Barcoding of Italian mosquitoes (BITMO): generation and validation of DNA barcoding reference libraries for native and alien species of Culicidae.

BACKGROUND: Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). METHODS: A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. RESULTS: Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. CONCLUSIONS: This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches.

Integrated de novo transcriptome of Culex pipiens mosquito larvae as a resource for genetic control strategies.

We present a de novo transcriptome of the mosquito vector Culex pipiens, assembled by sequences of susceptible and insecticide resistant larvae. The high quality of the assembly was confirmed by TransRate and BUSCO. A mapping percentage until 94.8% was obtained by aligning contigs to Nr, SwissProt, and TrEMBL, with 27,281 sequences that simultaneously mapped on the three databases. A total of 14,966 ORFs were also functionally annotated by using the eggNOG database. Among them, we identified ORF sequences of the main gene families involved in insecticide resistance. Therefore, this resource stands as a valuable reference for further studies of differential gene expression as well as to identify genes of interest for genetic-based control tools.

Sex-biased phenotypic plasticity affects sexual dimorphism patterns under changing environmental conditions.

Sexual dimorphism is almost ubiquitous in animals. A common pattern observed across multiple taxa involves differences in development time (sexual bimaturism) and body size (sexual size dimorphism) between conspecific males and females. Furthermore, a strict association of dimorphism at these traits has been documented in several taxa, where the sex showing shorter development time also has a smaller body size than the other sex. Growth and development are strongly dependent on environmental conditions during individual life-cycle in ectotherms, inducing considerable phenotypic plasticity. However, how phenotypic plasticity affects the association between sexual dimorphism in development time and body size remains unclear. Here, we tracked development time, body size, and body mass throughout the ontogeny of the mosquito Aedes mariae. The larval development of this species is strictly linked to Mediterranean Sea rock-pools, whose highly variable environmental conditions over minimal time frames make this organism-environment system ideal for exploring plasticity-led eco-evolutionary processes. We found differential plasticity between males and females, dissolving the link between dimorphism in development time and body size under increasing temperature and decreasing salinity conditions. These findings contrast with the current hypotheses proposed to explain the origin of the association between sexual bimaturism and sexual size dimorphism, highlighting the condition dependence of sexual dimorphism patterns and the need to consider phenotypic plasticity in future studies on their evolution.

MosChito rafts as a promising biocontrol tool against larvae of the common house mosquito, Culex pipiens.

Mosquito control is of paramount importance, in particular, in light of the major environmental alterations associated with human activities, from climate change to the altered distribution of pathogens, including those transmitted by Arthropods. Here, we used the common house mosquito, Culex pipiens to test the efficacy of MosChito raft, a novel tool for mosquito larval control. MosChito raft is a floating hydrogel matrix, composed of chitosan, genipin and yeast cells, as bio-attractants, developed for the delivery of a Bacillus thuringiensis israeliensis (Bti)-based bioinsecticide to mosquito larvae. To this aim, larvae of Cx. pipiens were collected in field in Northern Italy and a novel colony of mosquito species (hereafter: Trescore strain) was established. MosChito rafts, containing the Bti-based formulation, were tested on Cx. pipiens larvae from the Trescore strain to determine the doses to be used in successive experiments. Thus, bioassays with MosChito rafts were carried out under semi-field conditions, both on larvae from the Trescore strain and on pools of larvae collected from the field, at different developmental stages. Our results showed that MosChito raft is effective against Cx. pipiens. In particular, the observed mortality was over 50% after two days exposure of the larvae to MosChito rafts, and over 70-80% at days three to four, in both laboratory and wild larvae. In conclusion, our results point to the MosChito raft as a promising tool for the eco-friendly control of a mosquito species that is not only a nuisance insect but is also an important vector of diseases affecting humans and animals.

Using Gamma Irradiation to Predict Sperm Competition Mechanism in Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae): Insights for a Future Management Strategy.

