Phylogenomics and biogeography of sawflies and woodwasps (Hymenoptera, Symphyta).

Phylogenomic approaches have recently helped elucidate various insect relationships, but large-scale comprehensive analyses on relationships within sawflies and woodwasps are still lacking. Here, we infer the relationships and long-term biogeographic history of these hymenopteran groups using a large dataset of 354 UCE loci collected from 385 species that represent all major lineages. Early Hymenoptera started diversifying during the Early Triassic âˆ¼249 Ma and spread all over the ancient supercontinent Pangaea. We recovered Xyeloidea as a monophyletic sister group to other Hymenoptera and Pamphilioidea as sister to Unicalcarida. Within the diverse family Tenthredinidae, our taxonomically and geographically expanded taxon sampling highlights the non-monophyly of several traditionally defined subfamilies. In addition, the recent removal of Athalia and related genera from the Tenthredinidae into the separate family Athaliidae is supported. The deep historical biogeography of the group is characterised by independent dispersals and re-colonisations between the northern (Laurasia) and southern (Gondwana) palaeocontinents. The breakup of these landmasses led to ancient vicariance in several Gondwanan lineages, while interchange across the Northern Hemisphere has continued until the Recent. The little-studied African sawfly fauna is likewise a diverse mixture of groups with varying routes of colonization. Our results reveal interesting parallels in the evolution and biogeography of early hymenopterans and other ancient insect groups.

Cypress terpenes in sawfly larva of Susana cupressi (Hymenoptera: Symphyta: Tenthredinoidea).

Several sawfly species (Hymenoptera: Symphyta) possess larval stages with oesophageal diverticula in which plant compounds are sequestered and used for defence against predators. These organs are present in the larvae of Susana (Tenthredinidae) but remain poorly studied. Here, the aim was to analyse the diverticula extract of Susana cupressi by gas chromatography-mass spectrometry to better understand the ecology of this species. The foliage of the hostplant (Cupressus sempervirens), as well as the larval foregut, midgut, and haemolymph were also analysed. Complementary data were gathered by morphological observations, bioassays using ants, and genetic analyses to identify the studied Susana species. Altogether, 48 terpenes were identified, 30 being sesquiterpenes. The terpenes were generally detected in the foliage, but also in the diverticula, foregut, and midgut, whereas none of them in the haemolymph. The main compounds were alpha-cedrene, alpha-fenchene, alpha-pinene, alpha-terpinyl acetate, beta-myrcene, beta-pinene, cedrol, delta 3-carene, epi-bicyclosesquiphellandrene, germacrene D, limonene, sabinene, and terpinolene. The chemical profiles of these 13 compounds were significantly correlated between foliage-diverticula, diverticula-foregut and foregut-midgut, but not correlated for the three remaining possible comparisons. Alpha-pinene decreased and germacrene D increased from the foliage to the diverticula, which may reflect a specific sequestration of the latter terpene and its known deleterious effects on insects. We conclude that larvae of S. cupressi, similarly to those of diprionids, are well defended against predatory attacks by sequestering and regurgitating hostplant terpenes, including germacrene D.

Some sawfly larvae survive predator-prey interactions with pentatomid Picromerus bidens.

Most Asopinae stinkbugs (Hemiptera: Pentatomidae) prey on other insects, including sawfly larvae (Hymenoptera: Symphyta). Sawfly larvae of the Argidae and Pergidae contain toxic peptides, but whether they are defended against stinkbugs remains poorly studied. A literature survey indicates that no publication is devoted to laboratory tests specifically using these sawflies against stinkbugs. Here, laboratory bioassays were made with the stinkbug Picromerus bidens and four sawfly species at last larval instars: Arge ochropus (Argidae), Arge pagana (also tested at medium instars), Lophyrotoma zonalis (Pergidae), and Allantus rufocinctus (Tenthredinidae). Following 24 h of possible predator-prey interactions, no larvae of A. rufocinctus survived, whereas most or all larvae of the other sawfly species did survive and were still alive 48 h later. When feeding on an argid or pergid larva, the feeding periods lasted on average 6-20 s only, some bugs removing their rostrum and abruptly backing away. Full-grown larvae of A. pagana were attacked less than younger ones. It is likely that the tested Argidae and Pergidae are well defended against P. bidens by potent, internal antifeedants, while defensive body movements combined with a large body size play a secondary role.

