The evolution of antennal courtship in diplazontine parasitoid wasps (Hymenoptera, Ichneumonidae, Diplazontinae)

Background As predicted by theory, traits associated with reproduction often evolve at a comparatively high speed. This is especially the case for courtship behaviour which plays a central role in reproductive isolation. On the other hand, courtship behavioural traits often involve morphological and behavioural adaptations in both sexes; this suggests that their evolution might be under severe constraints, for instance irreversibility of character loss. Here, we use a recently proposed method to retrieve data on a peculiar courtship behavioural trait, i.e. antennal coiling, for 56 species of diplazontine parasitoid wasps. On the basis of a well-resolved phylogeny, we reconstruct the evolutionary history of antennal coiling and associated morphological modifications to study the mode of evolution of this complex character system. Results Our study reveals a large variation in shape, location and ultra-structure of male-specific modifications on the antennae. As for antennal coiling, we find either single-coiling, double-coiling or the absence of coiling; each state is present in multiple genera. Using a model comparison approach, we show that the possession of antennal modifications is highly correlated with antennal coiling behaviour. Ancestral state reconstruction shows that both antennal modifications and antennal coiling are highly congruent with the molecular phylogeny, implying low levels of homoplasy and a comparatively low speed of evolution. Antennal coiling is lost on two independent occasions, and never reacquired. A zero rate of regaining antennal coiling is supported by maximum parsimony, maximum likelihood and Bayesian approaches. Conclusions Our study provides the first comparative evidence for a tight correlation between male-specific antennal modifications and the use of the antennae during courtship. Antennal coiling in Diplazontinae evolved at a comparatively low rate, and was never reacquired in any of the studied taxa. This suggests that the loss of antennal coiling is irreversible on the timescale examined here, and therefore that evolutionary constraints have greatly influenced the evolution of antennal courtship in this group of parasitoid wasps. Further studies are needed to ascertain whether the loss of antennal coiling is irreversible on larger timescales, and whether evolutionary constraints have influenced courtship behavioural traits in a similar way in other groups.

The Role of Fresh versus Old Leaf Damage in the Attraction of Parasitic Wasps to Herbivore-Induced Maize Volatiles

The odor produced by a plant under herbivore attack is often used by parasitic wasps to locate hosts. Any type of surface damage commonly causes plant leaves to release so-called green leaf volatiles, whereas blends of inducible compounds are more specific for herbivore attack and can vary considerably among plant genotypes. We compared the responses of naïve and experienced parasitoids of the species Cotesia marginiventris and Microplitis rufiventris to volatiles from maize leaves with fresh damage (mainly green leaf volatiles) vs. old damage (mainly terpenoids) in a six-arm olfactometer. These braconid wasps are both solitary endoparasitoids of lepidopteran larvae, but differ in geographical origin and host range. In choice experiments with odor blends from maize plants with fresh damage vs. blends from plants with old damage, inexperienced C. marginiventris showed a preference for the volatiles from freshly damaged leaves. No such preference was observed for inexperienced M. rufiventris. After an oviposition experience in hosts feeding on maize plants, C. marginiventris females were more attracted by a mixture of volatiles from fresh and old damage. Apparently, C. marginiventris has an innate preference for the odor of freshly damaged leaves, and this preference shifts in favor of a blend containing a mixture of green leaf volatiles plus terpenoids, after experiencing the latter blend in association with hosts. M. rufiventris responded poorly after experience and preferred fresh damage odors. Possibly, after associative learning, this species uses cues that are more directly related with the host presence, such as volatiles from host feces, which were not present in the odor sources offered in the olfactometer. The results demonstrate the complexity of the use of plant volatiles by parasitoids and show that different parasitoid species have evolved different strategies to exploit these signals.

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

High Bee and Wasp Diversity in a Heterogeneous Tropical Farming System Compared to Protected Forest

It is a globally important challenge to meet increasing demands for resources and, at the same time, protect biodiversity and ecosystem services. Farming is usually regarded as a major threat to biodiversity due to its expansion into natural areas. We compared biodiversity of bees and wasps between heterogeneous small-scale farming areas and protected forest in northern coastal Belize, Central America. Malaise traps operated for three months during the transition from wet to dry season. Farming areas consisted of a mosaic of mixed crop types, open habitat, secondary forest, and agroforestry. Mean species richness per site (alpha diversity), as well as spatial and temporal community variation (beta diversity) of bees and wasps were equal or higher in farming areas compared to protected forest. The higher species richness and community variation in farmland was due to additional species that did not occur in the forest, whereas most species trapped in forest were also found in farming areas. The overall regional species richness (gamma diversity) increased by 70% with the inclusion of farming areas. Our results suggest that small-scale farming systems adjacent to protected forest may not only conserve, but even favour, biodiversity of some taxonomic groups. We can, however, not exclude possible declines of bee and wasp diversity in more intensified farmland or in landscapes completely covered by heterogeneous farming systems.

Stage-dependent strategies of host invasion in the egg-larval parasitoid Chelonus inanitus.

