Global changes in the odonate family ratios in response to the tropical forest degradation.

Odonates (dragonflies and damselflies) can indicate the ecological health of aquatic biota within the rich but vulnerable biodiversity of tropical forests. The reaction of odonates to deforestation can be measured by changes in coarse taxonomic ratios. Suborder Zygoptera are thermal conformers susceptible to overheating, having the affinity with shaded, intact sites. Anisoptera have exothermic regulation and better dispersal capacities, suggesting their association with more altered, open environments. Similarly, with an increasing degradation, the proportion of Anisoptera species in assemblages should increase. However, based on the data from different continents, the Zygoptera/Anisoptera ratio may be too simple, strongly biased, and not applicable at the global scale. The main reason is that the most diverse, abundant, and cosmopolitan families, Coenagrionidae (Zygoptera) and Libellulidae (Anisoptera), comprise a great proportion of habitat generalists with high migratory capacity and affinity with open habitats. In this study, we sampled odonates from three bioregions (Indomalaya, Afrotropics, and Neotropics) over the gradient of tropical forest degradation with a comparable sampling effort to assess the suitability of species richness and suborder-based (Zygoptera/Anisoptera) and family-based (Libellulidae/other Anisoptera and Coenagrionidae/other Zygoptera) ratios and their abundance-weighted versions for monitoring tropical forest degradation. Our results show that simple Odonata as well as Zygoptera and Anisoptera richness are poor indicators of the forest biota alteration. Family-level indices weighted by relative abundance, especially those involving Coenagrionidae, were more sensitive to changes in forest conditions compared to suborder-level indices. Collectively, our results suggest that for biomonitoring, where financial resources and time are commonly critical, family-level ratio metrics may be effective tools to indicate even slight alterations of aquatic biota resulting from forest degradation. Although these indices have the potential for broader application, their effectiveness across tropical bioregions warrants further validation.

Sum or mean in calculation of qualitative scoring methods using the Dragonfly Biotic Index, and an alternative approach facilitating conservation prioritization.

Qualitative scoring methods are tools for rapid freshwater health assessments. Total score is often calculated as the sum or mean of the values of the species involved, with minor nuances in interpretation, but with significant implications. We used the Dragonfly Biotic Index (DBI) calculated on Central European odonate species to demonstrate these implications. Each species within a community has a score ranging from 0 (widespread generalists) to 9 (sensitive specialists). A total score is calculated as the sum of the scores of all species (DBIsum) or is calculated by dividing by species richness (DBImean). Despite this duality, there has been little debate on either approach. Using simulated scenarios (high vs low richness, presence or absence of high- or low-scoring species), we tested the implications of DBIsum and DBImean and suggested a total score calculation for conservation prioritization based on permutation. This algorithm shows the percentile of a community compared to a set of randomly assembled communities of the same species richness. We also present the 'dragDBI' package for the statistical software R, a tool for more automated DBI-based environmental health assessments. Our permutational calculation is applicable to other macroinvertebrate-based scoring methods, such as the Biological Monitoring Working Party and the Average Score Per Taxon.

Mycobiomes of two distinct clades of ambrosia gall midges (Diptera: Cecidomyiidae) are species-specific in larvae but similar in nutritive mycelia.

IMPORTANCE: Ambrosia gall midges are endophagous insect herbivores whose larvae live enclosed within a single gall for their entire development period. They may exhibit phytomycetophagy, a remarkable feeding mode that involves the consumption of plant biomass and mycelia of their cultivated gall symbionts. Thus, AGMs are ideal model organisms for studying the role of microorganisms in the evolution of host specificity in insects. However, compared to other fungus-farming insects, insect-fungus mutualism in AGMs has been neglected. Our study is the first to use DNA metabarcoding to characterize the complete mycobiome of the entire system of the gall-forming insects as we profiled gall surfaces, nutritive mycelia, and larvae. Interestingly, larval mycobiomes were significantly different from their nutritive mycelia, although Botryosphaeria dothidea dominated the nutritive mycelia, regardless of the evolutionary separation of the tribes studied. Therefore, we confirmed a long-time hypothesized paradigm for the important evolutionary association of this fungus with AGMs.