The stink bug, Bagrada hilaris, is a pest of mainly Brassicaceae crops. It is native to Africa and Asia and was recently reported as invasive in the southwestern part of the USA and in South America. There are no mitigation programs in place that do not involve pesticides. Therefore, much attention has recently been paid to the study of this species in order to identify sustainable and effective control strategies, such as the Sterile Insect Technique (SIT). In order to evaluate the suitability of the SIT on this pest, the mechanism of post-copulatory sperm competition was investigated. This is a polyandrous species, and it is thus important to understand whether irradiated males are able to compete with wild, e.g., non-irradiated, males for sperm competition after matings. Sperm competition was studied by sequentially mating a healthy virgin female first with a non-irradiated male, and then with a γ-irradiated (Co-60) one, and again in the opposite order. Males were irradiated at three different doses: 60, 80, and 100 Gy. The fecundity and fertility of the females, in the two orders of mating, were scored in order to perform an initial assessment of the success of sperm competition with a P2 index. Sperm from the non-irradiated male were utilized at the lowest irradiation doses (60 and 80 Gy), whereas the irradiated sperm were preferentially utilized at the highest dose (100 Gy). Bagrada hilaris exhibited high variability in P2 indexes, indicating a sperm-mixing mechanism.

The ecological importance of hybridization.

Hybridization as an evolutionary process has been studied in depth over the past few decades. Research has focused on its role in shaping reproductive barriers, its adaptive value, and its genomic consequences. In contrast, our knowledge of ecological dimensions of hybridization is still in its infancy, despite hybridization being an inherently ecological interaction. Using examples from various organisms, we show that hybridization can affect and be affected by non-reproductive interactions, including predation, competition, parasitism, mutualism, commensalism, and organism-environment interactions, with significant implications for community structure and ecosystem functioning. However, since these dimensions of hybridization have mostly been revealed from studies designed to decipher other evolutionary processes, we argue that much of the eco-evolutionary importance of hybridization is yet to be discovered.

Applying Satyrization to Insect Pest Control: The Case of the Spotted Wing Drosophila, Drosophila suzukii Matsumura.

Drosophila suzukii represents one of the major agricultural pests worldwide. The identification of safety and long-lasting tools to suppress its populations is therefore crucial to mitigate the environmental and economic damages due to its occurrence. Here, we explore the possibility of using satyrization as a tool to control the abundance of D. suzukii. By using males of D. melanogaster, we realized courtship tests, spermathecae analysis, and multiple-choice experiments to assess the occurrence and extent of pre- and post-zygotic isolation between the two species, as well as the occurrence of fitness costs in D. suzukii females due to satyrization. Our results showed that: (i) D. melanogaster males successfully courted D. suzukii females; (ii) D. melanogaster males significantly affected the total courtship time of D. suzukii males, which reduced from 22.6% to 6.4%; (iii) D. melanogaster males were able to inseminate D. suzukii and reduce their offspring, inducing a high fitness cost. Reproductive interference occurs at different steps between D. melanogaster and D. suzukii, both alone and in combination with other area-wide control approaches.

MosChito rafts as effective and eco-friendly tool for the delivery of a Bacillus thuringiensis-based insecticide to Aedes albopictus larvae.

Adult mosquito females, through their bites, are responsible for the transmission of different zoonotic pathogens. Although adult control represents a pillar for the prevention of disease spread, larval control is also crucial. Herein we characterized the effectiveness of a suitable tool, named "MosChito raft", for the aquatic delivery of a Bacillus thuringiensis var. israelensis (Bti) formulate, a bioinsecticide active by ingestion against mosquito larvae. MosChito raft is a floating tool composed by chitosan cross-linked with genipin in which a Bti-based formulate and an attractant have been included. MosChito rafts (i) resulted attractive for the larvae of the Asian tiger mosquito Aedes albopictus, (ii) induced larval mortality within a few hours of exposure and, more importantly, (iii) protected the Bti-based formulate, whose insecticidal activity was maintained for more than one month in comparison to the few days residual activity of the commercial product. The delivery method was effective in both laboratory and semi-field conditions, demonstrating that MosChito rafts may represent an original, eco-based and user-friendly solution for larval control in domestic and peri-domestic aquatic habitats such as saucers and artificial containers in residential or urban environments.

Cuticle Modifications and Over-Expression of the Chitin-Synthase Gene in Diflubenzuron-Resistant Phenotype.

Insecticide resistance is a major threat challenging the control of harmful insect species. The study of resistant phenotypes is, therefore, pivotal to understand molecular mechanisms underpinning insecticide resistance and plan effective control and resistance management strategies. Here, we further analysed the diflubenzuron (DFB)-resistant phenotype due to the point-mutation I1043M in the chitin-synthase 1 gene (chs1) in the mosquito Culex pipiens. By comparing susceptible and resistant strains of Cx. pipiens through DFB bioassays, molecular analyses and scanning electron microscopy, we showed that the I1043M-resistant mosquitoes have: (i) a striking level of DFB resistance (i.e., resistance ratio: 9006); (ii) a constitutive 11-fold over-expression of the chs1 gene; (iii) enhanced cuticle thickness and cuticular chitin content. Culex pipiens is one of the most important vector species in Europe and the rapid spread of DFB resistance can threaten its control. Our results, by adding new data about the DFB-resistant phenotype, provide important information for the control and management of insecticide resistance.