Body distribution of toxic peptides in larvae of a pergid and an argid sawfly species.

Larvae of most Pergidae and Argidae (Symphyta: Tenthredinoidea) species contain toxic peptides such as pergidin and lophyrotomin. Here, larval hemolymph and organs of the pergid Lophyrotoma zonalis and the argid Arge pagana were analysed by liquid chromatography-tandem mass spectrometry. The major identified peptides were pergidin and 4-valinepergidin in L. zonalis, whereas pergidin and lophyrotomin in A. pagana. The storage period prior to chemical analysis was longer for the samples of the pergid than the argid species, which influenced peptide concentrations. In both species, however, the peptides occurred in decreasing order of concentration, first in the hemolymph, then in the integument, while minor amounts of the peptides were detected in other organs such as gut and fat body. By separating the cuticle of the pergid from the remaining integument, the peptides were found in equivalent amounts in each of these two body structures. The results suggest that the peptides play an important role in the defence of these sawfly larvae against predators.

Berberis sawfly contains toxic peptides not only at larval stage.

Livestock can die from grazing in areas where larvae of certain Argidae or Pergidae species containing toxic peptides occur in mass. However, it remains unknown whether other stages also contain these compounds. Here, single specimens of larvae, prepupae, and adults of Arge berberidis, plus samples of its cocoons and larval feces, were analyzed by liquid chromatography-tandem mass spectrometry. The four peptides, pergidin (Perg), 4-valinepergidin (VPerg), dephosphorylated pergidin (dpPerg), and lophyrotomin (LGln), were detected in each of the three stages. Peptide concentrations, in percentage fresh weight, increased from larval up to adult stages, with mean values from 0.044 to 0.125% for Perg, 0.008 to 0.023% for VPerg, and 0.064 to 0.116% for LGln, whereas dpPerg never exceeded 0.001%. The concentrations of this latter peptide averaged 0.002% in the cocoon built by the prepupa, and nearly no peptides were detected in larval feces. Moreover, the concentrations of the three main peptides (Perg, LGln, and VPerg) tended to be correlated with each other in larvae and especially in adults. It is likely that peptide production, purportedly by an endosymbiont, stops at prepupal stage and that concentration of the peptides increases from prepupa to adult due to a decrease of body weight.

Toxic Peptides in Populations of Two Pergid Sawflies, Potential Biocontrol Agents of Brazilian Peppertree.

Determination of the safety of agents prior to release is one of the most important research goals in biological control. In addition to concerns for the safety of non-target plants, determination of the potential toxic properties of new agents needs to be assessed. Numerous phytophagous insects are defended by chemicals against the attack of natural enemies. Some of these defensive compounds could pose an environmental risk if an agent is released. Here, larval populations of two pergid sawflies, Heteroperreyia hubrichi and H. jorgenseni, were analyzed by LC-MS/MS to investigate whether they contain alleged toxic peptides. The first species is a potential candidate for biological control of the invasive weed Brazilian peppertree in Florida and Hawaii. The chemical analyses revealed the presence of the peptides pergidin (Perg), 4-valinepergidin (VPerg), dephosphorylated pergidin (dpPerg), lophyrotomin (LGln and LGlu). The effect of sawfly population for each species was significantly influencing peptide concentration. All peptides occurred at lower concentrations compared with purportedly toxic species of this sawfly family. However, the concentrations of the peptides are of concern for the welfare of wildlife and livestock that would be exposed to these species. These results demonstrate that release of this biological control agent in the invaded range may pose an environmental threat.

Furostanol Saponins and Ecdysteroids from Plants of the Genus Helleborus as Phagostimulants and Predator Deterrents for Larvae of Two Monophadnus Sawfly Species.

