Pathogen-associated molecular patterns (PAMPs) derived from Leishmania and bacteria increase gene expression of antimicrobial peptides and gut surface proteins in sand flies.

The interaction between pathogens and vectors' physiology can impact parasite transmission. Studying this interaction at the molecular level can help in developing control strategies. We study leishmaniases, diseases caused by Leishmania parasites transmitted by sand fly vectors, posing a significant global public health concern. Lipophosphoglycan (LPG), the major surface glycoconjugate of Leishmania, has been described to have several roles throughout the parasite's life cycle, both in the insect and vertebrate hosts. In addition, the sand fly midgut possesses a rich microbiota expressing lipopolysaccharides (LPS). However, the effect of LPG and LPS on the gene expression of sand fly midgut proteins or immunity effectors has not yet been documented. We experimentally fed Lutzomyia longipalpis and Phlebotomus papatasi sand flies with blood containing purified LPG from Leishmania infantum, Leishmania major, or LPS from Escherichia coli. The effect on the expression of genes encoding gut proteins galectin and mucin, digestive enzymes trypsin and chymotrypsin, and antimicrobial peptides (AMPs) attacin and defensins was assessed by quantitative PCR (qPCR). The gene expression of a mucin-like protein in L. longipalpis was increased by L. infantum LPG and E. coli LPS. The gene expression of a galectin was increased in L. longipalpis by L. major LPG, and in P. papatasi by E. coli LPS. Nevertheless, the gene expression of trypsins and chymotrypsins did not significantly change. On the other hand, both L. infantum and L. major LPG significantly enhanced expression of the AMP attacin in both sand fly species and defensin in L. longipalpis. In addition, E. coli LPS increased the expression of attacin and defensin in L. longipalpis. Our study showed that Leishmania LPG and E. coli LPS differentially modulate the expression of sand fly genes involved in gut maintenance and defence. This suggests that the glycoconjugates from microbiota or Leishmania may increase the vector's immune response and the gene expression of a gut coating protein in a permissive vector.

RNAi-mediated gene silencing of Phlebotomus papatasi defensins favors Leishmania major infection.

Introduction: Production of different antimicrobial peptides (AMPs) is one of the insect's prominent defense strategies, regulated mainly by Toll and immune deficiency (IMD) humoral pathways. Here we focused mainly on two AMPs of Phlebotomus papatasi, vector of Leishmania major parasites, their association with the relish transcription factor and the effective participation on Leishmania infection. Methods and results: We further characterized the role of previously described gut-specific P. papatasi defensin (PpDef1) and identified the second defensin (PpDef2) expressed in various sand fly tissues. Using the RNAi-mediated gene silencing, we report that the silencing of PpDef1 gene or simultaneous silencing of both defensin genes (PpDef1 and PpDef2) resulted in increased parasite levels in the sand fly (detectable by PCR) and higher sand fly mortality. In addition, we knocked down relish, the sole transcription factor of the IMD pathway, to evaluate the association of the IMD pathway with AMPs expression in P. papatasi. We demonstrated that the relish gene knockdown reduced the expression of PpDef2 and attacin, another AMP abundantly expressed in the sand fly body. Conclusions: Altogether, our experiments show the importance of defensins in the sand fly response toward L. major and the role of the IMD pathway in regulating AMPs in P. papatasi.

Enterobacter sp. AA26 as a Protein Source in the Larval Diet of Drosophila suzukii.

The Spotted-Wing Drosophila fly, Drosophila suzukii, is an invasive pest species infesting major agricultural soft fruits. Drosophila suzukii management is currently based on insecticide applications that bear major concerns regarding their efficiency, safety and environmental sustainability. The sterile insect technique (SIT) is an efficient and friendly to the environment pest control method that has been suggested for the D. suzukii population control. Successful SIT applications require mass-rearing of the strain to produce competitive and of high biological quality males that will be sterilized and consequently released in the wild. Recent studies have suggested that insect gut symbionts can be used as a protein source for Ceratitis capitata larval diet and replace the expensive brewer's yeast. In this study, we exploited Enterobacter sp. AA26 as partial and full replacement of inactive brewer's yeast in the D. suzukii larval diet and assessed several fitness parameters. Enterobacter sp. AA26 dry biomass proved to be an inadequate nutritional source in the absence of brewer's yeast and resulted in significant decrease in pupal weight, survival under food and water starvation, fecundity, and adult recovery.