Historical samples reveal a combined role of agriculture and public-health applications in vector resistance to insecticides.

BACKGROUND: Insecticide resistance is the major threat to vector control and for the prevention of vector-borne diseases. Because almost all insecticides used against insect vectors are or have been used in agriculture, a connection between agricultural insecticide use and resistance in insect vectors has been hypothesized. However, it is challenging to find a causal link between past agricultural use of insecticides and current resistance in vector populations without historical data series. Here we investigated the relative contribution across time of agricultural and public-health insecticide applications in selecting for diflubenzuron (DFB) resistance in Culex pipiens populations. Using DNA sequencing, we looked for DFB resistant mutations in current and historical mosquito samples, dating back to the 1980s-1990s, when DFB was used in agriculture but not yet in mosquito control. RESULTS: In the samples collected before the introduction of DFB in vector control, we found the resistant mutation I1043M in rural regions but not any of the neighboring urban and natural areas, indicating that the selection pressure was derived by agriculture. However, after the introduction of DFB for vector control, the resistant mutations were found across all study areas showing that the initial selection from agriculture was further boosted by the selection pressure imposed by the mosquito control applications in the 2000s. CONCLUSIONS: Our findings support a combined role of agricultural and public-health use of insecticides in vector resistance across time and call for specific actions in integrated resistance management, including increased communication between agriculture and health practitioners. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cannibalism and Necrophagy Promote a Resource Loop and Benefit Larval Development in Insects of Temporary Waters.

Temporary aquatic habitats are contingent on the allochthonous inputs of plant and animal detritus, whose quality and availability can significantly affect the species developing in these habitats. Although animal detritus (i.e., invertebrate carcasses) is a high-quality food, it is an unpredictable and variable resource. On the contrary, conspecific individuals (dead or alive) are a nutritionally high-quality food source that is always available. In this context, conspecifics consumption, by cannibalism or necrophagy, can be a good strategy to overcome nutrient limitation and allow individual maintenance and development. Here, we tested this hypothesis by using the tiger mosquito Aedes albopictus. By carrying out laboratory and semi-field experiments, we first estimated the relative rate of cannibalism and necrophagy, under different larval densities. Then, we analyzed the effects of cannibalism and necrophagy on larval survival and adult yield. Consistent with our hypothesis, we found that cannibalism and necrophagy occurred under all experimental conditions, and that conspecific consumption had positive effects on individual development, as it significantly increased the rate of adult emergence and larval survival. Interestingly, about 50% of the initial cohort was consumed by conspecifics, suggesting that cannibalism and necrophagy can drive an important resources loop in temporary aquatic habitats.

Evolution of Adaptive Variation in the Mosquito Culex pipiens: Multiple Independent Origins of Insecticide Resistance Mutations.

Insecticide resistance is an informative model for studying the appearance of adaptive traits. Simultaneously, understanding how many times resistance mutations originate is essential to design effective resistance management. In the mosquito Culex pipiens, target-site resistance to the insecticide diflubenzuron (DFB) has been recently found in Italian and Turkish populations. Three point mutations confer it at the codon 1043 of the chitin synthase 1 gene (chs-1): I1043L, I1043M, and I1043F. Whether the resistant mutations originated independently from different susceptible alleles or sequentially from resistant alleles and whether resistant alleles from Italy and Turkey have originated once or multiple times remain unresolved. Here, we sequenced a fragment of the chs-1 gene carrying the resistant mutations and inferred the phylogenetic relationships among susceptible and resistant alleles. Confirming previous findings, we found the three mutations in Italy and the I1043M in Turkey. Notably, the I1043F was also found for the first time in Turkish samples, highlighting the need for extensive monitoring activities. Phylogenetic analyses are consistent with an independent origin of the I1043F, I1043M, and I1043L mutations from different susceptible alleles and with multiple independent origins of the Italian and Turkish I1043M and I1043F alleles.

Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance.

BACKGROUND: Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. Among the Cx. pipiens populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. CONCLUSIONS/SIGNIFICANCE: The findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.

