Laura Valerio

Host-feeding patterns of aedes albopictus (Diptera: Culicidae) in urban and rural contexts within Rome province, Italy

Knowledge of the frequency of contact between a mosquito species and its different hosts is essential to understand the role of each vector species in the transmission of diseases to humans and/or animals. However, no data are so far available on the feeding habits of Aedes albopictus in Italy or in other recently colonized temperate regions of Europe, due to difficulties in collecting blood-fed females of this diurnal and exophilic species. We analyzed Ae. albopictus host-feeding patterns in two urban and two rural sites within the area of Rome (Italy). Ae. albopictus was collected using sticky-traps and the blood-meal origin of 303 females was determined by direct dot-ELISA. The blood-fed sample, although representing only 4% of the total Ae. albopictus collected, demonstrates the useful application of sticky-trap in studying the feeding behavior of the species. The human blood index was significantly different among sites, ranging from 79-96% in urban sites to 23-55% in rural sites, where horses and bovines represented the most bitten hosts. The results obtained confirm the plastic feeding behavior shown by Ae. albopictus in its original range of distribution and highlights the high potential of this species as a vector of human pathogens in urban areas of Italy, where both humans and the mosquito itself may reach very high densities.

Field Cage Studies and Progressive Evaluation of Genetically-Engineered Mosquitoes

Background A genetically-engineered strain of the dengue mosquito vector Aedes aegypti, designated OX3604C, was evaluated in large outdoor cage trials for its potential to improve dengue prevention efforts by inducing population suppression. OX3604C is engineered with a repressible genetic construct that causes a female-specific flightless phenotype. Wild-type females that mate with homozygous OX3604C males will not produce reproductive female offspring. Weekly introductions of OX3604C males eliminated all three targeted Ae. aegypti populations after 10–20 weeks in a previous laboratory cage experiment. As part of the phased, progressive evaluation of this technology, we carried out an assessment in large outdoor field enclosures in dengue endemic southern Mexico. Methodology/Principal Findings OX3604C males were introduced weekly into field cages containing stable target populations, initially at 10∶1 ratios. Statistically significant target population decreases were detected in 4 of 5 treatment cages after 17 weeks, but none of the treatment populations were eliminated. Mating competitiveness experiments, carried out to explore the discrepancy between lab and field cage results revealed a maximum mating disadvantage of up 59.1% for OX3604C males, which accounted for a significant part of the 97% fitness cost predicted by a mathematical model to be necessary to produce the field cage results. Conclusions/Significance Our results indicate that OX3604C may not be effective in large-scale releases. A strain with the same transgene that is not encumbered by a large mating disadvantage, however, could have improved prospects for dengue prevention. Insights from large outdoor cage experiments may provide an important part of the progressive, stepwise evaluation of genetically-engineered mosquitoes.

Evidence of polyandry for Aedes aegypti in semifield enclosures.

Female Aedes aegypti are assumed to be primarily monandrous (i.e., mate only once in their lifetime), but true estimates of mating frequency have not been determined outside the laboratory. To assess polyandry in Ae. aegypti with first-generation progeny from wild mosquitoes, stable isotope semen-labeled males ((15)N or (13)C) were allowed to mate with unlabeled females in semifield enclosures (22.5 m(3)) in a dengue-endemic area in southern Mexico. On average, 14% of females were positive for both labels, indicating that they received semen from more than one male. Our results provide evidence of a small but potentially significant rate of multiple mating within a 48-hour period and provide an approach for future open-field studies of polyandry in this species. Polyandry has implications for understanding mosquito ecology, evolution, and reproductive behavior as well as genetic strategies for mosquito control.

A regulatory structure for working with genetically modified mosquitoes: lessons from Mexico.

