Rediscovery of a lost semi-aquatic Leaf Beetle in the Hula Valley, Israel (Coleoptera, Chrysomelidae, Donaciinae).

Between 1951-1958, most of the Hula Lake and its surrounding swamps in the Upper Jordan River (Rift) Valley of Israel were drained with the supposed purposes to eliminate malaria and to reclaim land for agriculture; both reasons later proved to be unnecessary decisions. With the paucity of biological knowledge of the Hula region, especially its aquatic invertebrates, accurate assessment of the environmental damage from this drainage is still being realized. Based on natural history museum collection specimen records, the pre-drainage presence of some aquatic insect species has been verified. Among these was Donaciabicolora, a member of a semi-aquatic subfamily (Donaciinae) of Leaf Beetles (Chrysomelidae) and whose Israeli populations were thought to have gone extinct because of the drainage of the Hula and other locations. Recently this species was rediscovered in two populations. However, the molecular identification of two of these recently collected specimens from one population revealed that the identity of this species is actually Donaciasimplex. In this work, the re-discovery of this species is detailed, and its conservation importance discussed.

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota.

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems.

European and Mediterranean Myzocallidini Aphid Species: DNA Barcoding and Remarks on Ecology with Taxonomic Modifications in An Integrated Framework.

The genus Myzocallis Passerini (Hemiptera, Aphididae, Calaphidinae, Myzocallidini) is a rather primitive group of aphids currently comprising 45 species and 3 subspecies, subdivided into ten subgenera, three of them having a West Palaearctic distribution. The majority of the species inhabit Fagales plants and some of them are considered pests. Despite their ecological interest and the presence of some taxonomic controversies, there are only a few molecular studies on the group. Here, the main aims were to develop a DNA barcodes library for the molecular identification of West Palaearctic Myzocallis species, to evaluate the congruence among their morphological, ecological and DNA-based delimitation, and verify the congruence of the subgeneric subdivision presently adopted by comparing the results with those obtained for other Panaphidini species. These study findings indicate that Myzocallis (Agrioaphis) leclanti, originally described as a subspecies of M. (A.) castanicola and M. (M.) schreiberi, considered as a subspecies of M. (M.) boerneri, should be regarded at a rank of full species, and the subgenera Agrioaphis, Lineomyzocallis, Neomyzocallis, Pasekia were elevated to the rank of genus, while Myzocallis remain as such.

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.

Unsupervised machine learning and geometric morphometrics as tools for the identification of inter and intraspecific variations in the Anopheles Maculipennis complex.

Geometric morphometric analysis was combined with two different unsupervised machine learning algorithms, UMAP and HDBSCAN, to visualize morphological differences in wing shape among and within four Anopheles sibling species (An. atroparvus, An. melanoon, An. maculipennis s.s. and An. daciae sp. inq.) of the Maculipennis complex in Northern Italy. Specifically, we evaluated: (1) wing shape variation among and within species; (2) the consistencies between groups of An. maculipennis s.s. and An. daciae sp. inq. identified based on COI sequences and wing shape variability; and (3) the spatial and temporal distribution of different morphotypes. UMAP detected at least 13 main patterns of variation in wing shape among the four analyzed species and mapped intraspecific morphological variations. The relationship between the most abundant COI haplotypes of An. daciae sp. inq. and shape ordination/variation was not significant. However, morphological variation within haplotypes was reported. HDBSCAN also recognized different clusters of morphotypes within An. daciae sp. inq. (12) and An. maculipennis s.s. (4). All morphotypes shared a similar pattern of variation in the subcostal vein, in the anal vein and in the radio-medial cross-vein of the wing. On the contrary, the marginal part of the wings remained unchanged in all clusters of both species. Any spatial-temporal significant difference was observed in the frequency of the identified morphotypes.  Our study demonstrated that machine learning algorithms are a useful tool combined with geometric morphometrics and suggest to deepen the analysis of inter and intra specific shape variability to evaluate evolutionary constrains related to wing functionality.

Disentangling phylogenetic relations and biogeographic history within the Cucujus haematodes species group (Coleoptera: Cucujidae).

