Lagria Villosa Research Articles

Bacterial ectosymbionts in cuticular organs chemically protect a beetle during molting stages

In invertebrates, the cuticle is the first and major protective barrier against predators and pathogen infections. While immune responses and behavioral defenses are also known to be important for insect protection, the potential of cuticle-associated microbial symbionts to aid in preventing pathogen entry during molting and throughout larval development remains unexplored. Here, we show that bacterial symbionts of the beetle Lagria villosa inhabit unusual dorsal invaginations of the insect cuticle, which remain open to the outer surface and persist throughout larval development. This specialized location enables the release of several symbiont cells and the associated protective compounds during molting. This facilitates ectosymbiont maintenance and extended defense during larval development against antagonistic fungi. One Burkholderia strain, which produces the antifungal compound lagriamide, dominates the community across all life stages, and removal of the community significantly impairs the survival probability of young larvae when exposed to different pathogenic fungi. We localize both the dominant bacterial strain and lagriamide on the surface of eggs, larvae, pupae, and on the inner surface of the molted cuticle (exuvia), supporting extended protection. These results highlight adaptations for effective defense of immature insects by cuticle-associated ectosymbionts, a potentially key advantage for a ground-dwelling insect when confronting pathogenic microbes.

Comparative efficacy of two plant extracts and cypermethrin against field insect pests of Amaranthus hybridus L.

Abstract. Insect infestations are major constraints to vegetable production in Nigeria causing low yields and poor quality of produce. A study was conducted at the experimental farm of the National Horticultural Research Institute (NIHORT) Ibadan, Nigeria, to compare the efficacy of ethanol seed extracts of Azadirachta indica A.Juss (neem), Annona muricata L(soursop) and cypermethrin against field insect pests of Amaranthus hybridus L. under rain fed conditions. Four major insect species were observed causing damage to A. hybridus during the study and they include: cotton bollworm (Heliothis armigera Hubner), okra leaf roller (Sylepta derogata Fabricius), lagriid beetle (Lagria villosa Fabricius) and variegated grasshopper (Zonocerus variegatus Linnaeus). Ethanol seed extracts of A. indica were as effective as cypermethrin and more effective than A. muricata seed extracts in protecting A. hybridus against insect damage. Plots treated with A. indica extracts and cypermethrin significantly (p<0.05) reduced leaf damage, observed insect density and enhanced leaf yield compared to plots treated with A. muricata and control. There was no significant difference (p>0.05) between A. indica and cypermethrin treated plots on observed insect density, leaf damage and leaf yield. A. indica has proved to be efficient as cypermethrin and can be successfully used for the control of field insect pests of A. hybridus.

Transmission of Bacterial Symbionts With and Without Genome Erosion Between a Beetle Host and the Plant Environment.

Many phytophagous insects harbor symbiotic bacteria that can be transmitted vertically from parents to offspring, or acquired horizontally from unrelated hosts or the environment. In the latter case, plants are a potential route for symbiont transfer and can thus foster a tripartite interaction between microbe, insect, and plant. Here, we focus on two bacterial symbionts of the darkling beetle Lagria villosa that belong to the genus Burkholderia; the culturable strain B. gladioli Lv-StA and the reduced-genome strain Burkholderia Lv-StB. The strains can be transmitted vertically and confer protection to the beetle’s eggs, but Lv-StA can also proliferate in plants, and both symbiont strains have presumably evolved from plant pathogens. Notably, little is known about the role of the environment for the transmission dynamics and the maintenance of the symbionts. Through manipulative assays, we demonstrate the transfer of the symbionts from the beetle to wheat, rice and soybean plants, as well as leaf litter. In addition, we confirm that aposymbiotic larvae can pick up Lv-StA from dry leaves and the symbiont can successfully establish in the beetle’s symbiotic organs. Also, we show that the presence of plants and soil in the environment improves symbiont maintenance. These results indicate that the symbionts of L. villosa beetles are still capable of interacting with plants despite signatures of genome erosion and suggest that a mixed-mode of bacterial transmission is likely key for the persistence of the symbiosis.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a <em>Burkholderia gladioli</em> Symbiont of <em>Lagria villosa</em> Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles.

