Does diet breadth affect the complexity of the phytophagous insect microbiota? The case study of Chrysomelidae.

Chrysomelidae is a family of phytophagous insects with a highly variable degree of trophic specialization. The aim of this study is to test whether species feeding on different plants (generalists) harbour more complex microbiotas than those feeding on a few or a single plant species (specialists). The microbiota of representative leaf beetle species was characterized with a metabarcoding approach targeting V1-V2 and V4 regions of the bacterial 16S rRNA. Almost all the analysed species harbour at least one reproductive manipulator bacteria (e.g., Wolbachia, Rickettsia). Two putative primary symbionts, previously isolated only from a single species (Bromius obscurus), have been detected in two species of the same subfamily, suggesting a widespread symbiosis in Eumolpinae. Surprisingly, the well-known aphid symbiont Buchnera is well represented in the microbiota of Orsodacne humeralis. Moreover, in this study, using Hill numbers to dissect the components of the microbiota diversity (abundant and rare bacteria), it has been demonstrated that generalist insect species harbour a more diversified microbiota than specialists. The higher microbiota diversity associated with a wider host-plant spectrum could be seen as an adaptive trait, conferring new metabolic potential useful to expand the diet breath, or as a result of environmental stochastic acquisition conveyed by diet.

Predator and parasitoid insects along elevational gradients: role of temperature and habitat diversity.

Elevational gradients are characterized by strong abiotic variation within small geographical distances and provide a powerful tool to evaluate community response to variation in climatic and other environmental factors. We explored how temperature and habitat diversity shape the diversity of holometabolous predator and parasitoid insects along temperate elevational gradients in the European Alps. We surveyed insect communities along 12 elevational transects that were selected to separate effects of temperature from those of habitat diversity. Pitfall traps and pan traps were placed every 100 m of elevation increment along the transects ranging from 120 to 2200 m a.s.l. Sampling took place once a month from June to September 2015. Four groups characterized by having at least one life stage behaving as predator or parasitoid were examined: tachinids (Diptera), hoverflies (Diptera), sphecids (Hymenoptera) and ground beetles (Coleoptera). Species richness and evenness changed with elevation, but the shape and direction of the elevation-diversity patterns varied between groups. The effect of temperature on species richness was positive for all groups except for hoverflies. Habitat diversity did not affect species richness, while it modulated the evenness of most groups. Often, elevational patterns of species richness and evenness were contrasting. Our study indicates that natural enemies characterized by diverse ecological requirements can be differentially affected by temperature and habitat diversity across the same elevational gradients. As climate warming is predicted to increase mean annual temperatures and exacerbate weather variability, it is also expected to strongly influence natural enemies and their ability to regulate herbivore populations.

Filling knowledge gaps in insect conservation by leveraging genetic data from public archives.

Insect decline has become a growing concern in recent years, with studies showing alarming declines in populations of several taxa. Our knowledge about genetic spatial patterns and evolutionary history of insects still exhibits significant gaps hindering our ability to effectively conserve and manage insect populations and species. Genetic data may provide valuable insights into the diversity and the evolutionary relationships of insects' species and populations. Public repositories, such as GenBank and BOLD, containing vast archives of genetic data with associated metadata, offer an irreplaceable resource for researchers contributing to our understanding of species diversity, population structure and evolutionary relationships. However, there are some issues in using these data, as they are often scattered and may lack accuracy due to inconsistent sampling protocols and incomplete information. In this paper we describe a curated georeferenced database of genetic data collected in GenBank and BOLD, for insects listed in the International Union for Conservation of Nature (IUCN) Italian Red Lists (dragonflies, bees, saproxylic beetles and butterflies). After querying these repositories, we performed quality control and data standardization steps. We created a dataset containing approximately 33 000 mitochondrial sequences and associated metadata about taxonomy, collection localities, geographic coordinates and IUCN Red List status for 1466 species across the four insect lists. We describe the current state of geographical metadata in queried repositories for species listed under different conservation status in the Italian Red Lists to quantify data gaps posing barriers to prioritization of conservation actions. Our curated dataset is available for data repurposing and analysis, enabling researchers to conduct comparative studies. We emphasize the importance of filling knowledge gaps in insect diversity and distribution and highlight the potential of this dataset for promoting other research fields like phylogeography, macrogenetics and conservation strategies. Our database can be downloaded through the Zenodo repository in SQL format. Database URL:  https://zenodo.org/records/8375181.

Paternity Analyses for the Planning of SIT Projects against the Red Palm Weevil.

The red palm weevil Rhynchophorus ferrugineus is an invasive pest from southeastern Asia and Melanesia that has spread widely across the Middle East and the Mediterranean Basin over the last 30 years. Its endophagous larvae cause huge amounts of damage to several palm tree species from the Arecaceae family. Many of these palms are economically important for agricultural and ornamental purposes. Therefore, a lot of attention has recently been focused on studying this species with the aim of identifying sustainable and effective eradication strategies. Sterile insect techniques are biological control strategies that are currently being investigated for their potential to eradicate this pest in selected invasion areas. Mating system features (e.g., polyandry and related features) can affect the success and suitability of these approaches. The main goal of this research was to assess the performance of a previously developed microsatellite panel in terms of the paternity assignment of progeny from laboratory mating experiments. Using a simulation approach, we evaluated the reliability of the microsatellite markers in the paternity tests both in complex laboratory experiment scenarios and on the progeny of wild-caught gravid females to help future studies on the RPW mating system. As a case study of the simulation results, we performed two double-mating experiments, genotyped the progeny and estimated the P2 values to compare to the expected progeny genotypes according to the crossing scheme of each experiment. The results of our simulations on laboratory experiments showed that it was possible to carry out paternity assignments for all progeny with reliable statistical confidence using our 13 microsatellites set. On the contrary the low genetic variability measured in red palm weevil populations in invaded areas made the resolution power of our loci too low to carry out paternity analyses on natural populations. Results of laboratory crossing were completely congruent with the expectations from the Mendelian laws.