Recent progress in the taxonomy of flat bark beetles (Cucujidae), specifically, in the genus Cucujus, has revealed great diversity in subtropical Asia, but the seemingly well-known temperate and boreal taxa need further attention because of their conservation status. Here, we used an integrative approach using morphology, DNA, and species distribution modelling to disentangle phylogenetic relations, verify the number of species, and understand the historical biogeography of Palearctic and Nearctic Cucujus beetles, particularly the C. haematodes species group. Species distinctiveness was supported for C. cinnaberinus, but present-day C. haematodes turned out to be a species complex made up of separate lineages in the western, middle and eastern parts of its Palearctic range. Cucujus muelleri was a member of that complex, being sister to Asian C. haematodes. Moreover, C. haematodes caucasicus was found to be phylogenetically closely related to Italian C. tulliae, and both to be sister to European C. haematodes. North American C. clavipes clavipes and C. c. puniceus resulted to be enough divergent to be considered different species. Interestingly, western American C. puniceus turned out to be closely related to the C. haematodes complex, whereas eastern American C. clavipes constituted a separate lineage, being distantly related to both C. puniceus and C. cinnaberinus. These patterns suggest former trans-continental connections among the ancestors of extant flat bark beetle species. Moreover, a divergent lineage of C. cinnaberinus was found in Calabria, which should be regarded at the very least as a subspecies. The ancestor of C. hameatodes group originated in mid-Miocene, and next, ca. 6.2 Mya, a line leading to C. cinnaberinus had split. Speciation of the American lineages occurred during Pliocene (4.4 Mya for C. clavipes and 3.3 Mya for C. puniceus). Species classified as C. haematodes, C. tulliae and C. muelleri, as well as distinct lineages within C. cinnaberinus split during mid Pleistocene (ca. 1.5 Mya). A comparison of species climatic requirements and their present distribution allowed to identify glacial refugia in south-eastern areas of North America (C. clavipes), south-western areas of North America (C. puniceus), and the Mediterranean and Caspian Sea Basins (European Cucujus species), or south-eastern areas of Asia and the foothills of the central Asian mountains (eastern C. haematodes). Subsequent climatic changes in the Holocene forced these beetles to move their ranges northwards along the coasts of the Pacific (C. puniceus) or Atlantic (C. clavipes), north-eastwards to central, northern, and eastern Europe (C. cinnaberinus and European C. haematodes) or Siberia (Asian C. haematodes). The combined use of molecular, morphological and climatic data allows a comprehensive understanding of the phylogenetic relations and past distributions of Cucujus beetles, highlighting the complexity of C. haematodes species group evolution.

Prevention and control of OQDS (olive quick decline syndrome) outbreaks caused by Xylella fastidiosa.

In Southern Italy, since 2013, there has been an ongoing Olive Quick Decline Syndrome (OQDS) outbreak, due to the bacterium Xylella fastidiosa, which has caused a dramatic impact from both socio-economic and environmental points of view. The main players involved in OQDS are represented by the insect vector, Philaenus spumarius, its host plants (olive trees and weeds) and the bacterium, X. fastidiosa. Current agronomic practices are mainly based on uprooting the sick olive trees and their surrounding ones, with later installment of olive cultivars more resistant to the bacterium infection. Unfortunately, both of these practices are having an undesirable impact on the environment (most of these olive trees were monumental ones) and on the economy. Based on a mathematical model expressed in terms of a nontrivial system of ordinary differential equations, our analysis has provided a clear picture of all possible steady states (feasible equilibria) and their stability properties, corresponding to a variety of different parameter scenarios; all of this has been illustrated by a set of computational experiments. A significant original contribution of this paper is the proof of the global asymptotic stability of each of the feasible equilibria under its existence assumptions, a fact that excludes multiple equilibria under the given conditions. It has emerged that the removal of a suitable amount of weed biomass (host plants of the juvenile stages of the insect vector of X. fastidiosa) from olive orchards and surrounding areas leads to the eradication of the epidemic, without requiring neither the removal nor the substitution of the existing olive trees.

Pathogenic potential of bacteria isolated from commercial biostimulants.

Microbial-based products are a promising alternative to agrochemicals in sustainable agriculture. However, little is known about their impact on human health even if some of them, i.e., Bacillus and Paenibacillus species, have been increasingly implicated in different human diseases. In this study, 18 bacteria were isolated from 2 commercial biostimulants, and they were genotypically and phenotypically characterized to highlight specific virulence properties. Some isolated bacteria were identified as belonging to the genus Bacillus by BLAST and RDP analyses, a genus in-depth studied for plant growth-promoting ability. Moreover, 16S rRNA phylogenetic analysis showed that seven isolates grouped with Bacillus species while two and four clustered, respectively, with Neobacillus and Peribacillus. Unusually, bacterial strains belonging to Franconibacter and Stenotrophomonas were isolated from biostimulants. Although Bacillus species are generally considered nonpathogenic, most of the species have shown to swim, swarm, and produced biofilms, that can be related to bacterial virulence. The evaluation of toxins encoding genes revealed that five isolates had the potential ability to produce the enterotoxin T. In conclusion, the pathogenic potential of microorganisms included in commercial products should be deeply verified, in our opinion. The approach proposed in this study could help in this crucial step.

