A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Population-specific responses to developmental temperature in the arboviral vector Aedes albopictus: Implications for climate change.

The increase of environmental temperature due to current global warming is not only favouring the expansion of the distribution range of many insect species, but it is also changing their phenology. Insect phenology is tightly linked to developmental timing, which is regulated by environmental temperatures. However, the degree to which the effects of developmental temperatures extend across developmental stages and their inter-stage relationships have not been thoroughly quantified in mosquitoes. Here, we used the mosquito Aedes albopictus, which is an aggressive invasive species and an arboviral vector, to study how developmental temperature influences fitness across developmental stages, thermal traits, energy reserves, transcriptome and Wolbachia prevalence in laboratory-reared populations originally collected from either temperate or tropical regions. We show that hatchability, larval and pupal viability and developmental speed are strongly influenced by temperature, and these effects extend to wing length, body mass, longevity and content of water, protein and lipids in adults in a population-specific manner. On the contrary, neither adult thermal preference nor heat resistance significantly change with temperature. Wolbachia density was generally lower in adult mosquitoes reared at 18°C than at other tested temperatures, and transcriptome analysis showed enrichment for functions linked to stress responses (i.e. cuticle proteins and chitin, cytochrome p450 and heat shock proteins) in mosquitoes reared at both 18 and 32°C. Our data showed an overall reduced vector fitness performance when mosquitoes were reared at 32°C, and the absence of isomorphy in the relationship between developmental stages and temperature in the laboratory population deriving from larvae collected in northern Italy. Altogether, these results have important implications for reliable model projections of the invasion potentials of Ae. albopictus and its epidemiological impact.

Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis.

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. KEY POINTS: • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites.

Cross Talk between Viruses and Insect Cells Cytoskeleton.

Viruses are excellent manipulators of host cellular machinery, behavior, and life cycle, with the host cell cytoskeleton being a primordial viral target. Viruses infecting insects generally enter host cells through clathrin-mediated endocytosis or membrane fusion mechanisms followed by transport of the viral particles to the corresponding replication sites. After viral replication, the viral progeny egresses toward adjacent cells and reaches the different target tissues. Throughout all these steps, actin and tubulin re-arrangements are driven by viruses. The mechanisms used by viruses to manipulate the insect host cytoskeleton are well documented in the case of alphabaculoviruses infecting Lepidoptera hosts and plant viruses infecting Hemiptera vectors, but they are not well studied in case of other insect-virus systems such as arboviruses-mosquito vectors. Here, we summarize the available knowledge on how viruses manipulate the insect host cell cytoskeleton, and we emphasize the primordial role of cytoskeleton components in insect virus motility and the need to expand the study of this interaction.

Genomics and Proteomics Analyses Revealed Novel Candidate Pesticidal Proteins in a Lepidopteran-Toxic Bacillus thuringiensis Strain.

Discovery and identification of novel insecticidal proteins in Bacillus thuringiensis (Bt) strains are of crucial importance for efficient biological control of pests and better management of insect resistance. In this study, the Bt strain KhF, toxic for Plodia interpunctella and Grapholita molesta larvae, underwent genomics and proteomics analyses to achieve a better understanding of the bases of its pathogenicity. The whole-genome sequencing results revealed that the KhF strain contained nine coding sequences with homologies to Bt insecticidal genes. The lepidopteran toxic mixture of spores and crystals of this Bt strain was subjected to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to assess the protein composition. The results of the proteomic analyses, combined with the toxin gene sequences, revealed that two of the main components of the crystals were two new candidate pesticidal proteins, named KhFA and KhFB. These proteins showed a similarity lower than 36% to the other known Bt toxins. The phylogenetic analysis showed that the KhFA and KhFB grouped with the newly denominated Xpp and Mpp (former ETX/Mtx) pesticidal protein groups, respectively. Altogether, this study has led to the discovery of two novel candidate pesticidal toxins in the lepidopteran toxic KhF strain.

The Independent Biological Activity of Bacillus thuringiensis Cry23Aa Protein Against Cylas puncticollis.

The Cry23Aa/Cry37Aa proteins from Bacillus thuringiensis (Bt) have been described toxic to Cylas puncticollis larvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity toward C. puncticollis larvae has been assessed. The results showed that both proteins were toxic for C. puncticollis larvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity against C. puncticollis larvae. Also, the binding behavior of Cry23Aa protein to midgut receptors of C. puncticollis larvae has been determined. According to our results, Cry23Aa binds to C. puncticollis brush border membrane vesicles (BBMV) specifically and independently of Cry37Aa. Due to the lack of common binding sites, Cry23Aa can be pyramided with Cry3Aa protein for better management of C. puncticollis.

Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells.

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.