Curation of a reference database of COI sequences for insect identification through DNA metabarcoding: COins.

DNA metabarcoding is a widespread approach for the molecular identification of organisms. While the associated wet-lab and data processing procedures are well established and highly efficient, the reference databases for taxonomic assignment can be implemented to improve the accuracy of identifications. Insects are among the organisms for which DNA-based identification is most commonly used; yet, a DNA-metabarcoding reference database specifically curated for their species identification using software requiring local databases is lacking. Here, we present COins, a database of 5' region cytochrome c oxidase subunit I sequences (COI-5P) of insects that includes over 532 000 representative sequences of >106 000 species specifically formatted for the QIIME2 software platform. Through a combination of automated and manually curated steps, we developed this database starting from all COI sequences available in the Barcode of Life Data System for insects, focusing on sequences that comply with several standards, including a species-level identification. COins was validated on previously published DNA-metabarcoding sequences data (bulk samples from Malaise traps) and its efficiency compared with other publicly available reference databases (not specific for insects). COins can allow an increase of up to 30% of species-level identifications and thus can represent a valuable resource for the taxonomic assignment of insects' DNA-metabarcoding data, especially when species-level identification is needed https://doi.org/10.6084/m9.figshare.19130465.v1.

Comparative analysis of sequences and secondary structures of the rRNA internal transcribed spacer 2 (ITS2) in pollen beetles of the subfamily Meligethinae (Coleoptera, Nitidulidae): potential use of slippage-derived sequences in molecular systematics.

A comparative analysis of ITS2 sequences and secondary structures in 89 species of pollen beetles of the subfamily Meligethinae (Coleoptera, Nitidulidae) was performed. The ITS2 folding pattern was highly conserved and comparable with the general model proposed for eukaryotes. Simple sequence repeats (SSRs) were responsible for most of the observed nucleotide variability (approximately 1-3%) and length variation (359-459bp). When plotted on secondary structures, SSRs mapped in expansion segments positioned at the apices of three ITS2 helices ('A', 'B' and 'D1') and appeared to have evolved under mechanisms of compensatory slippage. Homologies among SSRs nucleotides could not be unambiguously assigned, and thus were not useful to resolve phylogeny. However, slippage-derived motifs provided some preliminary genetic support for newly proposed taxonomic arrangements of several genera and subgenera of Meligethinae, corroborating existing morphological and ecological datasets.

Effects of the diet on the microbiota of the red palm weevil (Coleoptera: Dryophthoridae).

Rhynchophorus ferrugineus, also known as the red palm weevil, is regarded as the major pest of palm trees. Although studies of the microbiota associated with this species have been performed in recent years, little attention has been dedicated to the influence of the diet in shaping the host bacterial community. Here, we investigated the influence of food sources (i.e. palm tissues vs apple based substrate) on the microbial diversity associated with RPW, which was compared with the microbiota associated with wild individuals of the sister species Rhynchophorus vulneratus. The bacterial characterization was performed using a culture independent approach, i.e. the 16S rRNA pyrotag, and a culture dependent approach for a subset of the samples, in order to obtain bacterial isolates from RPW tissues. The bacterial community appeared significantly influenced by diet. Proteobacteria resulted to be the most abundant clade and was present in all the specimens of the three examined weevil groups. Within Proteobacteria, Enterobacteriaceae were identified in all the organs analysed, including hemolymph and reproductive organs. The apple-fed RPWs and the wild R. vulneratus showed a second dominant taxon within Firmicutes that was scarcely present in the microbiota associated with palm-fed RPWs. A comparative analysis on the bacteria associated with the palm tissues highlighted that 12 bacterial genera out of the 13 identified in the plant tissues were also present in weevils, thus indicating that palm tissues may present a source for bacterial acquisition.

Structure and evolution of the mitochondrial control region of the pollen beetle Meligethes thalassophilus (Coleoptera: Nitidulidae).

The organization of the mitochondrial DNA (mtDNA) control region (CR) of the pollen beetle Meligethes thalassophilus is described. This mtDNA CR represents the longest sequenced for beetles so far, since the entire nucleotide sequence ranges from approximately 5000 to approximately 5500 bp. The CR of M. thalassophilus is organized in three distinct domains: a conserved domain near the tRNAIle gene, a variable domain flanking the 12S rRNA gene, and a relatively large central tandem array made up of a variable number of approximately 170 bp repeats that is responsible for the intraspecific length variation observed. Like other CRs found in insects, the M. thalassophilus CR contains two long homopolymeric runs that may be involved in mtDNA replication. Furthermore, conserved stem-and-loop structures in the repetitive domain were identified and their possible role in generating length variation is examined. Intraspecific comparison of the tandem repeat elements of M. thalassophilus suggests mechanisms of concerted evolution leading to homogenization of the repetitive region. The utility of such an array of tandem repeats as a genetic marker for assessing population-level variability and evolutionary relationships among populations is discussed. Finally, the technical difficulties found in isolating the mtDNA CR in beetles are remarked upon.