Corrections and Additions to Descriptions of Some Species of the Subgenus Orthocladius s. str. (Diptera, Chironomidae, Orthocladiinae).

The larvae of some species of the subgenus Orthocladius s. str. (Diptera, Chironomidae) are here described for the first time with corrections and additions to the descriptions of adult males and pupal exuviae. The identification of larvae is generally not possible without association with pupal exuviae and/or adult males, so the descriptions here are based only on reared material or on pupae with the associated larval exuviae. Usually, Chironomidae larvae can be separated on the basis of morphometric characters, the most discriminant ones are: (1) the ratio between the width of median tooth of mentum (Dm) and the width of the first lateral tooth (Dl) = mental ratio (DmDl), (2) the ratio between the length of the first antennal segment (A1) and the combined length of segments 2-5 (A2-5) = antennal ratio (AR). The shape of mandible, maxilla, and other body parts are almost identical in all the species considered in this study. The larva of Orthocladius (Symposiocladius) lignicola is very characteristic and can be separated by the shape of mentum and the larvae of all the known species of Symposiocladius are characterized by the presence of large Lauterborn organs on antennae and of tufts of setae on abdominal segments. The larvae of Orthocladius (Orthocladius) oblidens and Orthocladius (Orthocladius) rhyacobius can be distinguished from other species basing on their large Dm and to each other by AR. A principal component analysis was carried out using 5 characters: (1) Dm, (2) Dl, (3) length of A1, (4) width of A1 (A1W), (5) combined length of segments 2-5 (A2-5). The most discriminant characters were Dm and A1, confirming that DmDl and AR can be used to separate species at larval stage, but the large superposition of morphometric characters in different species confirms that association with pupal exuviae is in any case needed to identify larvae. In future perspective, the development of reference DNA barcodes from specimens identified by specialists is recommended since possibly the best tool for larvae identification, but association of barcodes with morphotypes is in any case fundamental.

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.

Complete Genome Sequence of Rhynchophorus ferrugineus Endocytobiont "Candidatus Nardonella dryophthoridicola" Strain NardRF.

We report the complete genome sequence and annotation of "Candidatus Nardonella dryophthoridicola" strain NardRF, obtained by sequencing its host bacteriome, Rhynchophorus ferrugineus, using Oxford Nanopore technology.

Multi-country investigation of the diversity and associated microorganisms isolated from tick species from domestic animals, wildlife and vegetation in selected african countries.

In many areas of Africa, recent studies highlighted the great impact of ticks on animal and human health throughout the continent. On the other hand, very limited information on the bacterial endosymbionts of the African ticks and their pattern of co-infections with other bacteria are found in literature, notwithstanding their pivotal role in tick survival and vector efficiency. Thus, we investigated the distribution of selected pathogenic and symbiotic bacteria in hard ticks collected from wild, domestic animals and from vegetation in various ecological zones in Africa and their co-occurrence in the same tick host. Overall, 339 hard ticks were morphologically identified as belonging to the genera Amblyomma, Dermacentor, Hyalomma, Haemaphysalis, Ixodes and Rhipicephalus. Molecular screening provided information on pathogens circulation in Africa, detecting spotted fever group rickettsiae, Anaplasma spp., Ehrlichia ruminantium, Borrelia garinii, Babesia spp., Theileria spp. and Coxiella burnetii. Furthermore, our work provides insights on the African scenario of tick-symbiont associations, revealing the presence of Coxiella, Francisella and Midichloria across multiple tick populations. Coxiella endosymbionts were the most prevalent microorganisms, and that with the broadest spectrum of hosts, being detected in 16 tick species. Francisella was highly prevalent among the Hyalomma species tested and correlated negatively with the presence of Coxiella, showing a potential competitive interaction. Interestingly, we detected a positive association of Francisella with Rickettsia in specimens of Hy. rufipes, suggesting a synergistic interaction between them. Finally, Midichloria was the most prevalent symbiont in Rhipicephalus sanguineus sensu lato from Egypt.

Factors affecting the efficiency of molecular species delimitation in a species-rich insect family.

