Exploring protandry and pupal size selection for Aedes albopictus sex separation

Background: We explored the possibility to improve male/female separation (sexing) in Aedes albopictus by selection of two strains, one toward increasing sex dimorphism and another toward increasing protandry. In the laboratory we selected and crossed small males with large females to exploit dimorphism, and early pupating males with late pupating females to exploit protandry. Results: While selection for enhanced dimorphism was not a profitable character, the selection for enhanced protandry up to F10 produced significant improvement in the time interval between male and female pupation. By collecting the pupae at 24 h from the beginning of pupation, without any sieving operation, we obtained about 28.50% of pupae (calculated in relation to the estimated initial number of first instar larvae used), vs 26.49% we had in the control strain, and, more interestingly, when checking the sex ratio of these pupae we observed a presence of females of 0.92% vs 23.02% in the control strain. We also modified our egg hatching protocol from the previous standard procedure that required keeping the eggs in the glass hatching container overnight (for about 14-16 h) to a new protocol where eggs are kept in the hatching container for 4 h in order to obtain more synchronized larvae. This was possible without any reduction in the egg hatching rate. Conclusions: In Aedes albopictus it is possible to develop hyper-protandric strains useful to produce male pupae without applying other sexing systems. This represents a considerable achievement assisting the Sterile Insect Technique application, allowing improvement of the current sexing method based on mechanical separation. More investigations are under way in order to further enhance the male productivity capacity of the strain and to determine whether the selection has any impact on the male fitness parameters

Dynamics of mtDNA introgression during species range expansion. Insights from an experimental longitudinal study

Introgressive hybridization represents one of the long-lasting debated genetic consequences of species range expansion. Mitochondrial DNA has been shown to heavily introgress between interbreeding animal species that meet in new sympatric areas and, often, asymmetric introgression from local to the colonizing populations has been observed. Disentangling among the evolutionary and ecological processes that might shape this pattern remains difficult, because they continuously act across time and space. In this context, long-term studies can be of paramount importance. Here, we investigated the dynamics of mitochondrial introgression between two mosquito species (Aedes mariae and Ae. zammitii ) during a colonization event that started in 1986 after a translocation experiment. By analyzing 1,659 individuals across 25 years, we showed that introgression occurred earlier and at a higher frequency in the introduced than in the local species, showing a pattern of asymmetric introgression. Throughout time, introgression increased slowly in the local species, becoming reciprocal at most sites. The rare opportunity to investigate the pattern of introgression across time during a range expansion along with the characteristics of our study-system allowed us to support a role of demographic dynamics in determining the observed introgression pattern

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 ªfavorableº 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

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

Biological compatibility between two temperate lineages of brown dog ticks, Rhipicephalus sanguineus (sensu lato)

Background: The brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called "temperate" and "tropical" lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal). Methods: Ticks morphologically identified as Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted. Results: Crossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically. Conclusions: The temperate lineages of Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks

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

Population genomics of the Asian tiger mosquito, Aedes albopictus. Insights into the recent worldwide invasion

Aedes albopictus, the “Asian tiger mosquito,” is an aggressive biting mosquito native to Asia that has colonized all continents except Antarctica during the last ~30–40 years. The species is of great public health concern as it can transmit at least 26 arboviruses, including dengue, chikungunya, and Zika viruses. In this study, using double- digest Restriction site-Associated DNA (ddRAD) sequencing, we developed a panel of ~58,000 single nucleotide polymorphisms (SNPs) based on 20 worldwide Ae. albopic-tus populations representing both the invasive and the native range. We used this genomic- based approach to study the genetic structure and the differentiation of Ae. albopictus populations and to understand origin(s) and dynamics of the recent inva-sions. Our analyses indicated the existence of two major genetically differentiated population clusters, each one including both native and invasive populations. The de-tection of additional genetic structure within each major cluster supports that these SNPs can detect differentiation at a global and local scale, while the similar levels of genomic diversity between native and invasive range populations support the scenario of multiple invasions or colonization by a large number of propagules. Finally, our re-sults revealed the possible source(s) of the recent invasion in Americas, Europe, and Africa, a finding with important implications for vector- control strategies

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

Evidence of reinforcement of premating isolation between two species of the genus Ochthebius (Coleoptera : Hydraenidae)

The increase in premating reproductive isolation between recently diverged and potentially interbreeding taxa resulting from selection against hybridization (reinforcement) is one of the most contentious issues in evolutionary biology. After many years of debate, its plausibility under various conditions has been shown by theoretical studies and some cases have been documented. At present, interest is arising about the frequency and importance of reinforcement in nature. Ochthebius quadricollis and Ochthebius sp. A are two hydraenid beetles inhabiting marine rock pools in the Mediterranean basin. By molecular analysis of a contact zone between the two species along the Italian Tyrrhenian coast, full reproductive isolation between the two species was evidenced. However, the finding of introgressed specimens at some diagnostic loci suggested that gene flow occurred in the past but then ceased. In this article, by analyzing species composition of mating couples collected in sympatric localities, we show the existence of strong assortative mating between the two species in nature. In laboratory multiple-choice mating trials, sympatric populations showed greater assortative mating than allopatric populations. Reinforcement is suggested as the most parsimonious hypothesis to explain the evolution of discriminative mate recognition systems occurring among O. quadricollis and Ochthebius sp. A under sympatric, but not allopatric, populations