Microbial associates of the elm leaf beetle: uncovering the absence of resident bacteria and the influence of fungi on insect performance.

Microbial symbionts play crucial roles in the biology of many insects. While bacteria have been the primary focus of research on insect-microbe symbiosis, recent studies suggest that fungal symbionts may be just as important. The elm leaf beetle (ELB, Xanthogaleruca luteola) is a serious pest species of field elm (Ulmus minor). Using culture-dependent and independent methods, we investigated the abundance and species richness of bacteria and fungi throughout various ELB life stages and generations, while concurrently analyzing microbial communities on elm leaves. No persistent bacterial community was found to be associated with the ELB or elm leaves. By contrast, fungi were persistently present in the beetle's feeding life stages and on elm leaves. Fungal community sequencing revealed a predominance of the genera Penicillium and Aspergillus in insects and on leaves. Culture-dependent surveys showed a high prevalence of two fungal colony morphotypes closely related to Penicillium lanosocoeruleum and Aspergillus flavus. Among these, the Penicillium morphotype was significantly more abundant on feeding-damaged compared with intact leaves, suggesting that the fungus thrives in the presence of the ELB. We assessed whether the detected prevalent fungal morphotypes influenced ELB's performance by rearing insects on (i) surface-sterilized leaves, (ii) leaves inoculated with Penicillium spores, and (iii) leaves inoculated with Aspergillus spores. Insects feeding on Penicillium-inoculated leaves gained more biomass and tended to lay larger egg clutches than those consuming surface-sterilized leaves or Aspergillus-inoculated leaves. Our results demonstrate that the ELB does not harbor resident bacteria and that it might benefit from associating with Penicillium fungi.IMPORTANCEOur study provides insights into the still understudied role of microbial symbionts in the biology of the elm leaf beetle (ELB), a major pest of elms. Contrary to expectations, we found no persistent bacterial symbionts associated with the ELB or elm leaves. Our research thus contributes to the growing body of knowledge that not all insects rely on bacterial symbionts. While no persistent bacterial symbionts were detectable in the ELB and elm leaf samples, our analyses revealed the persistent presence of fungi, particularly Penicillium and Aspergillus on both elm leaves and in the feeding ELB stages. Moreover, when ELB were fed with fungus-treated elm leaves, we detected a potentially beneficial effect of Penicillium on the ELB's development and fecundity. Our results highlight the significance of fungal symbionts in the biology of this insect.

Whole-genome resequencing improves the utility of otoliths as a critical source of DNA for fish stock research and monitoring.

Fish ear bones, known as otoliths, are often collected in fisheries to assist in management, and are a common sample type in museum and national archives. Beyond their utility for ageing, morphological and trace element analysis, otoliths are a repository of valuable genomic information. Previous work has shown that DNA can be extracted from the trace quantities of tissue remaining on the surface of otoliths, despite the fact that they are often stored dry at room temperature. However, much of this work has used reduced representation sequencing methods in clean lab conditions, to achieve adequate yields of DNA, libraries and ultimately single-nucleotide polymorphisms (SNPs). Here, we pioneer the use of small-scale (spike-in) sequencing to screen contemporary otolith samples prepared in regular molecular biology (in contrast to clean) laboratories for contamination and quality levels, submitting for whole-genome resequencing only samples above a defined endogenous DNA threshold. Despite the typically low quality and quantity of DNA extracted from otoliths, we are able to produce whole-genome libraries and ultimately sets of filtered, unlinked and even putatively adaptive SNPs of ample numbers for downstream uses in population, climate and conservation genomics. By comparing with a set of tissue samples from the same species, we are able to highlight the quality and efficacy of otolith samples from DNA extraction and library preparation, to bioinformatic preprocessing and SNP calling. We provide detailed schematics, protocols and scripts of our approach, such that it can be adopted widely by the community, improving the use of otoliths as a source of valuable genomic data.

Environmental DNA, hydrochemistry and stable water isotopes as integrative tracers of urban ecohydrology.