In the context of global biodiversity loss, molecular species delimitation approaches can be very useful for accelerating species discovery through DNA taxonomy and inventory through DNA metabarcoding. In this study, the effect of some intrinsic factors on the efficiency of various single-marker species delimitation methods (fixed and variable nucleotide distance thresholds, ABGD, ASAP, GMYC, mPTP) was tested on more than 90 empirical data sets, derived from a set of 7,237 COI sequences attributed to 542 leaf beetles species (Coleoptera: Chrysomelidae). The considered factors were: (i) the number of haplotypes per species (as a proxy for genetic diversity), (ii) the geographic distance among conspecific collection localities (as a proxy of sampling width), (iii) the difficulty related to morphological identification of species, and (iv) the taxonomic rank. Distance-based methods, with on average more than 70% of match with morphological identification, outperformed those relying on phylogenetic trees, with less than 59%. A high number of haplotypes per species was found to have a negative effect on delimitation efficiency, whereas large geographic distances within species had a positive effect. All methods delimitations (except for GMYC) were significantly affected by the presence of species that are difficult to be identified, decreasing their efficiency. Finally, the only method influenced by the taxonomic rank of the data set was GMYC, showing lower efficiency in data sets at the genus than at higher levels. The observed biases we highlighted affecting efficiency could be accounted for when developing input data sets for species delimitation analyses to obtain a more reliable representation of biological diversity.

Comment on Phylogenetic analyses with four new Cretaceous bristletails reveal inter-relationships of Archaeognatha and Gondwana origin of Meinertellidae.

Archaeognatha (Machilida plus †Monura), with ~450 extant species worldwide, represent a key lineage for the understanding of insect evolution. In a recently published phylogenetic analysis on Archaeognatha, based on a total-evidence approach, a Machilidae key fossil (Gigamachilis triassicus) from the Middle Triassic Lagerstätte of Monte San Giorgio and a representative of †Monura (Dasyleptus triassicus) from the same fossiliferous site, were not considered. In the present study, the phylogenetic analyses performed by Zhang et al. (2018) using maximum parsimony and Bayesian inference were repeated including these two key taxa; in addition, Tricholepidion gertschi and Lepisma saccharina were included as representatives of Zygentoma. In the total-evidence Bayesian analysis, †Monura was found to be paraphyletic, whereas in the maximum parsimony analysis, performed only on morphological characters, they were monophyletic. Moreover, the attribution of G. triassicus to Machilidae is now confirmed by these analyses, indicating that members of the extant group of Machilidae were already present in the Middle Triassic, at least ~100 Myr before the previous estimate.

Recalibration of the insect evolutionary time scale using Monte San Giorgio fossils suggests survival of key lineages through the End-Permian Extinction.

Insects are a highly diverse group of organisms and constitute more than half of all known animal species. They have evolved an extraordinary range of traits, from flight and complete metamorphosis to complex polyphenisms and advanced eusociality. Although the rich insect fossil record has helped to chart the appearance of many phenotypic innovations, data are scarce for a number of key periods. One such period is that following the End-Permian Extinction, recognized as the most catastrophic of all extinction events. We recently discovered several 240-million-year-old insect fossils in the Mount San Giorgio Lagerstätte (Switzerland-Italy) that are remarkable for their state of preservation (including internal organs and soft tissues), and because they extend the records of their respective taxa by up to 200 million years. By using these fossils as calibrations in a phylogenomic dating analysis, we present a revised time scale for insect evolution. Our date estimates for several major lineages, including the hyperdiverse crown groups of Lepidoptera, Hemiptera: Heteroptera and Diptera, are substantially older than their currently accepted post-Permian origins. We found that major evolutionary innovations, including flight and metamorphosis, appeared considerably earlier than previously thought. These results have numerous implications for understanding the evolution of insects and their resilience in the face of extreme events such as the End-Permian Extinction.

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae).

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.

Phylogeography and species distribution modelling of Cryptocephalusbarii (Coleoptera: Chrysomelidae): is this alpine endemic species close to extinction?

The alternation of glacial and interglacial cycles of the Quaternary period contributed in shaping the current species distribution. Cold-adapted organisms experienced range expansion and contraction in response to the temperature decrease and increase, respectively. In this study, a fragment of the mitochondrial marker COI was used to investigate the phylogeography of Cryptocephalusbarii, a cold-adapted alpine leaf beetle species endemic of Orobie Alps, northern Italy. The relationships among populations, their divergence time, and the most probable migration model were estimated and are discussed in light of the Pleistocene climate oscillations. Through a species distribution modelling analysis, the current habitat suitability was assessed and the distribution in a future global warming scenario predicted. The main divergence events that led to the actual population structure took place from ~750,000 to ~150,000 years ago, almost following the pattern of the climate oscillations that led to the increase of the connections between the populations during cold periods and the isolation on massifs in warm periods. The most supported migration model suggests that the species survived to past adverse climatic conditions within refugia inside and at the limit of the actual range. The species distribution modelling analysis showed that C.barii is extremely sensitive to air temperature variations, thus the increase of temperature caused by global warming will reduce the suitable areas within the species range, leading to its possible extinction in the next 50 years. Cryptocephalusbarii is a representative case of how cold adapted and limited distributed species have been and could be affected by climate change, that highlights the implementation of conservation actions.

