Mechanisms of Isothiocyanate Detoxification in Larvae of Two Belowground Herbivores, Delia radicum and D. floralis (Diptera: Anthomyiidae).

Like aboveground herbivores, belowground herbivores are confronted with multiple plant defense mechanisms including complex chemical cocktails in plant tissue. Roots and shoots of Brassicaceae plants contain the two-component glucosinolate (GSL)-myrosinase defense system. Upon cell damage, for example by herbivore feeding, toxic and pungent isothiocyanates (ITCs) can be formed. Several aboveground-feeding herbivores have developed biochemical adaptation strategies to overcome the GSL-ITC defenses of their host plant. Whether belowground herbivores feeding on Brassica roots possess similar mechanisms has received little attention. Here, we analyze how two related belowground specialist herbivores detoxify the GSL-ITC defenses of their host plants. The larvae of the fly species Delia radicum and D. floralis are common pests and specialized herbivores on the roots of Brassicaceae. We used chemical analyses (HPLC-MS/MS and HPLC-UV) to examine how the GSL-ITC defense system is metabolized by these congeneric larvae. In addition, we screened for candidate genes involved in the detoxification process using RNAseq and qPCR. The chemical analyses yielded glutathione conjugates and amines. This indicates that both species detoxify ITCs using potentially the general mercapturic acid pathway, which is also found in aboveground herbivores, and an ITC-specific hydrolytic pathway previously characterized in microbes. Performance assays confirmed that ITCs negatively affect the survival of both species, in spite of their known specialization to ITC-producing plants and tissues, whereas ITC breakdown products are less toxic. Interestingly, the RNAseq analyses showed that the two congeneric species activate different sets of genes upon ITC exposure, which was supported by qPCR data. Based on our findings, we conclude that these specialist larvae use combinations of general and compound-specific detoxification mechanisms with differing efficacies and substrate preferences. This indicates that combining detoxification mechanisms can be an evolutionarily successful strategy to handle plant defenses in herbivores.

A high-quality functional genome assembly of Delia radicum L. (Diptera: Anthomyiidae) annotated from egg to adult.

Belowground herbivores are overseen and underestimated, even though they can cause significant economic losses in agriculture. The cabbage root fly Delia radicum (Anthomyiidae) is a common pest in Brassica species, including agriculturally important crops, such as oilseed rape. The damage is caused by the larvae, which feed specifically on the taproots of Brassica plants until they pupate. The adults are aboveground-living generalists feeding on pollen and nectar. Female flies are attracted by chemical cues in Brassica plants for oviposition. An assembled and annotated genome can elucidate which genetic mechanisms underlie the adaptation of D. radicum to its host plants and their specific chemical defences, in particular isothiocyanates. Therefore, we assembled, annotated and analysed the D. radicum genome using a combination of different next-generation sequencing and bioinformatic approaches. We assembled a chromosome-level D. radicum genome using PacBio and Hi-C Illumina sequence data. Combining Canu and 3D-DNA genome assembler, we constructed a 1.3 Gbp genome with an N50 of 242 Mbp and 6 pseudo-chromosomes. To annotate the assembled D. radicum genome, we combined homology-, transcriptome- and ab initio-prediction approaches. In total, we annotated 13,618 genes that were predicted by at least two approaches. We analysed egg, larval, pupal and adult transcriptomes in relation to life-stage specific molecular functions. This high-quality annotated genome of D. radicum is a first step to understanding the genetic mechanisms underlying host plant adaptation. As such, it will be an important resource to find novel and sustainable approaches to reduce crop losses to these pests.

Infection Patterns and Fitness Effects of Rickettsia and Sodalis Symbionts in the Green Lacewing Chrysoperla carnea.

Endosymbionts are widely distributed in insects and can strongly affect their host ecology. The common green lacewing (Chrysoperla carnea) is a neuropteran insect which is widely used in biological pest control. However, their endosymbionts and their interactions with their hosts have not been very well studied. Therefore, we screened for endosymbionts in natural and laboratory populations of Ch. carnea using diagnostic PCR amplicons. We found the endosymbiont Rickettsia to be very common in all screened natural and laboratory populations, while a hitherto uncharacterized Sodalis strain was found only in laboratory populations. By establishing lacewing lines with no, single or co-infections of Sodalis and Rickettsia, we found a high vertical transmission rate for both endosymbionts (>89%). However, we were only able to estimate these numbers for co-infected lacewings. Sodalis negatively affected the reproductive success in single and co-infected Ch. carnea, while Rickettsia showed no effect. We hypothesize that the fitness costs accrued by Sodalis infections might be more tolerable in the laboratory than in natural populations, as the latter are also prone to fluctuating environmental conditions and natural enemies. The economic and ecological importance of lacewings in biological pest control warrants a more profound understanding of its biology, which might be influenced by symbionts.