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identifymolecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette iscarried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

First record of the invasive Lagria villosa (Fabricius, 1781) (Coleoptera: Tenebrionidae: Lagriinae) in Europe.

The lagriid beetle Lagria villosa (Fabricius, 1781), an invasive species of African origin, is recorded for the first time in Europe. A single specimen was found in November 2020 in Turku (Finland) inside a box of table grapes from a local supermarket. This species, included in the EPPO Global Database and in the CABI Invasive Species Compendium, is widely recognized as a significant pest of crops.

Additive Positive Effects of Pollination on Bt and Non-Bt Soybean Cultivars by Honey Bee and Native Flower-Visiting

In addition to the increase in the number of pods produced, a well-conducted pollination also contributes to an increase in the number of grains per pod, improves the quality of the grains and seeds, and renders the grain ripening more uniform, thus, increasing the production at harvest. The objective of the present study was to quantify the benefits of flower-visiting insects in soybean production. The experiment was conducted with two soybean cultivars, one Bt and one non-Bt. During the flowering period, 150 plants of each cultivar were randomly selected, yielding 25 replicates (three plants per replicate) with free access to flower visitors, and another 25 replicates (also with three plants per replicate) that flower visitors could not access. During the flowering period, 549 specimens of flower-visiting insects were found in both cultivars, divided into eight orders, 30 families, and 92 species. The most abundant species were Apis mellifera (Linnaeus) (Hymenoptera: Apidae), Musca sp.1 (Linnaeus) (Diptera: Muscidae), and Lagria villosa (Fabricius) (Coleoptera: Tenebrionidae). In the treatment with flower visitors, grain weight increased by 84.22% in Bt cultivar and by 202.52% in non-Bt cultivar, compared with the area without the presence of flower visitors. The increase in the number of pods in Bt and non-Bt cultivars was 45.72% and 101.25% respectively, in the area open to flower visitors. The high increase in grain yield and number of pods observed in the area with free access to pollinator insects emphasizes the high importance of the pollination service performed by flower visitors to the soybean crop.

Insect‐Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non‐Canonical Polyketide Chain Termination

AbstractGenome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol‐derived C3 building block. The key role of an A‐factor synthase (AfsA)‐like offloading domain was corroborated by CRISPR‐Cas‐mediated gene editing, which facilitated precise excision within a PKS domain.

Insect-Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non-Canonical Polyketide Chain Termination.

Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol‐derived C3 building block. The key role of an A‐factor synthase (AfsA)‐like offloading domain was corroborated by CRISPR‐Cas‐mediated gene editing, which facilitated precise excision within a PKS domain.

Horizontal Gene Transfer to a Defensive Symbiont with a Reduced Genome in a Multipartite Beetle Microbiome.

Symbiotic mutualisms of bacteria and animals are ubiquitous in nature, running a continuum from facultative to obligate from the perspectives of both partners. The loss of functions required for living independently but not within a host gives rise to reduced genomes in many symbionts. Although the phenomenon of genome reduction can be explained by existing evolutionary models, the initiation of the process is not well understood. Here, we describe the microbiome associated with the eggs of the beetle Lagria villosa, consisting of multiple bacterial symbionts related to Burkholderia gladioli, including a reduced-genome symbiont thought to be the exclusive producer of the defensive compound lagriamide. We show that the putative lagriamide-producing symbiont is the only member of the microbiome undergoing genome reduction and that it has already lost the majority of its primary metabolism and DNA repair pathways. The key step preceding genome reduction in the symbiont was likely the horizontal acquisition of the putative lagriamide lga biosynthetic gene cluster. Unexpectedly, we uncovered evidence of additional horizontal transfers to the symbiont's genome while genome reduction was occurring and despite a current lack of genes needed for homologous recombination. These gene gains may have given the genome-reduced symbiont a selective advantage in the microbiome, especially given the maintenance of the large lga gene cluster despite ongoing genome reduction.IMPORTANCE Associations between microorganisms and an animal, plant, or fungal host can result in increased dependence over time. This process is due partly to the bacterium not needing to produce nutrients that the host provides, leading to loss of genes that it would need to live independently and to a consequent reduction in genome size. It is often thought that genome reduction is aided by genetic isolation-bacteria that live in monocultures in special host organs, or inside host cells, have less access to other bacterial species from which they can obtain genes. Here, we describe exposure of a genome-reduced beetle symbiont to a community of related bacteria with nonreduced genomes. We show that the symbiont has acquired genes from other bacteria despite going through genome reduction, suggesting that isolation has not yet played a major role in this case of genome reduction, with horizontal gene gains still offering a potential route for adaptation.