Urbanization and the persistent environmental changes present a major challenge for urban freshwaters and availability of water for humans and wildlife. In order to increase understanding of urban ecohydrology, we investigated the variability of planktonic bacteria and benthic diatoms - as two key biological indicators - coupled with insights from hydrochemistry and stable water isotopes across four urban streams characterized by different dominant water sources in Berlin, the German capital, over a period of one year (2021-2022). DNA metabarcoding results show that substantial spatio-temporal variability exists across urban streams in terms of microbial diversity and richness, with clear links to abiotic factors and nutrient concentrations. Bacterial communities showed clear distinction between effluent-impacted and non-effluent impacted streams as well as clear seasonal turnover. In-stream benthic diatom assemblages also showed robust seasonal variation as well as high species diversity. Our multiple-tracer approach is relevant for emerging questions regarding the increased use of treated effluent to supplement declining baseflows, the assessment of stream restoration projects and the impact of storm drainage and surface pollution on aquatic ecosystem health. eDNA analysis allows analysis of spatial and temporal patterns not feasibly studied with traditional analyses of macroinvertebrates. This can ultimately be leveraged for future water resource management and restoration planning and monitoring of urban freshwater systems across metropolitan areas.

Scaling-up RADseq methods for large datasets of non-invasive samples: Lessons for library construction and data preprocessing.

Genetic non-invasive sampling (gNIS) is a critical tool for population genetics studies, supporting conservation efforts while imposing minimal impacts on wildlife. However, gNIS often presents variable levels of DNA degradation and non-endogenous contamination, which can incur considerable processing costs. Furthermore, the use of restriction-site-associated DNA sequencing methods (RADseq) for assessing thousands of genetic markers introduces the challenge of obtaining large sets of shared loci with similar coverage across multiple individuals. Here, we present an approach to handling large-scale gNIS-based datasets using data from the spotted hyena population inhabiting the Ngorongoro Crater in Tanzania. We generated 3RADseq data for more than a thousand individuals, mostly from faecal mucus samples collected non-invasively and varying in DNA degradation and contamination level. Using small-scale sequencing, we screened samples for endogenous DNA content, removed highly contaminated samples, confirmed overlap fragment length between libraries, and balanced individual representation in a sequencing pool. We evaluated the impact of (1) DNA degradation and contamination of non-invasive samples, (2) PCR duplicates and (3) different SNP filters on genotype accuracy based on Mendelian error estimated for parent-offspring trio datasets. Our results showed that when balanced for sequencing depth, contaminated samples presented similar genotype error rates to those of non-contaminated samples. We also showed that PCR duplicates and different SNP filters impact genotype accuracy. In summary, we showed the potential of using gNIS for large-scale genetic monitoring based on SNPs and demonstrated how to improve control over library preparation by using a weighted re-pooling strategy that considers the endogenous DNA content.

Persisting roadblocks in arthropod monitoring using non-destructive metabarcoding from collection media of passive traps.

Background: Broad-scale monitoring of arthropods is often carried out with passive traps (e.g., Malaise traps) that can collect thousands of specimens per sample. The identification of individual specimens requires time and taxonomic expertise, limiting the geographical and temporal scale of research and monitoring studies. DNA metabarcoding of bulk-sample homogenates has been found to be faster, efficient and reliable, but the destruction of samples prevents a posteriori validation of species occurrences and relative abundances. Non-destructive metabarcoding of DNA extracted from collection medium has been applied in a limited number of studies, but further tests of efficiency are required with different trap types and collection media to assess the consistency of the method. Methods: We quantified the detection rate of arthropod species when applying non-destructive DNA metabarcoding with a short (127-bp) fragment of mitochondrial COI on two combinations of passive traps and collection media: (1) water with monopropylene glycol (H2O-MPG) used in window-flight traps (WFT, 53 in total); (2) ethanol with monopropylene glycol (EtOH-MPG) used in Malaise traps (MT, 27 in total). We then compared our results with those obtained for the same samples using morphological identification (for WFTs) or destructive metabarcoding of bulk homogenate (for MTs). This comparison was applied as part of a larger study of arthropod species richness in silver fir (Abies alba Mill., 1759) stands across a range of climate-induced tree dieback levels and forest management strategies. Results: Of the 53 H2O-MPG samples from WFTs, 16 produced no metabarcoding results, while the remaining 37 samples yielded 77 arthropod MOTUs in total, of which none matched any of the 343 beetle species morphologically identified from the same traps. Metabarcoding of 26 EtOH-MPG samples from MTs detected more arthropod MOTUs (233) than destructive metabarcoding of homogenate (146 MOTUs, 8 orders), of which 71 were shared MOTUs, though MOTU richness per trap was similar between treatments. While we acknowledge the failure of metabarcoding from WFT-derived collection medium (H2O-MPG), the treatment of EtOH-based Malaise trapping medium remains promising. We conclude however that DNA metabarcoding from collection medium still requires further methodological developments and cannot replace homogenate metabarcoding as an approach for arthropod monitoring. It can be used nonetheless as a complementary treatment when enhancing the detection of soft-bodied arthropods like spiders and Diptera.