Sawfly species of the genus Monophadnus are specialised on Ranunculaceae plants from which the larvae can sequester furostanol saponins into the haemolymph, mainly (25R)-26-[(α-L-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3ß-yl-O-ß-D-glucopyranosyl-(1→3)-O-[6-acetyl-ß-D-glucopyranosyl-(1→3)]-O-ß-D-glucopyranoside (compound 1). In this work, TLC, GC-MS, and HPLC-DAD-ESI/MS analyses together with feeding, repeated simulated attacks, and ant deterrence bioassays were conducted to extend the chemoecological knowledge about two sawfly species specialised on H. foetidus L. (Monophadnus species A) and H. viridis L. (Monophadnus species B). Larvae of Monophadnus species B were mostly feeding on the squares treated with the n-butanol fraction from H. foetidus, compound 1 being its primary non-nutritional stimulant. In contrast, all H. viridis fractions stimulated feeding, with n-hexane marginally more active. ß-sitosterol within n-hexane was determined as the nutritional stimulant. Quantitative analyses demonstrated that leaves of H. viridis but not H. foetidus contain the ecdysteroids 20-hydroxyecdysone and polypodine B. Moreover, the haemolymph of Monophadnus species B larvae reared on H. viridis contained the glycosides of polypodine B and 20-hydroxyecdysone at a concentration of 2.5 to 6.8 µmol/g fresh weight of haemolymph. This concentration is several thousand times higher than the concentration range of the aglycones in their host plant (3.63 × 10-4 to 2.23 × 10-4 µmol total ecdysteroids/g fresh weight of leaves), suggesting bioaccumulation. The larvae of both species fed on H. foetidus do not show any traces of ecdysteroids in their haemolymph, indicating a facultative role of these compounds in their defence as well as their inability to endogenously synthesise these compounds. The haemolymph containing ecdysteroids was a significant feeding deterrent against Myrmica rubra L. ant workers (one of their natural predators) at 0.8 mg/mL. The larvae kept effective deterrent levels of glycosylated ecdysteroids (≅175 mM) between simulated attacks on days 1 and 2, but the levels clearly decreased on day 3 (≅75 mM). Most larvae (89%) survived a first attack but only 23% a consecutive second one. As a conclusion, we report for the first time that two Monophadnus species feeding on H. viridis sequester phytoecdysteroids into the larval haemolymph in the form of glycosides. In addition, compound 1 possesses defensive and phagostimulant activities, and we present evidence for a combined effect of furostanol saponins and ecdysteroids as repellents against ants.

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

BACKGROUND: Many 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Integument and defence in larva and prepupa of a sawfly living on a semi-aquatic plant.

The larvae of the sawfly Rhadinoceraea micans live and feed on a semi-aquatic plant, Iris pseudacorus, and their integument is strongly hydrophobic. The hydrophobicity is part of a chemical defence strategy, easy bleeding, also known from congeners. The prepupae burrow into the soil where they form a cocoon in which they pupate, thus implying different micro-environmental conditions. The cuticle structure and wetting defensive effectiveness of R. micans were compared between larvae and prepupae. The two stages were similarly well defended against attacking ants by the bleeding of a deterrent hemolymph, whereas they were dissimilar in the cuticle surface that presented sculptures and wax crystals at the larval stage only. The integument of prepupae was less structured, and hydrophilic. Larvae of R. micans exhibit, among sawflies, an exceptional cuticle structuring and we assume that they occupy this particular niche of a semi-aquatic environment to avoid encounters with ground-dwelling predators whereas prepupae may benefit from the chemical defence acquired at larval stage.

Parameter Mapping Sonification of Human Olfactory Thresholds.

An objective of chemical ecology is to understand the chemical diversity across and within species, as well as the bioactivity of chemical compounds. We previously studied defensive volatiles from phytophagous insects that were subjected to parameter mapping sonification. The created sounds contained information about the repellent bioactivity of the volatiles, such as the repellence from the volatiles themselves when tested against live predators. Here, we applied a similar sonification process to data about human olfactory thresholds. Randomized mapping conditions were used and a peak sound pressure, Lpeak, was calculated from each audio file. The results indicate that Lpeak values were significantly correlated with the olfactory threshold values (e.g., rS = 0.72, t = 10.19, p < 0.001, Spearman rank-order correlation; standardized olfactory thresholds of 100 volatiles). Furthermore, multiple linear regressions used the olfactory threshold as a dependent variable. The regressions revealed that the molecular weight, the number of carbon and oxygen atoms, as well as the functional groups aldehyde, acid, and (remaining) double bond were significant determinants of the bioactivity, while the functional groups ester, ketone, and alcohol were not. We conclude that the presented sonification methodology that converts chemicals into sound data allows for the study of their bioactivities by integrating compound characteristics that are easily accessible.

Flavonoid glycosides and naphthodianthrones in the sawfly Tenthredo zonula and its host-plants, Hypericum perforatum and H. hirsutum.

