Jean-Luc Boevé

Sequestration of Host Plant Glucosinolates in the Defensive Hemolymph of the Sawfly Athalia rosae

Interactions between insects and glucosinolate-containing plant species have been investigated for a long time. Although the glucosinolate–myrosinase system is believed to act as a defense mechanism against generalist herbivores and fungi, several specialist insects use these secondary metabolites for host plant finding and acceptance and can handle them physiologically. However, sequestration of glucosinolates in specialist herbivores has been less well studied. Larvae of the turnip sawfly Athalia rosae feed on several glucosinolate-containing plant species. When larvae are disturbed by antagonists, they release one or more small droplets of hemolymph from their integument. This “reflex bleeding” is used as a defense mechanism. Specific glucosinolate analysis, by conversion to desulfoglucosinolates and analysis of these by high-performance liquid chromatography coupled to diode array UV spectroscopy and mass spectrometry, revealed that larvae incorporate and concentrate the plants characteristic glucosinolates from their hosts. Extracts of larvae that were reared on Sinapis alba contained sinalbin, even when the larvae were first starved for 22 hr and, thus, had empty guts. Hemolymph was analyzed from larvae that were reared on either S. alba, Brassica nigra, or Barbarea stricta. Leaves were analyzed from the same plants the larvae had fed on. Sinalbin (from S. alba), sinigrin (B. nigra), or glucobarbarin and glucobrassicin (B. stricta) were present in leaves in concentrations less than 1 μmol/g fresh weight, while the same glucosinolates could be detected in the larvaes hemolymph in concentrations between 10 and 31 μmol/g fresh weight, except that glucobrassicin was present only as a trace. In larval feces, only trace amounts of glucosinolates (sinalbin and sinigrin) could be detected. The glucosinolates were likewise found in freshly emerged adults, showing that the sequestered phytochemicals were transferred through the pupal stage.

Host plant derived feeding deterrence towards ants in the turnip sawfly Athalia rosae

Larvae of the sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Tenthredinidae) feed on several glucosinolate‐containing plants and have been shown to sequester the main glucosinolates of different hosts, namely sinalbin (p‐hydroxybenzylglucosinolate) from Sinapis alba L., sinigrin (allylglucosinolate) from Brassica nigra (L.) Koch, and glucobarbarin ((S)‐2‐hydroxy‐2‐phenylethylglucosinolate) from Barbarea stricta Andrz. (Brassicaceae). These plant metabolites are stored in the haemolymph, which is readily released when larvae are attacked by predators. In a dual‐choice bioassay the bio‐activity of sawfly haemolymph collected from larvae reared on different host plants (S. alba, B. nigra, and B. stricta) was tested against the ant Myrmica rubra L. (Hymenoptera: Formicidae). The haemolymph had a stronger deterrence effect when the corresponding sawfly larvae were reared on S. alba than when reared on B. nigra and B. stricta. Haemolymph of caterpillars of Pieris rapae L. (Lepidoptera: Pieridae) that had fed on S. alba was not deterrent to the ants. No sinalbin could be detected in their haemolymph. The glucosinolates sinalbin and sinigrin, offered in a concentration comparable to that in the sawfly haemolymph, were deterrent to the ants, but not as strongly as the corresponding haemolymph samples. This suggests, that glucosinolates are not the only compounds involved in the chemical defence of A. rosae. However, the presence of sequestered glucosinolates is already a sufficient defence towards predators such as ants, and their effectiveness is modulated by the host plant chemistry.

Gustatory perception and metabolic utilization of sugars by Myrmica rubra ant workers

The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra (L.) was studied. The sugars used included monosaccharides (fructose, glucose, galactose, mannose, rhamnose), disaccharides (sucrose, maltose, trehalose, melibiose, lactose) and trisaccharides (melizitose, raffinose, erlose). Single-sugar solutions were tested on ant workers in a long-term laboratory bioassay in which acceptance of the solutions and ant survival were recorded. The acceptance of the sugars was confirmed in a second bioassay in which feeding time was established. Enzymatic hydrolysis of sucrose, maltose and melibiose was investigated through HPLC analyses of workers fed these disaccharides. Sugar acceptance and feeding time were related to ant survival. Considering the monosaccharide units of which the sugars are composed, fructose seems especially suitable as a short-term energy source, while glucose appears to be used both directly and for storage. The presence of a galactose unit appears to reduce sugar suitability. It is suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase. The gustatory perception is correlated with the profitability of sugars in further metabolic processes.