Green Technology: Bacteria-Based Approach Could Lead to Unsuspected Microbe⁻Plant⁻Animal Interactions.

The recent and massive revival of green strategies to control plant diseases, mainly as a consequence of the Integrated Pest Management (IPM) rules issued in 2009 by the European Community and the increased consumer awareness of organic products, poses new challenges for human health and food security that need to be addressed in the near future. One of the most important green technologies is biocontrol. This approach is based on living organisms and how these biocontrol agents (BCAs) directly or indirectly interact as a community to control plant pathogens and pest. Although most BCAs have been isolated from plant microbiomes, they share some genomic features, virulence factors, and trans-kingdom infection abilities with human pathogenic microorganisms, thus, their potential impact on human health should be addressed. This evidence, in combination with the outbreaks of human infections associated with consumption of raw fruits and vegetables, opens new questions regarding the role of plants in the human pathogen infection cycle. Moreover, whether BCAs could alter the endophytic bacterial community, thereby leading to the development of new potential human pathogens, is still unclear. In this review, all these issues are debated, highlighting that the research on BCAs and their formulation should include these possible long-lasting consequences of their massive spread in the environment.

Thermotolerant isolates of Beauveria bassiana as potential control agent of insect pest in subtropical climates.

The use of Beauveria bassiana in biological control of agricultural pests is mainly hampered by environmental factors, such as elevated temperatures and low humidity. These limitations, further amplified in a global warming scenario, could nullify biological control strategies based on this fungus. The identification of thermotolerant B. bassiana isolates represents a possible strategy to overcome this problem. In this study, in order to maximize the probability in the isolation of thermotolerant B. bassiana, soil samples and infected insects were collected in warm areas of Syria. The obtained fungal isolates were tested for different biological parameters (i.e., growth rate, sporulation and spore germination) at growing temperatures ranging from 20°C to 35°C. Among these isolates (eight from insects and 11 from soil samples), the five with the highest growth rate, spore production and germination at 30°C were tested for their entomopathogenicity through in vivo assays on Ephestia kuehniella larvae. Insect mortality induced by the five isolates ranged from 31% to 100%. Two isolates, one from Phyllognathus excavatus and one from soil, caused 50% of the larval mortality in less than four days, reaching values exceeding 92% in ten days. These two isolates were molecularly identified as B. bassiana sensu stricto by using three markers (i.e., ITS, Bloc and EF1-α). Considering these promising results, further studies are ongoing, testing their efficiency in field conditions as control agents for agricultural insect pests in Mediterranean and Subtropical regions.

Evidence for a conserved microbiota across the different developmental stages of Plodia interpunctella.

Diversity and composition of lepidopteran microbiotas are poorly investigated, especially across the different developmental stages. To improve this knowledge, we characterize the microbiota among different developmental stages of the Indian meal moth, Plodia interpunctella, which is considered one of the major pest of commodities worldwide. Using culture-independent approach based on Illumina 16S rRNA gene sequencing we characterized the microbiota of four developmental stages: eggs, first-, and last-instar larvae, and adult. A total of 1022 bacterial OTUs were obtained, showing a quite diversified microbiota associated to all the analyzed stages. The microbiotas associated with P. interpunctella resulted almost constant throughout the developmental stages, with approximately 77% of bacterial OTUs belonging to the phylum of Proteobacteria. The dominant bacterial genus is represented by Burkholderia (∼64%), followed by Propionibacterium, Delftia, Pseudomonas, and Stenotrophomonas. A core bacterial community, composed of 139 OTUs, was detected in all the developmental stages, among which 112 OTUs were assigned to the genus Burkholderia. A phylogenetic reconstruction, based on the 16S rRNA, revealed that our Burkholderia OTUs clustered with Burkholderia cepacia complex, in the same group of those isolated from the hemipterans Gossyparia spuria and Acanthococcus aceris. The functional profiling, predicted on the base of the bacterial 16S rRNA, indicates differences in the metabolic pathways related to metabolism of amino acids between preimaginal and adult stages. We can hypothesize that bacteria may support the insect host during preimaginal stages.