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.

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.

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.

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.

Cannibalism in temporary waters: Simulations and laboratory experiments revealed the role of spatial shape in the mosquito Aedes albopictus.

Cannibalism is a commonly observed phenomenon in arthropod species having relevant consequences for population dynamics and individual fitness. It is a context-dependent behaviour and an understanding of the factors affecting cannibalism rate is crucial to highlight its ecological relevance. In mosquitoes, cannibalism between larval stages has been widely documented, and the role of density, food availability and length of contact between individuals also ascertained. However, although mosquitoes can develop in temporary water habitats with very heterogeneous topologies, the role of the site shape where cannibals and victims co-occur has been instead overlooked. In this paper, we investigated this issue by using a simulation approach and laboratory cannibalism experiments between old (third- and fourth-instars) and young (first-instar) larvae of the tiger mosquito Aedes albopictus. Three virtual spaces with different shapes were simulated and the number of larval encounters was estimated in each one to assess whether the spatial shape affected the number of encounters between cannibal and victims. Then, experimental trials in containers with similar shapes to those used in the simulations were performed, and the cannibalism rate was estimated at 24 and 48h. Our results showed that the spatial shape plays a role on cannibalism interactions, affecting the number of encounters between individuals. Indeed, in the experimental trials performed, we observed the highest cannibalism rate in the container with the highest number of encounters predicted by the simulations. Interestingly, we found also that spatial shape can affect cannibalism not only by affecting the number of encounters, but also the number of encounters "favorable" for cannibalistic events. Temporary waters are inhabited by several species other than mosquitoes. Our results, showing an influence of the spatial shape on cannibalism in Ae. albopictus larvae, add a new critical factor to those affecting ecological interactions in these habitats.

Gene expression modulation of ABC transporter genes in response to permethrin in adults of the mosquito malaria vector Anopheles stephensi.

Living organisms have evolved an array of genes coding for detoxifying enzymes and efflux protein pumps, to cope with endogenous and xenobiotic toxic compounds. The study of the genes activated during toxic exposure is relevant to the area of arthropod vector control, since these genes are one of the targets upon which natural selection acts for the evolution of insecticide resistance. ATP-binding cassette (ABC) transporters participate to insecticide detoxification acting as efflux pumps, that reduce the intracellular concentration of toxic compounds, or of their metabolic derivatives. Here we analyzed the modulation of the expression of six genes coding for ABC transporters, after the exposure of adult females and males of the mosquito Anopheles stephensi, a major malaria vector in Asia, to permethrin. Male and female mosquitoes were exposed to insecticide for one hour, then the expression profiles of the ABC transporter genes AnstABCB2, AnstABCB3, AnstABCB4, AnstABCBmember6, AnstABCC11, and AnstABCG4 were analysed after one and 24h. Our results showed that three genes (AnstABCB2, AnstABCBmember6, AnstABCG4) were up-regulated in both sexes; two of these (AnstABCBmember6 and AnstABCG4) have previously been shown to be up-regulated also in larval stages of An. stephensi, supporting a role for these genes in permethrin defence in larvae as well as in adults. Finally, the same ABC transporter genes were activated both in females and males; however, the timing of gene induction was different, with a prompter induction in females than in males.

The choreography of the chemical defensome response to insecticide stress: insights into the Anopheles stephensi transcriptome using RNA-Seq.

Animals respond to chemical stress with an array of gene families and pathways termed "chemical defensome". In arthropods, despite many defensome genes have been detected, how their activation is arranged during toxic exposure remains poorly understood. Here, we sequenced the transcriptome of Anopheles stephensi larvae exposed for six, 24 and 48 hours to the LD50 dose of the insecticide permethrin to monitor transcriptional changes of defensome genes across time. A total of 177 genes involved in insecticide defense were differentially expressed (DE) in at least one time-point, including genes encoding for Phase 0, I, II, III and antioxidant enzymes and for Heat Shock and Cuticular Proteins. Three major patterns emerged throughout time. First, most of DE genes were down-regulated at all time-points, suggesting a reallocation of energetic resources during insecticide stress. Second, single genes and clusters of genes turn off and on from six to 48 hours of treatment, showing a modulated response across time. Third, the number of up-regulated genes peaked at six hours and then decreased during exposure. Our results give a first picture of how defensome gene families respond against toxicants and provide a valuable resource for understanding how defensome genes work together during insecticide stress.

Transcriptome of larvae representing the Rhipicephalus sanguineus complex.

Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) is possibly the most widespread tick species worldwide, responsible for transmitting several vector-borne pathogens of medical and veterinary importance. Here, we explore the transcriptome of R. sanguineus s.l. larvae (Putignano strain). We sequenced total RNA from R. sanguineus s.l. larvae. A total of 15,566,986 short paired-end reads were de novo-assembled into 33,396 transcripts and then annotated and analyzed. Particular attention was paid to transcripts putatively encoding ATP-binding proteins, due to their importance as mechanisms of detoxification and acaricide resistance. Additionally, microsatellite loci were investigated, as these are useful markers for population genetic studies. The present data and analyses provide a comprehensive transcriptomic resource for R. sanguineus. The results presented here will aid further genetic and genomic studies of this important tick species.

Exon-intron structure and sequence variation of the calreticulin gene among Rhipicephalus sanguineus group ticks.

BACKGROUND: Calreticulin proteins (CRTs) are important components of tick saliva, which is involved in the blood meal success, pathogen transmission and host allergic responses. The characterization of the genes encoding for salivary proteins, such as CRTs, is pivotal to understand the mechanisms of tick-host interaction during blood meal and to develop tick control strategies based on their inhibition. In hard ticks, crt genes were shown to have only one intron with conserved position among species. In this study we investigated the exon-intron structure and variation of the crt gene in Rhipicephalus spp. ticks in order to assess the crt exon-intron structure and the potential utility of crt gene as a molecular marker. METHODS: We sequenced the exon-intron region of crt gene in ticks belonging to so-called tropical and temperate lineages of Rhipicephalus sanguineus (sensu lato), Rhipicephalus sp. I, Rhipicephalus sp. III, Rhipicephalus sp. IV, R. guilhoni, R. muhsamae and R. turanicus. Genetic divergence and phylogenetic relationships between the sequences obtained were estimated. RESULTS: All individuals belonging to the tropical lineage of R. sanguineus (s.l.), R. guilhoni, R. muhsamae, R. turanicus, Rhipicephalus sp. III and Rhipicephalus sp. IV analysed showed crt intron-present alleles. However, both crt intron-present and intron-absent alleles were found in Rhipicephalus sp. I and the temperate lineage of R. sanguineus (s.l.), showing the occurrence of an intraspecific intron presence-absence polymorphism. Phylogenetic relationships among the crt intron-present sequences showed distinct lineages for all taxa, with the tropical and temperate lineages of R. sanguineus (s.l.) being more closely related to each other. CONCLUSIONS: We expanded previous studies about the characterization of crt gene in hard ticks. Our results highlighted a previously overlooked variation in the crt structure among Rhipicephalus spp., and among hard ticks in general. Notably, the intron presence/absence polymorphism observed herein can be a candidate study-system to investigate the early stages of intron gain/loss before fixation at species level and some debated questions about intron evolution. Finally, the sequence variation observed supports the suitability of the crt gene for molecular recognition of Rhipicephalus spp. and for phylogenetic studies in association with other markers.

Intra-instar larval cannibalism in Anopheles gambiae (s.s.) and Anopheles stephensi (Diptera: Culicidae).

BACKGROUND: Cannibalism has been observed in a wide range of animal taxa and its importance in persistence and stability of populations has been documented. In anopheline malaria vectors the inter-instar cannibalism between fourth- and first-instar larvae (L4-L1) has been shown in several species, while intra-instar cannibalism remains poorly investigated. In this study we tested the occurrence of intra-instar cannibalism within larvae of second-, third- and fourth-instar (L2, L3 and L4) of Anopheles gambiae (s.s.) and An. stephensi. Experiments were set up under laboratory conditions and the effects of larval density, duration of the contact period among larvae and the presence of an older larva (i.e. a potential cannibal of bigger size) on cannibalism rate were analysed. Cannibalism was assessed by computing the number of missing larvae after 24 and 48 h from the beginning of the experiments and further documented by records with a GoPro videocamera. RESULTS: Intra-instar cannibalism was observed in all larval instars of both species with higher frequency in An. gambiae (s.s.) than in An. stephensi. In both species the total number of cannibalistic events increased from 0-24 to 0-48 h. The density affected the cannibalism rate, but its effect was related to the larval instar and to the presence of older larvae. Interestingly, the lower cannibalism rate between L4 larvae was observed at the highest density and the cannibalism rate between L3 larvae decreased when one L4 was added. CONCLUSIONS: The present study provides experimental evidence of intra-instar cannibalism in the malaria vectors An. gambiae (s.s.) and An. stephensi and highlights the possible occurrence of complex interactions between all larval instars potentially present in the breeding sites. We hypothesize that the high density and the presence of a potential cannibal of bigger size could affect the readiness to attack conspecifics, resulting into low risk larval behavior and lower cannibalism rate. The understanding of cannibalistic behavior and the factors affecting it is of utmost importance for malaria vectors, as nutrition during larval development can strongly affect the fitness of adult female mosquitoes and ultimately their vector ability.