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.

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.

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.

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.

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.

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.

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.

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.

A century of temporal stability of genetic diversity in wild bumblebees.

Since the 1950s, bumblebee (Bombus) species are showing a clear decline worldwide. Although many plausible drivers have been hypothesized, the cause(s) of this phenomenon remain debated. Here, genetic diversity in recent versus historical populations of bumblebee species was investigated by selecting four currently restricted and four currently widespread species. Specimens from five locations in Belgium were genotyped at 16 microsatellite loci, comparing historical specimens (1913-1915) with recent ones (2013-2015). Surprisingly, our results showed temporal stability of genetic diversity in the restricted species. Furthermore, both historical and recent populations of restricted species showed a significantly lower genetic diversity than found in populations of co-occurring widespread species. The difference in genetic diversity between species was thus already present before the alleged recent drivers of bumblebee decline could have acted (from the 1950's). These results suggest that the alleged drivers are not directly linked with the genetic variation of currently declining bumblebee populations. A future sampling in the entire distribution range of these species will infer if the observed link between low genetic diversity and population distribution on the Belgium scale correlates with species decline on a global scale.

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.

Sawflies of Ethiopia (Hymenoptera: Argidae, Tenthredinidae).

Sawflies were collected in Ethiopia during 2010-2013. Three species represent new records for the country: Arge deckerti Koch, 2005, Athalia excisa Koch, 2006 and Xenapates nigrifrons Koch, 2012. Arge flavifrons Mocsáry, 1909, syn. n. and A. transvaalensis Cameron, 1911, syn. n. are subjective synonyms of A. micheli (Buysson, 1900) that is re-described here. Athalia fumosa Gribodo, 1879 sp. rev. is recognized as a valid species and is removed from synonymy with A. scioensis Gribodo, 1879. Distega braunsi Enslin, 1911 syn. n. and D. brunniventris Enslin, 1913 syn. n. are subjective synonyms of D. montium Konow, 1907. Pseudoneacidiophora Koch, 1998 is a new junior synonym of Kivua Forsius, 1934 (syn. n.), resulting in the new combination Kivua pleuritica (comb. n.) for Athalia pleuritica Forsius, 1927. Kivua camerunensis nom. n. is proposed for P. bicolor Koch, 1998 (preoccupied in Kivua by K. bicolor (Pasteels, 1949) (Bicrista bicolor Pasteels)), the second species formerly included in Pseudoneacidiophora. The female of Distega abyssinica Pasteels, 1955 is described for the first time. An annotated and illustrated list including six distribution maps is given for Ethiopian sawflies. It is composed of 34 species belonging to the genus Arge (Argidae), and seven genera of Tenthredinidae: Athalia (Athaliinae), Kivua, Neacidiophora, Xenapates (Allantinae), Distega, Trisodontophyes (Blennocampinae), and Dulophanes (Selandriinae). Some ecological aspects of Athalia species are discussed, especially for the most abundantly collected A. vollenhoveni Gribodo, 1879.

DNA barcoding and male genital morphology reveal five new cryptic species in the West Palearctic bee Seladonia smaragdula (Vachal, 1895) (Hymenoptera: Apoidea: Halictidae).

Several forms or variants have long been recognized in the West Palearctic sweat bee Seladonia smaragdula (Vachal, 1895). Using DNA barcoding and morphological characters, primarily of the male genitalia, these variants are here recognized and described as five new species: S. gemmella Pauly sp. nov., S. submediterranea Pauly sp. nov., S. orientana Pauly & Devalez sp. nov., S. phryganica Pauly & Devalez sp. nov., and S. cretella Pauly & Devalez sp. nov. Also, we designate a lectotype for Halictus smaragdulus Vachal, consider Seladonia butea (Warncke, 1975) and S. morinella (Warncke, 1975) as nomina dubia, and discuss the identity of the Seladonia specimens from Australia currently determined as S. smaragdula.

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.

Taxonomy, phylogeny and host plants of some Abia sawflies (Hymenoptera, Cimbicidae).

We briefly review the taxonomy of Abia, and attempt to clarify their systematics by phylogenetic tree reconstructions inferred from three (nuclear and mitochondrial) genes of some West Palaearctic and Nearctic species. The main question which we asked is whether the distinction, made by several authors, of two genera within this group is justified. Based on the species here sampled, our results strongly support a clade recognised widely in earlier literature as Abia or Abia (Abia), but do not always support another clade, Zaraea or Abia (Zaraea), as monophyletic. In the interests of nomenclatural stability and for other practical reasons, the two nominal genera should be treated as synonyms. Host plant associations may be useful in the systematics of Abia species, but this topic requires further investigation and inclusion of more species in phylogenetic analyses.

Screening of Repellents against Vespid Wasps.

Vespid wasps are ecologically beneficial, but they can be a nuisance and dangerous to people due to their tendency to sting. Here, the aim was to screen samples of volatiles (i.e., essential oils and pure chemicals) for their repellency against wasps. The number of wasps (mainly Vespula vulgaris) present in a glass box with attractant and 5 µL sample was compared to the number of wasps in a similar box with attractant only. Both boxes were connected to a large glass container harboring 18-35 wasps. Among 66 tested samples, some essential oils from Lamiaceae and Asteraceae, as well as some pure natural compounds such as the monoterpenes (-)-terpinen-4-ol and isopulegol showed a significant repellency against vespids. Our results corroborate the potential of (mixtures of) volatiles in repelling these insects.

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.

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.