Defence effectiveness of easy bleeding sawfly larvae towards invertebrate and avian predators

Summary.Easy bleeding is a phenomenon discovered in some tenthredinid insects which possess a particularly low mechanical resistance of the integument, leading under mechanical stress to haemolymph exudation. It has a defensive effect against ants and wasps through harmful plant compounds which are sequestered in the haemolymph. Here we describe etho-ecological and some chemical aspects of the defence of easy bleeders and specify the range of predators to which easy bleeding might be effective. Beside a high haemolymph deterrence associated with low integument resistance across sawfly species, we also detected toxicity of the haemolymph of some species to workers of the ant Myrmica rubra. The behaviour of easy bleeders is to move slowly and, once disturbed, to become motionless, thereby probably impeding the tendency of a predator to attack. This behaviour had no beneficial effect for easy bleeders when attacked by the predatory bug Podisus maculiventris. Bugs could successfully and without harm prey on sawfly larvae without evoking easy bleeding. For the easy bleeder Athalia rosae, host plants with different secondary metabolite profiles, and, consequently, changes in haemolymph chemistry only slightly affected the feeding behaviour of the bugs. To test the effectiveness of easy bleeding towards a vertebrate predator, easy bleeders were offered to birds, Sturnus vulgaris. The body colouration of the sawfly larvae was of prime importance in determining the predator’s response when testing birds in a group. It is likely that easy bleeding is a defence strategy directed primarily towards foraging insects with biting-chewing mandibles and that it is much less active towards predatory insects with piercing-sucking mandibles as well as birds. The involvement of chemical and/or physical cues in the strategy is discussed with respect to these types of predators.

Invertebrate and avian predators as drivers of chemical defensive strategies in tenthredinid sawflies

BackgroundMany insects are chemically defended against predatory vertebrates and invertebrates. Nevertheless, our understanding of the evolution and diversity of insect defenses remains limited, since most studies have focused on visual signaling of defenses against birds, thereby implicitly underestimating the impact of insectivorous insects. In the larvae of sawflies in the family Tenthredinidae (Hymenoptera), which feed on various plants and show diverse lifestyles, two distinct defensive strategies are found: easy bleeding of deterrent hemolymph, and emission of volatiles by ventral glands. Here, we used phylogenetic information to identify phylogenetic correlations among various ecological and defensive traits in order to estimate the relative importance of avian versus invertebrate predation.ResultsThe mapping of 12 ecological and defensive traits on phylogenetic trees inferred from DNA sequences reveals the discrete distribution of easy bleeding that occurs, among others, in the genus Athalia and the tribe Phymatocerini. By contrast, occurrence of ventral glands is restricted to the monophyletic subfamily Nematinae, which are never easy bleeders. Both strategies are especially effective towards insectivorous insects such as ants, while only Nematinae species are frequently brightly colored and truly gregarious. Among ten tests of phylogenetic correlation between traits, only a few are significant. None of these involves morphological traits enhancing visual signals, but easy bleeding is associated with the absence of defensive body movements and with toxins occurring in the host plant. Easy bleeding functions through a combination of attributes, which is corroborated by an independent contrasts test indicating a statistically significant negative correlation between species-level integument mechanical resistance and hemolymph feeding deterrence against ants.ConclusionsOur analyses evidence a repeated occurrence of easy bleeding, and no phylogenetic correlation including specific visual signals is significant. We conclude that the evolution of chemically-based defenses in tenthredinids may have been driven by invertebrate as much as by avian predation. The clear-cut visual signaling often encountered in the Nematinae would be linked to differential trends of habitat use by prey and predators. Further studies on (prey) insect groups should include visual signals and other traits, as well as several groups of natural enemies, to better interpret their relative significance and to refine our understanding of insect chemical defenses.

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.