Concentration-dependent effect of plant secondary metabolites on bacterial and fungal microbiomes in caterpillar guts.

IMPORTANCE: The caterpillar gut is an excellent model system for studying host-microbiome interactions, as it represents an extreme environment for microbial life that usually has low diversity and considerable variability in community composition. Our study design combines feeding caterpillars on a natural and artificial diet with controlled levels of plant secondary metabolites and uses metabarcoding and quantitative PCR to simultaneously profile bacterial and fungal assemblages, which has never been performed. Moreover, we focus on multiple caterpillar species and consider diet breadth. Contrary to many previous studies, our study suggested the functional importance of certain microbial taxa, especially bacteria, and confirmed the previously proposed lower importance of fungi for caterpillar holobiont. Our study revealed the lack of differences between monophagous and polyphagous species in the responses of microbial assemblages to plant secondary metabolites, suggesting the limited role of the microbiome in the plasticity of the herbivore diet.

Seasonal Shifts in Bacterial and Fungal Microbiomes of Leaves and Associated Leaf-Mining Larvae Reveal Persistence of Core Taxa Regardless of Diet.

Microorganisms are key mediators of interactions between insect herbivores and their host plants. Despite a substantial interest in studying various aspects of these interactions, temporal variations in microbiomes of woody plants and their consumers remain understudied. In this study, we investigated shifts in the microbiomes of leaf-mining larvae (Insecta: Lepidoptera) and their host trees over one growing season in a deciduous temperate forest. We used 16S and ITS2 rRNA gene metabarcoding to profile the bacterial and fungal microbiomes of leaves and larvae. We found pronounced shifts in the leaf and larval microbiota composition and richness as the season progressed, and bacteria and fungi showed consistent patterns. The quantitative similarity between leaf and larval microbiota was very low for bacteria (~9%) and decreased throughout the season, whereas fungal similarity increased and was relatively high (~27%). In both leaves and larvae, seasonality, along with host taxonomy, was the most important factor shaping microbial communities. We identified frequently occurring microbial taxa with significant seasonal trends, including those more prevalent in larvae (Streptococcus, Candida sake, Debaryomyces prosopidis, and Neoascochyta europaea), more prevalent in leaves (Erwinia, Seimatosporium quercinum, Curvibasidium cygneicollum, Curtobacterium, Ceramothyrium carniolicum, and Mycosphaerelloides madeirae), and frequent in both leaves and larvae (bacterial strain P3OB-42, Methylobacterium/Methylorubrum, Bacillus, Acinetobacter, Cutibacterium, and Botrytis cinerea). Our results highlight the importance of considering seasonality when studying the interactions between plants, herbivorous insects, and their respective microbiomes, and illustrate a range of microbial taxa persistent in larvae, regardless of their occurrence in the diet. IMPORTANCE Leaf miners are endophagous insect herbivores that feed on plant tissues and develop and live enclosed between the epidermis layers of a single leaf for their entire life cycle. Such close association is a precondition for the evolution of more intimate host-microbe relationships than those found in free-feeding herbivores. Simultaneous comparison of bacterial and fungal microbiomes of leaves and their tightly linked consumers over time represents an interesting study system that could fundamentally contribute to the ongoing debate on the microbial residence of insect gut. Furthermore, leaf miners are ideal model organisms for interpreting the ecological and evolutionary roles of microbiota in host plant specialization. In this study, the larvae harbored specific microbial communities consisting of core microbiome members. Observed patterns suggest that microbes, especially bacteria, may play more important roles in the caterpillar holobiont than generally presumed.

Highway stormwater ponds as islands of Odonata diversity in an agricultural landscape.