Drosophila suzukii (Diptera: Drosophilidae): A Decade of Research Towards a Sustainable Integrated Pest Management Program.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) also known as spotted-wing drosophila (SWD), is a pest native to Southeast Asia. In the last few decades, the pest has expanded its range to affect all major European and American fruit production regions. SWD is a highly adaptive insect that is able to disperse, survive, and flourish under a range of environmental conditions. Infestation by SWD generates both direct and indirect economic impacts through yield losses, shorter shelf life of infested fruit, and increased production costs. Fresh markets, frozen berries, and fruit export programs have been impacted by the pest due to zero tolerance for fruit infestation. As SWD control programs rely heavily on insecticides, exceedance of maximum residue levels (MRLs) has also resulted in crop rejections. The economic impact of SWD has been particularly severe for organic operations, mainly due to the limited availability of effective insecticides. Integrated pest management (IPM) of SWD could significantly reduce chemical inputs but would require substantial changes to horticultural management practices. This review evaluates the most promising methods studied as part of an IPM strategy against SWD across the world. For each of the considered techniques, the effectiveness, impact, sustainability, and stage of development are discussed.

Mass-Rearing of Drosophila suzukii for Sterile Insect Technique Application: Evaluation of Two Oviposition Systems.

Drosophila suzukii (Diptera: Drosophilidae) is an invasive pest of a wide range of commercial soft-skinned fruits. To date, most management tactics are based on spraying of conventional and/or organic insecticides, baited traps, and netting exclusion. Interest has been expressed in using the sterile insect technique (SIT) as part of area-wide integrated pest management (AW-IPM) programs to control D. suzukii infestations. Mass-rearing protocols are one of the prerequisites for successful implementation of the SIT. To establish mass-rearing methods for this species, two different egg-collection systems were developed and compared with respect to the number of eggs produced, egg viability, pupa and adult recovery, adult emergence rate, and flight ability. Female flies kept in cages equipped with a wax panel produced significantly more eggs with higher viability and adult emergence rate, as compared to the netted oviposition system. The wax panel system was also more practical and less laborious regarding the collection of eggs. Furthermore, the wax panel oviposition system can be adapted to any size or design of an adult cage. In conclusion, this system bears great promise as an effective system for the mass production of D. suzukii for SIT.

Irradiation dose response under hypoxia for the application of the sterile insect technique in Drosophila suzukii.

Treating insects with a lower oxygen atmosphere before and during exposure to radiation can mitigate some of the negative physiological effects due to the irradiation. The irradiation of pupae under oxygen-reduced environment such as hypoxia or anoxia is routinely used in the sterile insect technique (SIT) of some tephritid species as it provides radiological protection. This treatment allows to have the sterile pupae already in sealed containers facilitating the shipment. SIT is an environment friendly control tactic that could be used to manage populations of Drosophila suzukii in confined areas such as greenhouses. The objectives of this study were to assess the effect of irradiation on the reproductive sterility in D. suzukii males and females under low-oxygen atmosphere (hypoxia) and atmosphere conditions (normoxia). Additionally, we assessed the differences in radiological sensitivity of pupae treated under hypoxia and normoxia conditions. Finally, the effect on emergence rate and flight ability of the irradiated D. suzukii adults exposed to doses that induced >99% of sterility were assessed. Pupae needed a 220 Gy irradiation dose to achieve >99% of egg hatch sterility in males irrespective of the atmosphere condition. For females the same level of sterility was achieved already at 75 Gy and 90 Gy for the normoxia and hypoxia treatments, respectively. Radiation exposure at 170 and 220 Gy under the two atmosphere treatments did not have any effect on the emergence rate and flight ability of D. suzukii males and females. Therefore, hypoxia conditions can be used as part of an area-wide insect pest management program applying SIT to facilitate the protocols of packing, irradiation and shipment of sterile D. suzukii pupae.