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid which lays its eggs into eggs of Spodoptera littoralis (Noctuidae); the parasitoid larva then develops in the haemocoel of the host larva. Host embryonic development lasts approx. 3.5 days while parasitoid embryonic development lasts approx. 16 h. All stages of host eggs can be successfully parasitized, and we show here that either the parasitoid larva or the wasp assures that the larva eventually is located in the host's haemocoel. (1) When freshly laid eggs, up to almost 1-day-old, are parasitized, the parasitoid hatches while still in the yolk and enters the host either after waiting or immediately through the dorsal opening. (2) When 1-2-day-old eggs are parasitized, the host embryo has accomplished final dorsal closure and is covered by an embryonic cuticle when the parasitoid hatches; in this case the parasitoid larva bores with its moving abdominal tip into the host. (3) When 2.5-3.5-day-old eggs are parasitized, the wasp oviposits directly into the haemocoel of the host embryo; from day 2 to 2.5 the embryo is still very small and the wasps, after probing, often restrain from oviposition for a few hours.

A tritrophic signal that attracts parasitoids to host-damaged plants withstands disruption by non-host herbivores

Background: Volatiles emitted by herbivore-infested plants are highly attractive to parasitoids and therefore have been proposed to be part of an indirect plant defense strategy. However, this proposed function of the plant-provided signals remains controversial, and it is unclear how specific and reliable the signals are under natural conditions with simultaneous feeding by multiple herbivores. Phloem feeders in particular are assumed to interfere with plant defense responses. Therefore, we investigated how attack by the piercing-sucking cicadellid Euscelidius variegatus influences signaling by maize plants in response to the chewing herbivore Spodoptera littoralis.Results: The parasitoid Cotesia marginiventris strongly preferred volatiles of plants infested with its host S. littoralis. Overall, the volatile emissions induced by S. littoralis and E. variegatus were similar, but higher levels of certain wound-released compounds may have allowed the wasps to specifically recognize plants infested by hosts. Expression levels of defense marker genes and further behavioral bioassays with the parasitoid showed that neither the physiological defense responses nor the attractiveness of S. littoralis infested plants were altered by simultaneous E. variegatus attack.Conclusions: Our findings imply that plant defense responses to herbivory can be more robust than generally assumed and that ensuing volatiles convey specific information about the type of herbivore that is attacking a plant, even in complex situations with multiple herbivores. Hence, the results of this study support the notion that herbivore-induced plant volatiles may be part of a plant's indirect defense stratagem. © 2010 Erb et al; licensee BioMed Central Ltd.

The prospect of applying chemical elicitors and plant strengtheners to enhance the biological control of crop pests

An imminent food crisis reinforces the need for novel strategies to increase crop yields worldwide. Effective control of pest insects should be part of such strategies, preferentially with reduced negative impact on the environment and optimal protection and utilization of existing biodiversity. Enhancing the presence and efficacy of native biological control agents could be one such strategy. Plant strengthener is a generic term for several commercially available compounds or mixtures of compounds that can be applied to cultivated plants in order to ‘boost their vigour, resilience and performance’. Studies into the consequences of boosting plant resistance against pests and diseases on plant volatiles have found a surprising and dramatic increase in the plants' attractiveness to parasitic wasps. Here, we summarize the results from these studies and present new results from assays that illustrate the great potential of two commercially available resistance elicitors. We argue that plant strengtheners may currently be the best option to enhance the attractiveness of cultivated plants to biological control agents. Other options, such as the genetic manipulation of the release of specific volatiles may offer future solutions, but in most systems, we still miss fundamental knowledge on which key attractants should be targeted for this approach.

The maize lipoxygenase, ZmLOX10 , mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, majorregulatory genes and the signals that modulate these defense metabolites are vastly understudied, especiallyin important agro-economic monocot species. Here we show that products and signals derived from a singleZea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbiv-ory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substratefor the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the spe-cialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indi-cating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression ofJA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling isrequired for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to par-tially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produceGLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness toparasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic linkto the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compro-mised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence thatLOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene toagro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.

Less is More: Treatment with BTH and Laminarin Reduces Herbivore-Induced Volatile Emissions in Maize but Increases Parasitoid Attraction

Chemical plant strengtheners find increasing use in agriculture to enhance resistance against pathogens. In an earlier study, it was found that treatment with one such resistance elicitor, BTH (benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester), increases the attractiveness of maize plants to a parasitic wasp. This surprising additional benefit of treating plants with BTH prompted us to conduct a series of olfactometer tests to find out if BTH and another commercially available plant strengthener, Laminarin, increase the attractiveness of maize to three important parasitic wasps, Cotesia marginventris, Campoletis sonorensis, and Microplitis rufiventris. In each case, plants that were sprayed with the plant strengtheners and subsequently induced to release volatiles by real or mimicked attack by Spodoptera littoralis caterpillars became more attractive to the parasitoids than water treated plants. The elicitors alone or in combination with plants that were not induced by herbivory were not attractive to the wasps. Interestingly, plants treated with the plant strengtheners did not show any consistent increase in volatile emissions. On the contrary, treated plants released less herbivore-induced volatiles, most notably indole, which has been reported to interfere with parasitoid attraction. The emission of the sesquiterpenes (E)-β-caryophyllene, β-bergamotene, and (E)-β-farnesene was similarly reduced by the treatment. Expression profiles of marker genes showed that BTH and Laminarin induced several pathogenesis related (PR) genes. The results support the notion that, as yet undetectable and unidentified compounds, are of major importance for parasitoid attraction, and that these attractants may be masked by some of the major compounds in the volatile blends. This study confirms that elicitors of pathogen resistance are compatible with the biological control of insect pests and may even help to improve it.

