBac transposition occurred at its canonical target TTAA sequence. These results, which demonstrate the first successful stable transposon-mediated germline transformation in Hymenoptera, will expand the usefulness of the piggyBac vector.     

Estimation of allele numbers at the sex-determining locus in a field population of the turnip sawfly (Athalia rosae)

Hymenopteran insects (sawflies, ants, bees, and wasps) have an unusual genetic system called haplodiploidy, where parthenogenetically produced haploid eggs become males, and fertilized, diploid eggs become females. Several hypotheses have been proposed to explain the mechanism of such sex determination, including control at a single polymorphic locus. From experiments of mother-son mating and using a genetic marker, we show that a single multiallele locus controls sex determination in the turnip sawfly (Athalia rosae). We estimated the number of alleles at this single locus in a field population by analyzing the rate of diploid males in the field and the rate of diploid males by random crossing in the laboratory. Only one diploid male was discovered in 1306 diploid larvae collected in the field. However, the number of alleles calculated by random crossing in the laboratory was 45-50. We suggest that the effective population size may be much larger than that from the areas where we collected larvae, and that there are mechanisms for avoiding inbreeding, including protogyny, dispersion, and sperm displacement by second-mated males.     

A linkage map of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta) based on random amplified polymorphic DNAs

A linkage map was constructed for the sawfly, Athalia rosae (Hymenoptera), based on the segregation of random amplified polymorphic DNA (RAPD) markers and a visible mutation, yellow fat body (yfb). Forty haploid male progeny (20 yfb and 20+) from a single diploid female parent (yfb/+) were examined. Sixty-one of the 180 arbitrary primers tested by polymerase chain reaction (PCR) produced one or more RAPD bands. A total of 79 RAPD markers were detected. Of these, seven showed significant deviation from the expected 1:1 ratio, and were therefore excluded from further analysis. The remaining 72 RAPD markers and the marker mutation, yfb, were subjected to linkage analysis. Sixty RAPD markers and the yfb marker were organized into 16 linkage groups, spanning a distance of 517.2 cM. Twelve RAPD markers showed no linkage relationship to any group. Thirteen gel-purified RAPD bands were cloned and sequenced to generate the sequence-tagged sites (STSs). A single locus was represented by two markers, with one of them having a short internal deletion.     

Expansion-repression mechanism for scaling the Dpp activation gradient in Drosophila wing imaginal discs

Maintaining a proportionate body plan requires the adjustment or scaling of organ pattern with organ size. Scaling is a general property of developmental systems, yet little is known about its underlying molecular mechanisms. Using theoretical modeling, we examine how the Dpp activation gradient in the Drosophila wing imaginal disc scales with disc size. We predict that scaling is achieved through an expansion-repression mechanism [1] whose mediator is the widely diffusible protein Pentagone (Pent). Central to this mechanism is the repression of pent expression by Dpp signaling, which provides an effective size measurement, and the Pent-dependent expansion of the Dpp gradient, which adjusts the gradient with tissue size. We validate this mechanism experimentally by demonstrating that scaling requires Pent and further, that scaling is abolished when pent is ubiquitously expressed. The expansion-repression circuit can be readily implemented by a variety of molecular interactions, suggesting its general utilization for scaling morphogen gradients during development.     

Participation of testicular spermatids in development upon intracytoplasmic injection into eggs of the sawfly, Athalia rosae (Insecta, hymenoptera)

Fertilization by intracytoplasmic injection of mature sperm into mature eggs has previously been achieved in the sawfly, Athalia rosae (Insecta, Hymenoptera). In the present study, we examined the potential of spermatids, premature male gametes, for participating in development. When round spermatids and elongating spermatids from pupal testes were injected into the anterior end of mature eggs, about 5% of the total injected eggs developed into chimeric embryos (independent participation in development of the egg and spermatid nuclei). Some of them developed further, hatched, and pupated, with 1-2% of the total injected eggs becoming haploid chimeric male adults in which both the egg-derived and injected spermatid-derived nuclei contributed to the germline. No fertilized embryos were obtained by these injections. Elongated spermatids (immature sperm) from newly eclosed adult male testes upon injection did produce fertilized embryos that developed into normal diploid females (about 7% of the total injected). These results indicate that insect spermatids (round and elongating) have the potential to participate in development, but only independently of the egg nucleus. J. Exp. Zool. 286:181-192, 2000.     

Host shifts from Lamiales to Brassicaceae in the sawfly genus Athalia

Plant chemistry can be a key driver of host shifts in herbivores. Several species in the sawfly genus Athalia are important economic pests on Brassicaceae, whereas other Athalia species are specialized on Lamiales. These host plants have glucosides in common, which are sequestered by larvae. To disentangle the possible direction of host shifts in this genus, we examined the sequestration specificity and feeding deterrence of iridoid glucosides (IGs) and glucosinolates (GSs) in larvae of five species which either naturally sequester IGs from their hosts within the Plantaginaceae (Lamiales) or GSs from Brassicaceae, respectively. Furthermore, adults were tested for feeding stimulation by a neo-clerodane diterpenoid which occurs in Lamiales. Larvae of the Plantaginaceae-feeders did not sequester artificially administered p-hydroxybenzylGS and were more deterred by GSs than Brassicaceae-feeders were by IGs. In contrast, larvae of Brassicaceae-feeders were able to sequester artificially administered catalpol (IG), which points to an ancestral association with Lamiales. In line with this finding, adults of all tested species were stimulated by the neo-clerodane diterpenoid. Finally, in a phylogenetic tree inferred from genetic marker sequences of 21 Athalia species, the sister species of all remaining 20 Athalia species also turned out to be a Lamiales-feeder. Fundamental physiological pre-adaptations, such as the establishment of a glucoside transporter, and mechanisms to circumvent activation of glucosides by glucosidases are therefore necessary prerequisites for successful host shifts between Lamiales and Brassicaceae.     

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. Our data indicate that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients.     

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing

The sensitivity of the posterior crossvein in the pupal wing of Drosophila to reductions in the levels and range of BMP signaling has been used to isolate and characterize novel regulators of this pathway. We show here that crossveinless d (cv-d) mutations, which disrupt BMP signaling during the development of the posterior crossvein, mutate a lipoprotein that is similar to the vitellogenins that comprise the major constituents of yolk in animal embryos. Cv-d is made in the liver-like fat body and other tissues, and can diffuse into the pupal wing via the hemolymph. Cv-d binds to the BMPs Dpp and Gbb through its Vg domain, and to heparan sulfate proteoglycans, which are well-known for their role in BMP movement and accumulation in the wing. Cv-d acts over a long range in vivo, and does not have BMP co-receptor-like activity in vitro. We suggest that, instead, it affects the range of BMP movement in the pupal wing, probably as part of a lipid-BMP-lipoprotein complex, similar to the role proposed for the apolipophorin lipid transport proteins in Hedgehog and Wnt movement.