Stormwater management ponds, which are constructed to retain excess runoff and pollutants from traffic, play an important role in the freshwater biodiversity in highly modified areas. However, their roles in agricultural and semi-natural landscapes remain largely unexplored. In this study, we used Odonata as a bioindicator to compare a set of highway stormwater ponds and surrounding ponds within an agricultural and semi-natural landscape to examine the extent to which stormwater ponds act as biodiversity refuges. We analyzed the differences in environmental parameters and the richness, compositions, and conservation values of the odonate communities of stormwater and surrounding ponds. We also examined the factors controlling the differences in the communities of both pond types. The stormwater ponds were smaller, less eutrophicated, less shaded by trees, less stocked with fish, and less connected with other waterbodies than the surrounding ponds. However, they had a higher plant diversity and pH values and were more densely overgrown with vegetation. Compared with surrounding ponds, stormwater ponds had a higher Odonata richness and ß-diversity, but their taxonomic distinctness was significantly lower. Therefore, stormwater ponds hosted more variable communities but their assemblages were taxonomically similar. Indicator species were only identified in stormwater ponds. Furthermore, stormwater ponds harbored more species with higher conservation values. The most important factors affecting the differences between stormwater and surrounding ponds were the trophic state, relative tree shading, and fish stocking intensity. With their increase, the richness and rarity decreased. Our results highlight the potential of stormwater ponds to enhance the biodiversity outside urban areas by providing specific habitat conditions that are unique to the surrounding agricultural landscape. In addition, we suggest management practices that can be used to enhance their biodiversity conservation function.

Individual variability in habitat selection by aquatic insects is driven by taxonomy rather than specialisation.

Habitat selection, the choice of a habitat based on its perceived quality, is a key mechanism structuring freshwater communities. To date, individual variability in habitat selection has been neglected, and specialisation has never been considered in this type of studies. We examined the individual differences in the habitat selection of backswimmers (Notonectidae) and diving beetles (Dytiscidae). From each family, we selected one habitat generalist able to coexist with fish (Notonecta glauca, Dytiscus marginalis), and one species specialised to fishless habitats (Notonecta obliqua, Acilius sulcatus). We performed a mesocosm experiment quantifying the consistency in individuals' decisions in response to fish and vegetation structure, in relation to sex and specialisation. Neither the overall pattern of preferences nor consistency in individuals' decisions differed between specialists and generalists or between the sexes, but both were consistent within families. At the population level, backswimmers preferred fishless pools with submersed and floating macrophytes, while diving beetles showed no clear preferences. Individual decisions of backswimmers were consistent and likely driven by conspecific/heterospecific attraction. In diving beetles, individual decisions were primarily density-dependent. Our results reinforce the significance of habitat selectivity for aquatic community assembly, while suggesting a range of mechanisms driving variability in individual behaviour.

Fungi are more transient than bacteria in caterpillar gut microbiomes.

Despite an increasing number of studies on caterpillar (Insecta: Lepidoptera) gut microbiota, bacteria have been emphasized more than fungi. Therefore, we lack data on whether fungal microbiota is resident or transient and shaped by factors similar to those of bacteria. We sampled nine polyphagous caterpillar species from several tree species at multiple sites to determine the factors shaping leaf and gut bacterial and fungal microbiota as well as the extent to which caterpillars acquire microbiota from their diet. We performed 16S and ITS2 DNA metabarcoding of the leaves and guts to determine the composition and richness of the respective microbiota. While spatial variables shaped the bacterial and fungal microbiota of the leaves, they only affected fungi in the guts, whereas the bacteria were shaped primarily by caterpillar species, with some species harboring more specific bacterial consortia. Leaf and gut microbiota significantly differed; in bacteria, this difference was more pronounced. The quantitative similarity between leaves and guts significantly differed among caterpillar species in bacteria but not fungi, suggesting that some species have more transient bacterial microbiota. Our results suggest the complexity of the factors shaping the gut microbiota, while highlighting interspecific differences in microbiota residency within the same insect functional group.

Specialization directs habitat selection responses to a top predator in semiaquatic but not aquatic taxa.