Herbivore-Induced Plant Volatiles Can Serve as Host Location Cues for a Generalist and a Specialist Egg Parasitoid

Herbivore-induced plant volatiles are important host finding cues for larval parasitoids, and similarly, insect oviposition might elicit the release of plant volatiles functioning as host finding cues for egg parasitoids. We hypothesized that egg parasitoids also might utilize HIPVs of emerging larvae to locate plants with host eggs. We, therefore, assessed the olfactory response of two egg parasitoids, a generalist, Trichogramma pretiosum (Tricogrammatidae), and a specialist, Telenomus remus (Scelionidae) to HIPVs. We used a Y-tube olfactometer to tests the wasps’ responses to volatiles released by young maize plants that were treated with regurgitant from caterpillars of the moth Spodoptera frugiperda (Noctuidae) or were directly attacked by the caterpillars. The results show that the generalist egg parasitoid Tr. pretiosum is innately attracted by volatiles from freshly-damaged plants 0–1 and 2–3 h after regurgitant treatment. During this interval, the volatile blend consisted of green leaf volatiles (GLVs) and a blend of aromatic compounds, mono- and homoterpenes, respectively. Behavioral assays with synthetic GLVs confirmed their attractiveness to Tr. pretiosum. The generalist learned the more complex volatile blends released 6–7 h after induction, which consisted mainly of sesquiterpenes. The specialist T. remus on the other hand was attracted only to volatiles emitted from fresh and old damage after associating these volatiles with oviposition. Taken together, these results strengthen the emerging pattern that egg and larval parasitoids behave in a similar way in that generalists can respond innately to HIPVs, while specialists seems to rely more on associative learning.

Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering

Polydnaviruses (genera Ichnovirus and Bracovirus) have a segmented genome of circular double-stranded DNA molecules, replicate in the ovary of parasitic wasps and are essential for successful parasitism of the host. Here we show the first detailed analysis of various segments of a bracovirus, the Chelonus inanitus virus (CiV). Four segments were sequenced and two of them, CiV12 and CiV14, were found to be closely related while CiV14.5 and CiV16.8 were unrelated. CiV12, CiV14.5 and CiV16.8 are unique while CiV14 occurs also nested in another larger segment. All four segments are predicted to contain genes and predictions could be substantiated in most cases. Comparison with databases revealed no significant similarities at either the nucleotide or amino acid level. Inverted repeats with identities between 77% and 92% and lengths between 26 bp and 100 bp were found on all segments outside of predicted genes. Hybridization experiments indicate that CiV12 and CiV14 are both flanked by other virus segments, suggesting that proviral CiV segments are clustered in the genome of the wasp. The integration/excision site of CiV14 was analysed and compared to that of CiV12. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found. A model to illustrate where the terminal repeats might recombine to yield the circular molecule is presented. Excision of CiV12 and CiV14 is restricted to the female and sets in at a very specific time-point in pupal-adult development.

Presence of polydnavirus transcripts in an egg-larval parasitoid and its lepidopterous host

The parasitoid Chelonus inanitus (Braconidae, Hymenoptera) oviposits into eggs of Spodoptera littoralis (Noctuidae, Lepidoptera) and, along with the egg, also injects polydnaviruses and venom, which are prerequisites for successful parasitoid development. The parasitoid larva develops within the embryonic and larval stages of the host, which enters metamorphosis precociously and arrests development in the prepupal stage. Polydnaviruses are responsible for the developmental arrest and interfere with the host's endocrine system in the last larval instar. Polydnaviruses have a segmented genome and are transmitted as a provirus integrated in the wasp's genome. Virions are only formed in female wasps and no virus replication is seen in the parasitized host. Here it is shown that very small amounts of viral transcripts were found in parasitized eggs and early larval instars of S. littoralis. Later on, transcript quantities increased and were highest in the late last larval instar for two of the three viral segments tested and in the penultimate to early last larval instar for the third segment. These are the first data on the occurrence of viral transcripts in the host of an egg-larval parasitoid and they are different from data reported for hosts of larval parasitoids, where transcript levels are already high shortly after parasitization. The analysis of three open reading frames by RT-PCR revealed viral transcripts in parasitized S. littoralis and in female pupae of C. inanitus, indicating the absence of host specificity. For one open reading frame, transcripts were also seen in male pupae, suggesting transcription from integrated viral DNA.