An antifungal polyketide associated with horizontally acquired genes supports symbiont-mediated defense in Lagria villosa beetles

Microbial symbionts are often a source of chemical novelty and can contribute to host defense against antagonists. However, the ecological relevance of chemical mediators remains unclear for most systems. Lagria beetles live in symbiosis with multiple strains of Burkholderia bacteria that protect their offspring against pathogens. Here, we describe the antifungal polyketide lagriamide, and provide evidence supporting that it is produced by an uncultured symbiont, Burkholderia gladioli Lv-StB, which is dominant in field-collected Lagria villosa. Interestingly, lagriamide is structurally similar to bistramides, defensive compounds found in marine tunicates. We identify a gene cluster that is probably involved in lagriamide biosynthesis, provide evidence for horizontal acquisition of these genes, and show that the naturally occurring symbiont strains on the egg are protective in the soil environment. Our findings highlight the potential of microbial symbionts and horizontal gene transfer as influential sources of ecological innovation.

New insect pest at the culture of peach: Biology of Lagria villosa Fabricius, 1783 (Coleoptera: Tenebrionidae) fed with peach

The Idiamim ( Lagria villosa Fabricius, 1783) is an opportunist detritivore insect, but recently the young stage this inscet has been fed into peaches, making them unfit for consumption, in Chapeco, Santa Catarina. Given the lack of information on this species of insect, the aim of this study was evaluate the biology of this insect fed with peach cultivar BRS Libra in physiological maturity. The study was conducted in the Fruit Laboratory of the Federal University of Fronteira Sul, in Chapeco, Santa Catarina. The insects were collected in the adult stage in the field and isolated to obtain the eggs, and from that, we evaluated the duration of young stage and number of instars, reproductive period and fecundity, egg viability, incubation period, longevity and overall cycle. The food was efficient for the development of Idiamin in the laboratory, but the oviposition and egg viability were negatively affected.

English

One of the strategies of Integrated Pest Management (IPM)-soybean is the use of genetically modified plants. However, there are concerns about the unknown effects associated with this technology on non-target organisms. The objective of this study was to determine the population dynamics of species of floral-visiting insects in Bacillus thuringiensis (Bt) and non-Bt soybean. In the experiment, two soybean treatments were assessed, one of soy DM 6563 Intacta and the other BMX Potência RR. Floral-visiting insects were collected in the flowering period every 15 min/h for 12 h/day, every 3 to 4 days. 549 floral-visiting insects (Class Insecta) were collected, comprising 8 orders, 30 families, and 92 species. Of these, 279 were present in the cultivar DM 6563 Intacta and 270 in the cultivar BMX Potência. The most abundant species were Apis mellifera (35.15%), Musca species 1 (10.01%), and Lagria villosa (5.28%). Species composition was similar for the two cultivars. The highest number of species visited the flowers of the cultivars between 8:00 and 11:00 am, while the lowest intensity of visitation occurred between 12:00 and 15:00 pm. After diversity was determined, it was observed that cultivar BMX Potência (non-Bt) had a higher diversity than cultivar DM 6563 Intacta (Bt). Key words: Pollinators, species diversity, transgenic.

Rapid assessment of insect fauna based on local knowledge: comparing ecological and ethnobiological methods