Larvae of the sawfly Tenthredo zonula are specialized on Hypericum. Whether the sawfly is able to sequester plant metabolites was unknown. Aerial materials of Hypericum perforatum and H. hirsutum, as well as dissected larvae and prepupae of T. zonula, were analyzed by HPLC to determine the presence and content of flavonoid glycosides (rutin, hyperoside, isoquercitrin, and quercitrin) and naphthodianthrones (pseudohypericin and hypericin). All flavonoid glycosides were detected in both Hypericum species, with hyperoside as major compound in H. perforatum (ca. 1.7 µmol/g fresh weight, FW) and isoquercitrin in H. hirsutum (0.7 µmol/g FW). Naphthodianthrones were present at low concentrations (0.02 µmol/g FW) in the former, and almost undetected in the latter species. In the body parts (i.e., hemolymph, digestive tract, salivary glands, or miscellaneous organs) of T. zonula, the surveyed compounds were detected more frequently in prepupae than in larvae. The compounds were not present in every sample, and flavonoid glycosides especially occurred in highly variable amounts, with maximal concentrations of 41 µg rutin/prepupa in salivary glands, 8 µg hyperoside/prepupa in hemolymph (= 0.36 µmol/g FW), 32 µg isoquercitrin/prepupa in salivary glands, and 63 µg quercitrin/larva in miscellaneous organs (mainly composed of the integument). We conclude that flavonoid glycosides are sequestered since they were detected in organs other than the digestive tract of larvae, and because prepupae are a non-feeding stage. The naphthodianthrone pseudohypericin, but not hypericin, occurred generally in the digestive tract (up to 0.25 µg/larva). Both naphthodianthrones and related unidentified compounds, but not flavonoid glycosides, were found in the larval excrement. The highly variable distributions of flavonoid glycosides and naphthodianthrones in T. zonula larvae and prepupae make it difficult to determine the ecological significance of these metabolites.

Behavior and body size modulate the defense of toxin-containing sawfly larvae against ants.

The sawfly larvae of most Argidae and Pergidae (Hymenoptera: Symphyta) species contain toxic peptides, and these along with other traits contribute to their defense. However, the effectiveness of their defense strategy, especially against ants, remains poorly quantified. Here, five Arge species, A. berberidis, A. nigripes, A. ochropus, A. pagana, A. pullata, plus three Pergidae species, Lophyrotoma analis, Lophyrotoma zonalis, Philomastix macleaii, were tested in laboratory bioassays on ant workers mainly of Myrmica rubra. The experiments focused on short-term predator-prey interactions, sawfly survival rate after long-term interactions, and feeding deterrence of the sawfly hemolymph. The larvae of Arge species were generally surrounded by few ants, which rarely bit them, whereas larvae of Pergidae, especially P. macleaii, had more ants around with more biting. A detailed behavioral analysis of Arge-ant interactions revealed that larval body size and abdomen raising behavior were two determinants of ant responses. Another determinant may be the emission of a volatile secretion by non-eversible ventro-abdominal glands. The crude hemolymph of all tested species, the five Arge species and L. zonalis, was a strong feeding deterrent and remained active at a ten-fold dilution. Furthermore, the study revealed that the taxon-specific behavior of ants, sting or spray, impacted the survival of A. pagana but not the large body-sized A. pullata. The overall results suggest that the ability of Arge and Pergidae larvae to defend against ants is influenced by the body size and behavior of the larvae, as well as by chemicals.

Chemical composition: Hearing insect defensive volatiles.

Chemical signals mediate major ecological interactions in insects. However, using bioassays only, it is difficult to quantify the bioactivity of complex mixtures, such as volatile defensive secretions emitted by prey insects, and to assess the impact of single compounds on the repellence of the entire mixture. To represent chemical data in a different perceptive mode, we used a process of sonification by parameter mapping of single molecules, which translated chemical signals into acoustic signals. These sounds were then mixed at dB levels reflecting the relative concentrations of the molecules within species-specific secretions. Repellence of single volatiles, as well as mixtures of volatiles, against predators were significantly correlated with the repulsiveness of their respective auditory translates against humans, who mainly reacted to sound pressure. Furthermore, sound pressure and predator response were associated with the number of different molecules in a secretion. Our transmodal approach, from olfactory to auditory perception, offers further prospects for chemo-ecological research and data representation.