Comparative morphology and molecular phylogeny of Apicoporus n. Gen.: a new genus of marine benthic dinoflagellates formerly classified within Amphidinium.

The composition of the dinoflagellate genus Amphidinium is currently polyphyletic and includes several species in need of re-evaluation using modern morphological and phylogenetic methods. We investigated a broad range of uncultured morphotypes extracted from marine sediments in the Eastern Pacific Ocean that were similar in morphology to Amphidinium glabrum Hoppenrath and Okolodkov. To determine the number of distinct species associated with this phenotypic diversity, we collected LM, SEM, TEM and small subunit ribosomal DNA sequence information from different morphotypes, including the previously described A. glabrum. Both comparative morphological and molecular phylogenetic data supported the establishment of a new genus, Apicoporus n. gen., including at least two species, A. glaber n. comb., and A. parvidiaboli n. sp. Apicoporus is characterized by having amphiesmal pores and an apical pore covered by a hook-like protrusion; neither of these characters has been observed in other athecate dinoflagellates. The posterior end of Apicoporus parvidiaboli possessed varying degrees of "horn formation", ranging from slight to prominent. By contrast, the posterior end of Apicoporus glaber was distinctively rounded and lacked evidence of horn formation. Although these species were previously interpreted to be obligate heterotrophs, TEM and epifluorescence microscopy demonstrated that some cells of both species had unusually small but otherwise typical dinoflagellate plastids. The number and density of plastids in any particular cell varied significantly in the genus, but the plastids were almost always concentrated at the posterior end of the cells or around the nucleus. The presence of cryptic photosynthetic plastids in these benthic species suggests that photosynthesis might be much more widespread in dinoflagellates than is currently assumed.

MORPHOLOGY AND MOLECULAR PHYLOGENY OF ANKISTRODINIUM GEN. NOV. (DINOPHYCEAE), A NEW GENUS OF MARINE SAND-DWELLING DINOFLAGELLATES FORMERLY CLASSIFIED WITHIN AMPHIDINIUM(1).

The classical athecate dinoflagellate genera (Amphidinium, Gymnodinium, Gyrodinium) have long been recognized to be polyphyletic. Amphidinium sensu lato is the most diverse of all marine benthic dinoflagellate genera; however, following the redefinition of this genus ∼100 species remain now of uncertain or unknown generic affiliation. In an effort to improve our taxonomic and phylogenetic understanding of one of these species, namely Amphidinium semilunatum, we re-investigated organisms from several distant sites around the world using light and scanning electron microscopy and molecular phylogenetic methods. Our results enabled us to describe this species within a new heterotrophic genus, Ankistrodinium. Cells of A. semilunatum were strongly laterally flattened, rounded-quadrangular to oval in lateral view, and possessed a small asymmetrical epicone. The sulcus was wide and characteristically deeply incised on the hypocone running around the antapex and reaching the dorsal side. The straight acrobase with hook-shaped end started at the sulcal extension and continued onto the epicone. The molecular phylogenetic results clearly showed that A. semilunatum is a distinct taxon and is only distantly related to species within the genus Amphidinium sensu stricto. The nearest sister group to Ankistrodinium could not be reliably determined.