BackgroundThe rapid assessment of biodiversity making use of surveys of local knowledge has been successful for different biological taxa. However, there are no reports on the testing of such tools for sampling insect fauna. The present study aimed to evaluate the efficiency of different ethnobiological techniques for rapid sampling of insect fauna.MethodsField research for the conventional survey of insect fauna was conducted on a private farm (9 ° 43'38.95 "S, 37 ° 45'11.97" W) , where there was intensive cultivation of okra (Abelmoschus esculentus L. (Moench)). The survey of local entomological knowledge was conducted among all the producers of okra living in the rural villages Pereira, Santa Luzia, and Nassau de Souza, within the Jacaré Curituba irrigated settlement scheme. The combined use of the techniques “free list” and projective interviews was analyzed, using two types of visual stimuli: stock photos and an entomological box.ResultsDuring the conventional survey of insect fauna, the species Bemisia tabaci biotype B, Aphis gossypii, Phenacoccus sp., Icerya purchasi and Lagria villosa were the primary pests found in the okra crop. Regarding the survey of insect pests, the results were convergent in both techniques (conventional sampling and free list). Comparing the interview with visual stimuli (pictures) and specimen witnesses (entomological box) revealed that the latter was more effective.ConclusionTechniques based on the recording and analysis of local knowledge about insects are effective for quick sampling of pest insects, but ineffective in sampling predator insects. The utilization of collected insects, infested branches, or photos of the symptoms of damage caused by pests in projective interviews is recommended.

Occurrence of insects causing injuries to the yacon crop

This research aimed to catalog insects that cause injury to the yacon (Smallanthus sonchifolius, família Asteraceae) crop, in order to identify those who may cause damage. The study was carried out in Alegre, Espírito Santo State, Brazil. Cultivation began in May 2013 and the harvest was done 180 days later. Three species from the Coleoptera order: Lagria villosa (Coleoptera: Lagriidae); Cerotoma arcuata and Diabrotica speciosa (Coleoptera: Chrysomelidae) and two species from the Lepidoptera order: Spodoptera eridania (Lepidoptera: Noctuidae) and Chlosyne lacinia saundersii (Lepidoptera: Nymphalidae) were observed causing damage to the yacon crop. The caterpillar C. lacinia saundersii, known as sunflower caterpillar, was the most important species, being observed throughout the yacon cycle.

High-Resolution Physical Chromosome Mapping of Multigene Families in Lagria villosa (Tenebrionidae): Occurrence of Interspersed Ribosomal Genes in Coleoptera

The organization and mapping of multigene families can produce useful genetic markers, and its use may elucidate the mechanisms of karyotype variation and genomic organization in different groups of eukaryotes. To date, few species of Coleoptera have been analyzed using FISH for the location of multigene families. The purpose of this study was to use high-resolution chromosome mapping to establish the genomic organization of the 18S rDNA, 5S rDNA and histone H3 gene families in Lagria villosa. FISH was performed using 18S rDNA, 5S rDNA and histone H3 probes prepared via PCR labeling. Fiber-FISH for 18S and 5S rDNA indicated that both ribosomal elements are colocalized in the short arm of chromosome 4. Additionally, FISH, using the histone H3 probe, revealed that this sequence is found in only one autosomal pair and did not colocalize with rDNA. Fiber-FISH with 5S and 18S probes, used to improve the mapping resolution of these regions, showed that both genes are closely interspersed with varying amounts of both DNA classes.

Insect Pests of Fluted pumpkin (Telfairia occidentalis Hook.E) and Their Control with. Neem seed Extracts in Owerri West Area of Imo State

Field experiments were conducted to determine the insect pests of fluted pumpkin, Telfairia occidentalis at different stages of plant growth and their control with neem (Azadirachta indica A. Juss) seed extract. At different stages of growth, the crop was predominantly attacked by grasshoppers such as Zonocerus variegatus Linnaeus and Oedaleus nigeriensis Uvarov. During the early stage of growth (1-4 weeks after transplanting (WAT)), Z variegatus had the highest incidence (1.70 insects) followed by O. nigeriensis (1.60), Pachnoda marginella Fabricus (1.10), Lagria villosa Fabricius (0.80), Homorocoryphus nitidulus Vicinus (0.70) and Epilachna chrysomelina Fabricus (0.50) in that order. Z. variegatus and O. nigeriensis also recorded the highest mean numbers at 5 – 8 and 9 – 12 WAT,which indicated their abundance in the field. Unsprayed plots had higher mean number of insects than plots sprayed with crude neem extracts. At 5 – 8 and 9 -12 WAT, unsprayed plots had the highest mean number of insect pests (16.80 and 17.30) as against the plots sprayed with neem extracts (9.90 and 13.30) respectively. At 10 WAT, the unsprayed plots also had the highest mean number of damaged leaves (4.80) when compared with plots sprayed with neem extract (2.0). It can, therefore, be safely inferred that Z. variegatus, O. nigeriensis, P marginella and L. villosa are the major insect pests of T. occidentalis in Owerri West Area of lmo State which incidence could be greatly reduced by the application of neem seed extracts.