How common is ecological speciation in plant-feeding insects? A 'Higher' Nematinae perspective.

BACKGROUND: Ecological speciation is a process in which a transiently resource-polymorphic species divides into two specialized sister lineages as a result of divergent selection pressures caused by the use of multiple niches or environments. Ecology-based speciation has been studied intensively in plant-feeding insects, in which both sympatric and allopatric shifts onto novel host plants could speed up diversification. However, while numerous examples of species pairs likely to have originated by resource shifts have been found, the overall importance of ecological speciation in relation to other, non-ecological speciation modes remains unknown. Here, we apply phylogenetic information on sawflies belonging to the 'Higher' Nematinae (Hymenoptera: Tenthredinidae) to infer the frequency of niche shifts in relation to speciation events. RESULTS: Phylogenetic trees reconstructed on the basis of DNA sequence data show that the diversification of higher nematines has involved frequent shifts in larval feeding habits and in the use of plant taxa. However, the inferred number of resource shifts is considerably lower than the number of past speciation events, indicating that the majority of divergences have occurred by non-ecological allopatric speciation; based on a time-corrected analysis of sister species, we estimate that a maximum of c. 20% of lineage splits have been triggered by a change in resource use. In addition, we find that postspeciational changes in geographic distributions have led to broad sympatry in many species having identical host-plant ranges. CONCLUSION: Our analysis indicates that the importance of niche shifts for the diversification of herbivorous insects is at present implicitly and explicitly overestimated. In the case of the Higher Nematinae, employing a time correction for sister-species comparisons lowered the proportion of apparent ecology-based speciation events from c. 50-60% to around 20%, but such corrections are still lacking in other herbivore groups. The observed convergent but asynchronous shifting among dominant northern plant taxa in many higher-nematine clades, in combination with the broad overlaps in the geographic distributions of numerous nematine species occupying near-identical niches, indicates that host-plant shifts and herbivore community assembly are largely unconstrained by direct or indirect competition among species. More phylogeny-based studies on connections between niche diversification and speciation are needed across many insect taxa, especially in groups that exhibit few host shifts in relation to speciation.

Ecophysiology of dorsal versus ventral cuticle in flattened sawfly larvae.

Platycampus larvae are highly cryptic leaf feeders characterised by a dorso-ventrally flattened body, the dorsal integument resembling a shield. Dorsal and ventral cuticles from Platycampus luridiventris were compared by histology and gel electrophoresis. By Azan-staining, a red and a blue layer were distinguished in the dorsal cuticle, while the ventral cuticle showed one, almost uniform blue layer, as in both cuticles of control species. The two cuticles from P. luridiventris had similar amounts and sodium dodecyl sulphate-polyacrylamide gel electrophoresis profiles of soluble proteins, but not insoluble proteins. One insoluble protein (MW approximately 41 kDa) was visible as a large band in the ventral cuticle only. It is likely that this protein renders the cuticle elastic, and that the dorsal, red layer is the exocuticle, mainly composed of insoluble proteins. We discuss eco-physiological implications of the exocuticle in insects. Further, data from the literature indicate that the defence strategy in P. luridiventris larvae relies on being visually cryptic towards avian predators and tactically cryptic towards arthropod predators and parasitoids. Crypsis in both senses is favoured by the shield effect, itself based on an abnormally thick dorsal exocuticle. Although the larvae are external feeders, they may be considered as hidden from an ecological perspective.

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.

Endogenous toxins and the coupling of gregariousness to conspicuousness in Argidae and Pergidae sawflies.

Phytophagous insects tend to be either cryptic and solitary, or brightly colored and gregarious, as a defense against vertebrate predators. Here, we tested whether potent defensive chemicals produced de novo by larvae of Argidae and Pergidae sawflies have influenced the evolutionary relationship between larval appearance and levels of gregariousness. Phylogeny-based correlation analyses indicated only a weak trend for solitary species to be cryptic, and for gregarious ones to be conspicuous. Numerous Argidae were cryptic-solitary or conspicuous-gregarious, whereas most Pergidae were conspicuous-gregarious. Both families also included not truly gregarious but aggregated species, i.e. with individuals more evenly distributed on the host plant. By considering two specific morphological traits, predominant body coloration and contrasting spots on body, each one was (weakly) associated with appearance but none with gregariousness, which reflects the functional relevance of appearance as a whole. Furthermore, Argidae can display alternate appearances during successive larval instars. Finally, an independent contrasts test showed no obvious correlation between two major toxic peptides. Our results point towards diversely combined patterns of linked ecological traits in these insects. By assuming that warning coloration is more warranted against vertebrate than invertebrate predators, we suggest that the occurrence itself of toxins allowed this diversity via differing predator guilds and environmental factors, to which these insects were confronted during evolution.