Functional Variation in Dipteran Gut Bacterial Communities in Relation to Their Diet, Life Cycle Stage and Habitat.

True flies and mosquitos (Diptera) live in habitats and consume diets that pose specific demands on their gut bacterial communities (GBCs). Due to diet specializations, dipterans may have highly diverse and species-specific GBCs. Dipterans are also confronted with changes in habitat and food sources over their lifetime, especially during life history processes (molting, metamorphosis). This may prevent the development of a constant species- or diet-specific GBC. Some dipterans are vectors of several human pathogens (e.g., malaria), which interact with GBCs. In this review, we explore the dynamics that shape GBC composition in some Diptera species on the basis of published datasets of GBCs. We thereby focus on the effects of diet, habitats, and life cycle stages as sources of variation in GBC composition. The GBCs reported were more stage-specific than species- or diet-specific. Even though the presence of GBCs has a large impact on the performance of their hosts, the exact functions of GBCs and their interactions with other organisms are still largely unknown, mainly due to the low number of studies to date. Increasing our knowledge on dipteran GBCs will help to design pest management strategies for the reduction of insecticide resistance, as well as for human pathogen control.

Same Difference? Low and High Glucosinolate Brassica rapa Varieties Show Similar Responses Upon Feeding by Two Specialist Root Herbivores.

Glucosinolates (GSLs) evolved in Brassicaceae as chemical defenses against herbivores. The GSL content in plants is affected by both abiotic and biotic factors, but also depends on the genetic background of the plant. Since the bitter taste of GSLs can be unfavorable for both livestock and human consumption, several plant varieties with low GSL seed or leaf content have been bred. Due to their lower GSL levels, such varieties can be more susceptible to herbivore pests. However, low GSL varieties may quickly increase GSL levels upon herbivore feeding by activating GSL biosynthesis, hydrolysis, or transporter genes. To analyze differences in herbivore-induced GSL responses in relation to constitutive GSL levels, we selected four Brassica rapa varieties, containing either low or high root GSL levels. Plants were infested either with Delia radicum or Delia floralis larvae. The larvae of both root flies are specialists on Brassica plants. Root samples were collected after 3, 5, and 7 days. We compared the effect of root herbivore damage on the expression of GSL biosynthesis (CYP79A1, CYP83B2), transporter (GTR1A2, GTR2A2), and GSL hydrolysis genes (PEN2, TGG2) in roots of low and high GSL varieties in conjugation with their GSL levels. We found that roots of high GSL varieties contained higher levels of aliphatic, indole, and benzyl GSLs than low GSL varieties. Infestation with D. radicum larvae led to upregulation of indole GSL synthesis genes in low and high GSL varieties. High GSL varieties showed no or later responses than low varieties to D. floralis herbivory. Low GSL varieties additionally upregulated the GSL transporter gene expression. Low GSL varieties did not show a stronger herbivore-induced response than high GSL varieties, which indicates that there is no trade-off between constitutive and induced GSLs.

Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa.

The optimal defense theory predicts that plants invest most energy in those tissues that have the highest value, but are most vulnerable to attacks. In Brassica species, root-herbivory leads to the accumulation of glucosinolates (GSLs) in the taproot, the most valuable belowground plant organ. Accumulation of GSLs can result from local biosynthesis in response to herbivory. In addition, transport from distal tissues by specialized GSL transporter proteins can play a role as well. GSL biosynthesis and transport are both inducible, but the role these processes play in GSL accumulation during root-herbivory is not yet clear. To address this issue, we performed two time-series experiments to study the dynamics of transport and biosynthesis in local and distal tissues of Brassica rapa. We exposed roots of B. rapa to herbivory by the specialist root herbivore Delia radicum for 7 days. During this period, we sampled above- and belowground plant organs 12 h, 24 h, 3 days and 7 days after the start of herbivory. Next, we measured the quantity and composition of GSL profiles together with the expression of genes involved in GSL biosynthesis and transport. We found that both benzyl and indole GSLs accumulate in the taproot during root-herbivory, whereas we did not observe any changes in aliphatic GSL levels. The rise in indole GSL levels coincided with increased local expression of biosynthesis and transporter genes, which suggest that both biosynthesis and GSL transport play a role in the accumulation of GSLs during root herbivory. However, we did not observe a decrease in GSL levels in distal tissues. We therefore hypothesize that GSL transporters help to retain GSLs in the taproot during root-herbivory.