Habitat selectivity has become an increasingly acknowledged mechanism shaping the structure of freshwater communities; however, most studies have focused on the effect of predators and competitors, neglecting habitat complexity and specialization. In this study, we examined the habitat selection of semiaquatic (amphibians: Bufonidae; odonates: Libellulidae) and aquatic organisms (true bugs: Notonectidae; diving beetles: Dytiscidae). From each family, we selected one habitat generalist species able to coexist with fish (Bufo bufo, Sympetrum sanguineum, Notonecta glauca, Dytiscus marginalis) and one species specialized in fishless habitats (Bufotes viridis, Sympetrum danae, Notonecta obliqua, Acilius sulcatus). In a mesocosm experiment, we quantified habitat selection decisions in response to the non-consumptive presence of fish (Carassius auratus) and vegetation structure mimicking different successional stages of aquatic habitats (no macrophytes; submerged and floating macrophytes; submerged, floating, and littoral-emergent macrophytes). No congruence between habitat specialists and generalists was observed, but a similar response to fish and vegetation structure defined both semiaquatic and aquatic organisms. While semiaquatic generalists did not distinguish between fish and fishless pools, specialists avoided fish-occupied pools and had a preferred vegetation structure. In aquatic taxa, predator presence affected habitat selection only in combination with vegetation structure, and all species preferred fishless pools with floating and submerged macrophytes. Fish presence triggered avoidance only in the generalist bug N. glauca. Our results highlight the significance of habitat selectivity for structuring freshwater ecosystems and illustrate how habitat selection responses to a top predator are dictated by specialization and life history.

Wing wettability gradient in a damselfly Lestes sponsa (Odonata: Lestidae) reflects the submergence behaviour during underwater oviposition.

The phenomenon of hydrophobicity of insect cuticles has received great attention from technical fields due to its wide applicability to industry or medicine. However, in an ecological/evolutionary context such studies remain scarce. We measured spatial differences in wing wettability in Lestes sponsa (Odonata: Lestidae), a damselfly species that can submerge during oviposition, and discussed the possible functional significance. Using dynamic contact angle (CA) measurements together with scanning electron microscopy (SEM), we investigated differences in wettability among distal, middle and proximal wing regions, and in surface nanostructures potentially responsible for observed differences. As we moved from distal towards more proximal parts, mean values of advancing and receding CAs gradually increased from 104° to 149°, and from 67° to 123°, respectively, indicating that wing tips were significantly less hydrophobic than more proximal parts. Moreover, values of CA hysteresis for the respective wing parts decreased from 38° to 26°, suggesting greater instability of the structure of the wing tips. Accordingly, compared with more proximal parts, SEM revealed higher damage of the wax nanostructures at the distal region. The observed wettability gradient is well explained by the submergence behaviour of L. sponsa during underwater oviposition. Our study thus proposed the existence of species-dependent hydrophobicity gradient on odonate wings caused by different ovipositional strategies.

Phenotypic plasticity in specialists: How long-spined larval Sympetrum depressiusculum (Odonata: Libellulidae) responds to combined predator cues.

Phenotypic plasticity is a common defensive strategy in species experiencing variable predation risk, such as habitat generalists. Larvae of generalist dragonflies can elongate their abdominal spines in environments with fish, but long spines render larvae susceptible to invertebrate predators. Long-spined specialists adapted to fish-heavy habitats are not expected to have phenotypic plasticity in this defence trait, but no empirical studies have been undertaken. Moreover, in comparison to prey responding to multiple predators that induce similar phenotypes, relatively little is known regarding how species react to combinations of predators that favour opposing traits. We examined plasticity of larval dragonfly Sympetrum depressiusculum, a long-spined habitat specialist. In a rearing experiment, larvae were exposed to four environments: (i) no predator control, (ii) fish cues (Carassius auratus), (iii) invertebrate cues (Anax imperator), as well as (iv) a combination of (ii) and (iii). Compared with the control, fish but not invertebrate cues resulted in longer spines for two (one lateral, one dorsal) of the six spines measured. Interestingly, the combined-cue treatment led to the elongation of all four dorsal spines compared with the fish treatment alone, whereas lateral spines showed no response. Our experiment provided evidence of morphological plasticity in a long-spined specialist dragonfly. We showed that nearly all spines can elongate, but also react differently under specific predator settings. Therefore, while spine plasticity evolved in direct response to a single predator type (fish), plasticity was maintained against invertebrate predators as long as fish were also present. Selective spine induction under the combined condition suggests that S. depressiusculum can successfully survive in environments with both predators. Therefore, phenotypic plasticity may be an effective strategy for habitat generalists and specialists. Although more studies are necessary to fully understand how selection shapes the evolution of phenotypic plasticity, we demonstrated that in dragonflies, presence or absence of a specific predator is not the only factor that determines plastic defence responses.