Anti-predator defence mechanisms in sawfly larvae of Arge (Hymenoptera, Argidae).

Larvae of the sawfly Arge (Hymenoptera, Argidae) are exposed to predators such as ants. Their defence mechanisms, which have been almost unstudied, were investigated by behavioural observations coupled to a morphological approach and by testing the bioactivity of several body parts. Arge larvae raised their abdomen when contacted by Myrmica rubra workers. The ants rarely bit a larva and generally retreated immediately, sometimes without contacting it. Most of those few ants that bit a larva then showed an uncoordinated walk. Crude hemolymph from a common species, A. pagana, was a feeding deterrent towards ants. Hemolymph extracts remained active up to a concentration of 0.8 microg DW extract per microlitre solution, and were more active than integument and gut extracts. We also observed ants paralysed by extracts, especially from the gut. It is likely that this toxicity is due to a polypeptide, lophyrotomin, which is known to occur in A. pullata. Six or seven non-eversible ventro-abdominal glands occurred in all species studied (A. fuscipes, A. nigripes, A. ochropus, A. pagana, A. pullata, A. ustulata). These glands contain volatiles. We consider both types of chemicals to be important in defence, and we propose that the paralysing effect is a common feature among Arge species.

Searching for particular traits of sawfly (Hymenoptera: Tenthredinidae) larvae that emit hemolymph as a defence against predators.

Easy bleeding is a defence strategy that allows the larvae of some Tenthredinidae sawfly species to emit deterrent hemolymph when attacked by a predator. However, a drawback of this defence is that hemolymph is frequently in contact with the exterior, thus potentially subjected to multiple microbial infections at any body's integumental spot. Here we aimed to identify physiological traits that are linked to easy bleeding. First, larvae of several sawfly species were subjected to daily experimental losses of hemolymph equivalent to 10% of their body weight, and changes in body weight and survival were recorded. Easy bleeders' survival rates were better compared to non-easy bleeders. Second, testing hemolymph melanisation revealed that nearly all sawfly hemolymph samples did not melanise over a 24h period. Third, inhibition zone tests against live Escherichia coli were conducted using hemolymph collected 24-48h after a sterile wounding and an infection with Micrococcus luteus, as well as from control, untouched individuals. Sterile wounding induced similar antibacterial activities compared to those detected in the control group. However, the activity was significantly enhanced upon infection in some species, similarly to other insects. Thus, easy bleeders have a tendency to compensate for hemolymph loss resulting from predator-prey interactions, whereas a non-melanising hemolymph is probably a characteristic of sawflies, and the antimicrobial activity can be high but is comparable in easy bleeders versus other insects.

Field Method for Testing Repellency of an Icaridin-Containing Skin Lotion against Vespid Wasps.

Vespid wasps are ecologically beneficial predators of insects but their stings also pose a human health risk. Current control methods based on killing vespids are suboptimal. Here, the repellent effect against Vespula vulgaris of a 20% icaridin skin lotion was evaluated under field conditions. An experimental setup was designed in which six artificial skin pieces (10 × 10 cm) were video-recorded for 1 h, to count each min the numbers of flying and feeding vespids. Prior to monitoring, five pieces were successively smeared with 2 mg of cream per cm², in 30 min intervals, from t = -120 min to 0. The sixth sheet remained untreated to serve as a control. One milliliter of an attractant, fruit jam, was deposited on each of the six surfaces at t = 0. The control surface was free of any flying or feeding vespid during an average period of 25 min, whereas the other five surfaces (treated at t = -120, -90, -60, -30, and 0 min) remained vespid-free for 39, 40, 45, 49, and 51 min, respectively. The skin lotion remained significantly active for at least 2 h. The experimental methodology is adjustable and allows the study of repellents against vespids in semi-natural conditions.