Effects of combined drug treatments on Plasmodium falciparum: In vitro assays with doxycycline, ivermectin and efflux pump inhibitors.

There is great concern regarding the rapid emergence and spread of drug-resistance in Plasmodium falciparum, the parasite responsible for the most severe form of human malaria. Parasite populations resistant to some or all the currently available antimalarial treatments are present in different world regions. Considering the need for novel and integrated approaches to control malaria, combinations of drugs were tested on P. falciparum. The primary focus was on doxycycline, an antibiotic that specifically targets the apicoplast of the parasite. In combination with doxycycline, three different drugs known to inhibit efflux pumps (verapamil, elacridar and ivermectin) were tested, with the assumption that they could increase the intracellular concentration of the antibiotic and consequently its efficacy against P. falciparum. We emphasize that elacridar is a third-generation ABC transporters inhibitor, never tested before on malaria parasites. In vitro experiments were performed on asexual stages of two strains of P. falciparum, chloroquine-sensitive (D10) and chloroquine-resistant (W2). Incubation times on asynchronous or synchronous cultures were 72h or 96h, respectively. The antiplasmodial effect (i.e. the IC50) was determined by measuring the activity of the parasite lactate dehydrogenase, while the interaction between drugs was determined through combination index (CI) analyses. Elacridar achieved an IC50 concentration comparable to that of ivermectin, approx. 10-fold lower than that of verapamil, the other tested ABC transporter inhibitor. CI results showed synergistic effect of verapamil plus doxycycline, which is coherent with the starting hypothesis, i.e. that ABC transporters represent potential targets, worth of further investigations, towards the development of companion molecules useful to enhance the efficacy of antimalarial drugs. At the same time, the observed antagonistic effect of doxycycline in combination with ivermectin or elacridar highlighted the importance of drug testing, to avoid the de-facto generation of a sub-dosage, a condition that facilitates the development of drug resistance.

Ancient hybridization and mtDNA introgression behind current paternal leakage and heteroplasmy in hybrid zones.

Hybridization between heterospecific individuals has been documented as playing a direct role in promoting paternal leakage and mitochondrial heteroplasmy in both natural populations and laboratory conditions, by relaxing the egg-sperm recognition mechanisms. Here, we tested the hypothesis that hybridization can lead to mtDNA heteroplasmy also indirectly via mtDNA introgression. By using a phylogenetic approach, we showed in two reproductively isolated beetle species, Ochthebius quadricollis and O. urbanelliae, that past mtDNA introgression occurred between them in sympatric populations. Then, by developing a multiplex allele-specific PCR assay, we showed the presence of heteroplasmic individuals and argue that their origin was through paternal leakage following mating between mtDNA-introgressed and pure conspecific individuals. Our results highlight that mtDNA introgression can contribute to promote paternal leakage, generating genetic novelty in a way that has been overlooked to date. Furthermore, they highlight that the frequency and distribution of mtDNA heteroplasmy can be deeply underestimated in natural populations, as i) the commonly used PCR-Sanger sequencing approach can fail to detect mitochondrial heteroplasmy, and ii) specific studies aimed at searching for it in populations where mtDNA-introgressed and pure individuals co-occur remain scarce, despite the fact that mtDNA introgression has been widely documented in several taxa and populations.

Gene silencing through RNAi and antisense Vivo-Morpholino increases the efficacy of pyrethroids on larvae of Anopheles stephensi.

BACKGROUND: Insecticides are still at the core of insect pest and vector control programmes. Several lines of evidence indicate that ABC transporters are involved in detoxification processes against insecticides, including permethrin and other pyrethroids. In particular, the ABCG4 gene, a member of the G subfamily, has consistently been shown to be up-regulated in response to insecticide treatments in the mosquito malaria vector Anopheles stephensi (both adults and larvae). METHODS: To verify the actual involvement of this transmembrane protein in the detoxification process of permethrin, bioassays on larvae of An. stephensi, combining the insecticide with a siRNA, specifically designed for the inhibition of ABCG4 gene expression were performed. Administration to larvae of the same siRNA, labeled with a fluorescent molecule, was effected to investigate the systemic distribution of the inhibitory RNA into the larval bodies. Based on siRNA results, similar experiments using antisense Vivo-Morpholinos (Vivo-MOs) were effected. These molecules, compared to siRNA, are expected to guarantee a higher stability in environmental conditions and in the insect gut, and present thus a higher potential for future in-field applications. RESULTS: Bioassays using two different concentrations of siRNA, associated with permethrin, led to an increase of larval mortality, compared with results with permethrin alone. These outcomes confirm that ABCG4 transporter plays a role in the detoxification process against the selected insecticide. Moreover, after fluorescent labelling, it was shown the systemic dissemination of siRNA in different body districts of An. stephensi larvae, which suggest a potential systemic effect of the molecule. At the same time, results of Vivo-MO experiments were congruent with those obtained using siRNA, thus confirming the potential of ABCG4 inhibition as a strategy to increase permethrin susceptibility in mosquitoes. For the first time, Vivo-MOs were administered in water to larvae, with evidence for a biological effect. CONCLUSIONS: Targeting ABCG4 gene for silencing through both techniques resulted in an increased pyrethroid efficacy. These results open the way toward the possibility to exploit ABCG4 inhibition in the context of integrated programmes for the control An. stephensi mosquitoes and malaria transmission.