Functional transcriptome analyses of Drosophila suzukii antennae reveal mating-dependent olfaction plasticity in females.

Insect olfaction modulates basal behaviors and it is often influenced by the physiological condition of each individual such as the reproductive state. Olfactory plasticity can be achieved by modifications at both peripheral and central nervous system levels. Here we performed a genome-wide transcriptomic analysis of the main olfactory organ, the antenna, to investigate how gene expression varies with female mating status in Drosophila suzukii, a destructive and invasive soft fruit pest. We observed a wide mating-induced up-regulation of chemosensory-related genes in females, especially odorant receptor (Or) genes. We then used a candidate gene approach to define the comprehensive dataset of antenna-expressed chemosensory receptors and binding proteins, which showed many similarities with Drosophila melanogaster. Candidate gene approach was also used to finely quantify differential expression at Or isoform level, suggesting post-mating transcriptional modulation of genes involved in the peripheral olfactory system. We identified 27 up-regulated Or transcripts encoded by 25 genes, seven of them were duplications specific to D. suzukii lineage. Post-mating olfactory modulation was further supported by electroantennogram recordings that showed a differential response according to mating status to one out of eight odors tested (isoamyl-acetate). Our study characterizes the transcriptional mechanisms driven by mating in D. suzukii female antennae. Understanding the role of genes differentially expressed in virgin or mated females will be crucial to better understand host finding and the crop-damaging oviposition behavior of this species.

Sterile insect technique and Wolbachia symbiosis as potential tools for the control of the invasive species Drosophila suzukii.

Drosophila suzukii, a vinegar fly originated from Southeast Asia, has recently invaded western countries, and it has been recognized as an important threat of a wide variety of several commercial soft fruits. This review summarizes the current information about the biology and dispersal of D. suzukii and discusses the current status and prospects of control methods for the management of this pest. We highlight current knowledge and ongoing research on innovative environmental-friendly control methods with emphasis on the sterile insect technique (SIT) and the incompatible insect technique (IIT). SIT has been successfully used for the containment, suppression or even eradication of populations of insect pests. IIT has been proposed as a stand-alone tool or in conjunction with SIT for insect pest control. The principles of SIT and IIT are reviewed, and the potential value of each approach in the management of D. suzukii is analyzed. We thoroughly address the challenges of SIT and IIT, and we propose the use of SIT as a component of an area-wide integrated pest management approach to suppress D. suzukii populations. As a contingency plan, we suggest a promising alternative avenue through the combination of these two techniques, SIT/IIT, which has been developed and is currently being tested in open-field trials against Aedes mosquito populations. All the potential limiting factors that may render these methods ineffective, as well as the requirements that need to be fulfilled before their application, are discussed.

Genetic variability in Italian populations of Drosophila suzukii.

BACKGROUND: Drosophila suzukii is a highly destructive pest species, causing substantial economic losses in soft fruit production. To better understand migration patterns, gene flow and adaptation in invaded regions, we studied the genetic structure of D. suzukii collected across Italy, where it was first observed in 2008. In particular, we analysed 15 previously characterised Simple Sequence Repeat (SSR) markers to estimate genetic differentiation across the genome of 278 flies collected from nine populations. RESULTS: The nine populations showed high allelic diversity, mainly due to very high heterozygosity. The high Polymorphism Information Content (PIC) index values (ranging from 0.68 to 0.84) indicated good discrimination power for the markers. Negative fixation index (F IS) values in seven of the populations indicated a low level of inbreeding, as suggested by the high number of alleles. STRUCTURE, Principal Coordinate and Neighbour Joining analysis also revealed that the Sicilian population was fairly divergent compared to other Italian populations. Moreover, migration was present across all populations, with the exception of the Sicilian one, confirming its isolation relative to the mainland. CONCLUSIONS: This is the first study characterising the genetic structure of the invasive species D. suzukii in Italy. Our analysis showed extensive genetic homogeneity among D. suzukii collected in Italy. The relatively isolated Sicilian population suggests a largely human-mediated migration pattern, while the warm climate in this region allows the production of soft fruit, and the associated D. suzukii reproductive season occurring much earlier than on the rest of the peninsula.