Sperm morphology and phylogeny of lagriids (Coleoptera, Tenebrionidae)

The systematics of tenebrionids remain unclear, principally at the subfamily level, as is the case of the Lagriinae. Considering that sperm morphology has contributed to the various insect group phylogenies, in this work we describe the structure and ultrastructure of these cells in Lagria villosa. Sperm in this species exhibit a strong morphological similarity to those of Tenebrio molitor and Tribolium castaneum, the only two species of Tenebrionidae with previously described sperm. In tenebrionids, the flagellar components offer good diagnostic characters, e.g. the symmetry of mitochondrial derivatives in L. villosa differentiates sperm of this species from those of Te. molitor and Tr. castaneum. However, the lateral association of the nucleus with flagellar components, the form of accessory bodies, and the presence of material connecting the axoneme to the accessory bodies and mitochondrial derivatives indicate that the three species form a sister group. Therefore, the sperm morphology of L. villosa support lagriid beetles as a subfamily (Lagriinae) of Tenebrionidae.

Soil organisms associated to the weed suppressant Crotalaria juncea (fabaceae) and its importance as a refuge for natural enemies

Soil organisms play an important role in organic crops of Crotalaria juncea (Fabaceae) and are associated with the natural conservation of the environment. The present study was aimed to investigate the population of soil organisms in the organic culture of C. juncea, as well as its importance as a refuge for natural enemies. Dalbulus maidis (Hemiptera: Cicadellidae), Diabrotica sp. (Coleoptera: Chrysomelidae), Doru luteipes (Dermaptera: Forficulidae), Gryllus assimilis (Orthoptera: Gryllidae), Lagria villosa (Coleoptera: Lagriidae), Melanotus sp. (Coleoptera: Elateridae), Meloidogyne incognita (Tylenchida: Heteroderidae), Nephila clavipes (Araneae: Nephilidae), Orius insidiosus (Hemiptera: Anthocoridae), Pheidole sp. (Hymenoptera: Myrmicidae), Phyllophaga sp. (Coleoptera: Scarabeidae), Procornitermes sp. (Isoptera: Termitidae), Solenopsis sp. (Hymenoptera: Formicidae), and Utetheisa ornatrix (Lepidoptera: Arctiidae) were identified in C. juncea. The organisms that were found during a 3-month period in 144 trenches in C. juncea were pest species (84.47%) and natural enemies (15.53%) as well. Natural enemies had an average of 11.89 individuals per 1.08 m³ of soil cultivated with C. juncea. The abundance of organisms in the pod stage (5.49%) of C. juncea was lower than that in the vegetative (83.50%) and flowering (11.01%) stages. Crotalaria juncea plants can be used as part of a crop system for Integrated Pest Management.

Use of serological techniques for determination of Spodoptera frugiperda(J E Smith) predators (Lepidoptera: Noctuidae)

Spodoptera frugiperda (J E Smith) is an important pest of several crops, but especially on maize in Brazil. The implementation of biological control measures hinges on the identification of its predators and other natural enemies. As a means of identifying predators, antibodies against S. frugiperda eggs were generated by inoculating rabbits with macerated S. frugiperda eggs, and the production of antibodies against S. frugiperda egg proteins was verifi ed by double immunodiffusion (DID). These antibodies were then utilized in another serological technique, counterimmunoeletrophoresis (CIE), to identify insects that could have ingested S. frugiperda eggs. Macerates of entire insects collected in maize plantations and of individual parts of their digestive tract, including the crop, were the source of antigens in the CIE, while predators fed S. frugiperda eggs in the laboratory served as the control. Antibodies produced by the inoculated rabbits were effective in detecting S. frugiperda egg proteins, especially if crop macerates were used as antigens. Among the species of insects collected from maize plantations, Lagria villosa Fabricius (Coleoptera: Lagriidae) and a species of Lygaeidae (Hemiptera) were identified as possible S. frugiperda predators.