Insecticide Exposure Triggers a Modulated Expression of ABC Transporter Genes in Larvae of Anopheles gambiae s.s.

Insecticides remain a main tool for the control of arthropod vectors. The urgency to prevent the insurgence of insecticide resistance and the perspective to find new target sites, for the development of novel molecules, are fuelling the study of the molecular mechanisms involved in insect defence against xenobiotic compounds. In this study, we have investigated if ATP-binding cassette (ABC) transporters, a major component of the defensome machinery, are involved in defence against the insecticide permethrin, in susceptible larvae of the malaria vector Anopheles gambiae sensu stricto. Bioassays were performed with permethrin alone, or in combination with an ABC transporter inhibitor. Then we have investigated the expression profiles of five ABC transporter genes at different time points following permethrin exposure, to assess their expression patterns across time. The inhibition of ABC transporters increased the larval mortality by about 15-fold. Likewise, three genes were up-regulated after exposure to permethrin, showing different patterns of expression across the 48 h. Our results provide the first evidences of ABC transporters involvement in defence against a toxic in larvae of An. gambiae s.s. and show that the gene expression response is modulated across time, being continuous, but stronger at the earliest and latest times after exposure.

Paternal leakage and mtDNA heteroplasmy in Rhipicephalus spp. ticks.

Paternal leakage of mitochondrial DNA (mtDNA) and heteroplasmy have been recently described in several animal species. In arthropods, by searching in the Scopus database, we found only 23 documented cases of paternal leakage. Therefore, although arthropods represent a large fraction of animal biodiversity, this phenomenon has been investigated only in a paucity of species in this phylum, thus preventing a reliable estimate of its frequency. Here, we investigated the occurrence of paternal leakage and mtDNA heteroplasmy in ticks belonging to one of the most significant tick species complexes, the so-called Rhipicephalus sanguineus sensu lato. By developing a multiplex allele-specific PCR assay targeting a fragment of the 12S rRNA ribosomal region of the mtDNA, we showed the occurrence of paternal leakage and mtDNA heteroplasmy in R. sanguineus s.l. ticks originated from experimental crosses, as well as in individuals collected from the field. Our results add a new evidence of paternal leakage in arthropods and document for the first time this phenomenon in ticks. Furthermore, they suggest the importance of using allele-specific assays when searching for paternal leakage and/or heteroplasmy, as standard sequencing methods may fail to detect the rare mtDNA molecules.

Hybridization and extensive mitochondrial introgression among fire salamanders in peninsular Italy.

Discordance between mitochondrial and nuclear patterns of population genetic structure is providing key insights into the eco-evolutionary dynamics between and within species, and their assessment is highly relevant to biodiversity monitoring practices based on DNA barcoding approaches. Here, we investigate the population genetic structure of the fire salamander Salamandra salamandra in peninsular Italy. Both mitochondrial and nuclear markers clearly identified two main population groups. However, nuclear and mitochondrial zones of geographic transition between groups were located 600 km from one another. Recent population declines in central Italy partially erased the genetic imprints of past hybridization dynamics. However, the overall pattern of genetic variation, together with morphological and fossil data, suggest that a rampant mitochondrial introgression triggered the observed mitonuclear discordance, following a post-glacial secondary contact between lineages. Our results clearly show the major role played by reticulate evolution in shaping the structure of Salamandra salamandra populations and, together with similar findings in other regions of the species' range, contribute to identify the fire salamander as a particularly intriguing case to investigate the complexity of mechanisms triggering patterns of mitonuclear discordance in animals.