Defense by Volatiles in Leaf-Mining Insect Larvae

The defense strategy of an insect toward natural enemies can include a trait that appears at first sight to contradict its defensive function. We explored phylogeny, chemistry, and defense efficiency of a peculiar group of hymenopteran sawfly larvae where this contradiction is obvious. Pseudodineurini larvae live in leaf mines that protect them from some enemies. Disturbed larvae also emit a clearly perceptible lemon-like odor produced by ventral glands, although the mine hampers the evaporation of the secretion. The mine could also lead to autointoxication of a larva by its own emitted volatiles. Citral was the major component in all Pseudodineurini species, and it efficiently repels ants. We conclude that full-grown larvae that leave their mine to pupate in the soil benefit from citral by avoiding attacks from ground-dwelling arthropods such as ants. In some species, we also detected biosynthetically related compounds, two 8-oxocitral diastereomers (i.e., (2E,6E)- and (2E,6Z)-2,6-dimethylocta-2,6-dienedial). Synthetic 8-oxocitral proved to be a potent fungicide, but not an ant repellent. The discrete distribution of 8-oxocitral was unrelated to species grouping in the phylogenetic tree. In contrast, we discovered that its presence was associated with species from humid and cold zones but absent in species favoring warm and dry environments. The former should be protected by 8-oxocitral when faced with a fungal infestation while crawling into the soil. Our work shows the importance of integrating knowledge about behavior, morphology, and life history stages for understanding the complex evolution of insects and especially their defense strategies.

Toxic Peptides Occur Frequently in Pergid and Argid Sawfly Larvae

Toxic peptides containing D-amino acids are reported from the larvae of sawfly species. The compounds are suspected to constitute environmental contaminants, as they have killed livestock grazing in areas with congregations of such larvae, and related larval extracts are deleterious to ants. Previously, two octapeptides (both called lophyrotomin) and three heptapeptides (pergidin, 4-valinepergidin and dephosphorylated pergidin) were identified from three species in the family Pergidae and one in Argidae. Here, the hypothesis of widespread occurrence of these peptides among sawflies was tested by LC-MS analyses of single larvae from eight pergid and 28 argid species, plus nine outgroup species. At least two of the five peptides were detected in most sawfly species, whereas none in any outgroup taxon. Wherever peptides were detected, they were present in each examined specimen of the respective species. Some species show high peptide concentrations, reaching up to 0.6% fresh weight of 4-valinepergidin (1.75 mg/larva) in the pergid Pterygophorus nr turneri. All analyzed pergids in the subfamily Pterygophorinae contained pergidin and 4-valinepergidin, all argids in Arginae contained pergidin and one of the two lophyrotomins, whereas none of the peptides was detected in any Perginae pergid or Sterictiphorinae argid (except in Schizocerella pilicornis, which contained pergidin). Three of the four sawfly species that were previously known to contain toxins were reanalyzed here, resulting in several, often strong, quantitative and qualitative differences in the chemical profiles. The most probable ecological role of the peptides is defense against natural enemies; the poisoning of livestock is an epiphenomenon.

Easily Damaged Integument of Some Sawflies (Hymenoptera) is Part of a Defence Strategy Against Predators

During evolution insects have developed an impressive set of mechanisms to defend themselves against natural enemies. This includes adaptations in their phenology, behaviour, morphology, physiology, and chemistry (Evans and Schmidt 1990). Typically, at least two such traits occur jointly, so that one can speak about a real defence strategy. Yet, defensive traits per definition increase an insect’s fitness, but taken alone one trait may be disadvantageous. A new kind of defence strategy was discovered recently in larvae of some sawflies (which are herbivorous Hymenoptera) of the family Tenthredinidae. So far unknown in other insects, it was called easy bleeding and defined as “a low mechanical resistance of the whole body integument, by which slight mechanical damage of the integument is enough to provoke the release of hemolymph at this given spot” (Boeve and Schaffner 2003; Fig. 2.1).

Host specificity and host recognition in a chemically-defended herbivore, the tenthredinid sawfly Rhadinoceraea nodicornis

The sawfly Rhadinoceraea nodicornis Konow (Hymenoptera: Tenthredinidae) is a member of a closely related group of species, the tribe Phymatocerini, which feed on the Liliales and Ranunculales. It is known to sequester steroid alkaloids from its host plants, species in the genus Veratrum (Liliales: Melanthiaceae), and to use them as a defence against predators. There are known chemical relationships between the hosts of R. nodicornis and hosts of related sawfly species. We tested whether the R. nodicornis larvae would accept hosts of closely‐ and more distantly‐related sawflies, but found that they accepted only plant species in the genus Veratrum. This specificity was apparently innate, as it was independent of early larval experience. A feeding bioassay showed that the steroid alkaloids from Veratrum nigrum were phagostimulatory for R. nodicornis larvae, suggesting that they may be involved in host recognition. We discuss the possibility that the evolution of recognition of specific compounds may represent the mechanism of host radiation within the Phymatocerini.