Green lacewings (Neuroptera: Chrysopidae) are commonly associated with a diversity of rickettsial endosymbionts.

BACKGROUND: Bacterial symbionts transmitted from mothers to offspring are found in the majority of arthropods. Numerous studies have illustrated their wide impact on host biology, such as reproduction, behavior, and physiology One of the most common inherited symbionts is Rickettsia spp. (Alphaproteobacteria, Rickettsiales), which are found in about one-quarter of terrestrial arthropods, as well as in other invertebrates. In insect populations, Rickettsia spp. have been reported to cause reproductive modifications and fecundity-enhancing effects. Here, we investigated the incidence and genetic diversity of Rickettsia symbionts in green lacewings (Neuroptera, Chrysopidae), which are best known for their use as biological control agents against crop pests. RESULTS: We screened 18 species of green lacewings and allies for Rickettsia and found the symbiont in 10 species, infecting 20-100% of sampled individuals. Strain characterization based on multiple bacterial loci revealed an unprecedented diversity of Rickettsia associated with lacewings, suggesting multiple independent acquisitions. Further, the detected Rickettsia lineages are restricted to a specific lineage (i.e., species or genus) of investigated lacewings, and these associations are stable across multiple sampled locations and points in time. CONCLUSIONS: We conclude that Rickettsia-lacewing symbioses are common and evolutionarily stable. The role of these symbionts remains to be identified, but is potentially important to optimizing their use in biological pest control.

Syllidae mitochondrial gene order is unusually variable for Annelida.

Complete mitochondrial genomes of five syllids (Streptosyllis sp., Eusyllis blomstrandi, Myrianida brachycephala, Typosyllis antoni and Typosyllis sp.) have been obtained using Illumina sequencing. Together with two previous studied taxa (Ramisyllis multicaudata and Trypanobia cryptica), the analysed sequences represent most of the main lineages within the family Syllidae (Anoplosyllinae, Eusyllinae, Autolytinae and Syllinae). The genomic features, gene order and phylogenetic relationships are examined. Unusual for annelids, syllid mitochondrial genomes are highly variable in their gene order. Considering genomic features, such as length, skewness, gene content, and codon bias, most similar to the rest of annelids are the genomes of E. blomstrandi and M. brachycephala, while Streptosyllis sp. and the analysed sylline taxa (R. multicaudata, T. cryptica, T. antoni and Typosyllis sp.) are the most dissimilar. Two methionine tRNA's (trnM) have been found in T. antoni and Typosyllis sp. The mt genomes of these latter taxa are the longest with numerous non-coding regions. The 13 protein coding genes, as well as the rRNA's are used to perform phylogenetic analyses that recovered the relationships within the family explored before by previous authors. The gene order in Syllidae shows very different patterns. E. blomstrandi and M. prolifera show a similar pattern to the one found in Pleistoannelida; however this might have changed at least twice within Syllidae: in Streptosyllis sp. and within Syllinae. All analysed Syllinae show different gene orders, thereby illustrating more variability as all other pleistoannelids analysed so far. The information provided herein allows a more accurate reconstruction of the possible evolutionary scenarios in Syllidae.

Wolbachia distribution in selected beetle taxa characterized by PCR screens and MLST data.

Wolbachia (Alphaproteobacteria) is an inherited endosymbiont of arthropods and filarial nematodes and was reported to be widespread across insect taxa. While Wolbachia's effects on host biology are not understood from most of these hosts, known Wolbachia-induced phenotypes cover a spectrum from obligate beneficial mutualism to reproductive manipulations and pathogenicity. Interestingly, data on Wolbachia within the most species-rich order of arthropods, the Coleoptera (beetles), are scarce. Therefore, we screened 128 species from seven beetle families (Buprestidae, Hydraenidae, Dytiscidae, Hydrophilidae, Gyrinidae, Haliplidae, and Noteridae) for the presence of Wolbachia. Our data show that, contrary to previous estimations, Wolbachia frequencies in beetles (31% overall) are comparable to the ones in other insects. In addition, we used Wolbachia MLST data and host phylogeny to explore the evolutionary history of Wolbachia strains from Hydraenidae, an aquatic lineage of beetles. Our data suggest that Wolbachia from Hydraenidae might be largely host genus specific and that Wolbachia strain phylogeny is not independent to that of its hosts. As this contrasts with most terrestrial Wolbachia-arthropod systems, one potential conclusion is that aquatic lifestyle of hosts may result in Wolbachia distribution patterns distinct from those of terrestrial hosts. Our data thus provide both insights into Wolbachia distribution among beetles in general and a first glimpse of Wolbachia distribution patterns among aquatic host lineages.