Vertical canopy gradient shaping the stratification of leaf-chewer-parasitoid interactions in a temperate forest.

Knowledge about herbivores and their parasitoids in forest canopies remains limited, despite their diversity and ecological importance. Thus, it is important to understand the factors that shape the herbivore-parasitoid community structure, particularly the effect of vertical gradient. We investigated a quantitative community dataset of exposed and semiconcealed leaf-chewing larvae and their parasitoids along a vertical canopy gradient in a temperate forest. We sampled target insects using an elevated work platform in a 0.2 ha broadleaf deciduous forest plot in the Czech Republic. We analyzed the effect of vertical position among three canopy levels (first [lowest], second [middle], and third [highest]) and tree species on community descriptors (density, diversity, and parasitism rate) and food web structure. We also analyzed vertical patterns in density and parasitism rate between exposed and semiconcealed hosts, and the vertical preference of the most abundant parasitoid taxa in relation to their host specificity. Tree species was an important determinant of all community descriptors and food web structure. Insect density and diversity varied with the vertical gradient, but was only significant for hosts. Both host guilds were most abundant in the second level, but only the density of exposed hosts declined in the third level. Parasitism rate decreased from the first to third level. The overall parasitism rate did not differ between guilds, but semiconcealed hosts suffered lower parasitism in the third level. Less host-specific taxa (Ichneumonidae, Braconidae) operated more frequently lower in the canopy, whereas more host-specific Tachinidae followed their host distribution. The most host-specific Chalcidoidea preferred the third level. Vertical stratification of insect density, diversity, and parasitism rate was most pronounced in the tallest tree species. Therefore, our study contradicts the general paradigm of weak arthropod stratification in temperate forest canopies. However, in the network structure, vertical variation might be superseded by variation among tree species.

Performance of DNA metabarcoding, standard barcoding, and morphological approach in the identification of host-parasitoid interactions.

Understanding interactions between herbivores and parasitoids is essential for successful biodiversity protection and monitoring and for biological pest control. Morphological identifications employ insect rearing and are complicated by insects' high diversity and crypsis. DNA barcoding has been successfully used in studies of host-parasitoid interactions as it can substantially increase the recovered real host-parasitoid diversity distorted by overlooked species complexes, or by species with slight morphological differences. However, this approach does not allow the simultaneous detection and identification of host(s) and parasitoid(s). Recently, high-throughput sequencing has shown high potential for surveying ecological communities and trophic interactions. Using mock samples comprising insect larvae and their parasitoids, we tested the potential of DNA metabarcoding for identifying individuals involved in host-parasitoid interactions to different taxonomic levels, and compared it to standard DNA barcoding and morphological approaches. For DNA metabarcoding, we targeted the standard barcoding marker cytochrome oxidase subunit I using highly degenerate primers, 2*300 bp sequencing on a MiSeq platform, and RTAX classification using paired-end reads. Additionally, using a large host-parasitoid dataset from a Central European floodplain forest, we assess the completeness and usability of a local reference library by confronting the number of Barcoding Index Numbers obtained by standard barcoding with the number of morphotypes. Overall, metabarcoding recovery was high, identifying 92.8% of the taxa present in mock samples, and identification success within individual taxonomic levels did not significantly differ among metabarcoding, standard barcoding, and morphology. Based on the current local reference library, 39.4% parasitoid and 90.7% host taxa were identified to the species level. DNA barcoding estimated higher parasitoid diversity than morphotyping, especially in groups with high level of crypsis. This study suggests the potential of metabarcoding for effectively recovering host-parasitoid diversity, together with more accurate identifications obtained from building reliable and comprehensive reference libraries, especially for parasitoids.