Policy Platform A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico Janine M. Ramsey 1 , J. Guillermo Bond 1 , Maria Elena Macotela 1 , Luca Facchinelli 2,3 , Laura Valerio 2,4 , David M. Brown 5 , Thomas W. Scott 2 , Anthony A. James 5,6 * 1 Centro Regional de Investigacio´n en Salud Pu´blica, Instituto Nacional de Salud Pu´blica, Tapachula, Chiapas, Me´xico, 2 Department of Entomology, University of California, Davis, California, United States of America, 3 Department of Experimental Medicine, Functional Genomics Center, University of Perugia, Perugia, Italy, 4 Pasteur Institute–Cenci Bolognetti Foundation, University of Rome Sapienza, Rome, Italy, 5 Department of Microbiology and Molecular Genetics, University of California, Irvine, California, United States of America, 6 Department of Molecular Biology and Biochemistry, University of California, Irvine, California, United States of America Introduction Sustainable and effective control of dengue is hampered due to a number of factors, including the lack of evidence- based, locally relevant interventions; insuf- ficient information regarding key compo- nents of virus transmission and vector ecology; failure to implement precise and efficient surveillance systems; inefficient healthcare systems; ineffective health pro- motion and outreach resulting in lack of community dialogue and participation; and a paucity of efficient diagnostic strategies and clinical attention [1]. In- creased research efforts in response to the complexity of this problem have focused on the development of novel technologies that would enhance existing tools for vector-borne disease prevention [2–4]. Genetic strategies to reduce or replace mosquito populations and thereby inter- rupt transmission of dengue viruses are among the new approaches being consid- ered [5–7]. Many of these approaches take advantage of molecular genetic tools to engineer traits that cause lethal pheno- types or confer resistance to the pathogen in the mosquito. Genetic strategies are being advanced through a series of overlapping domains that inform the decision making on feasibility, safety, efficacy, and acceptabil- ity. Although the need to focus on science- based regulation using a risk-assessment framework is gaining support [8], there has been a relative lack of attention on broader community regulations that are explicitly or indirectly required to bring a genetics-based product to the field [3,9– 11]. An evidence-based approach would facilitate the integration, efficacy, and acceptability of policy for an intervention strategy. We addressed the regulatory challenges associated with testing a strain of Aedes aegypti engineered to result in population suppression in contained field trials in southwestern Mexico [12]. This large research effort (designated hereafter as the ‘‘Project’’) combined elements of scientific and social discovery and devel- opment as the basis for moving a new technology from the laboratory to the field. Unlike the rollout of other public- health products such as drugs, vaccines, and insecticides, no pipelines exist to move candidate genetically modified mosquitoes (GMMs) from the laboratory through safety and efficacy trials to field deploy- ment. This lack of a preexisting structure made it necessary for the scientists in the Project to play critical, unbiased roles in formulating the product development pathway. The challenge offered a unique opportunity for potential end users and beneficiaries of the technology to be involved from the beginning in product discovery and development. This ap- proach ensures that requirements for safety and efficacy are included as design features engineered into the modified mosquito strains [13]. It is incumbent on the researchers to identify gaps and assist in development of regulatory norms that should be applied to the products they create. These norms include not only statutory regulations but also a broader regulatory environment that addresses the needs and concerns of all communities in which the product will be applied. We describe here the regulatory and social structures used for obtaining approvals in Mexico. This review of our approach is intended to stimulate analysis and dia- logue that will help refine regulatory practices of genetic-based strategies for vector-borne disease control. Regulatory Domains for the Discovery and Development of Genetically Modified Mosquitoes An initial challenge of the Project was identifying relevant communities [14]. No consensus existed as to what comprises a relevant community to engage for a GMMs research project, nor were there any widely accepted methods for identify- ing their members. We adopted a defini- tion (modified from [15]) in which the community consists of all those individuals who share the identified risks and/or will benefit from the outcome of the proposed research project. In this context, the community coalesces as a result of the project and evolves continuously as it progresses through conceptualization, dis- cussion, and implementation [16]. The community is formed ultimately by those individuals, groups, organizations, and agencies that have legitimate interest in the research, and therefore they must be engaged in an effective and timely man- ner. Successfully moving a novel technology from the laboratory to practical application Citation: Ramsey JM, Bond JG, Macotela ME, Facchinelli L, Valerio L, et al. (2014) A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico. PLoS Negl Trop Dis 8(3): e2623. doi:10.1371/journal.pntd.0002623 Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America Published March 13, 2014 Copyright: s 2014 Ramsey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research and Policy for Infectious Disease Dynamics program of the Science and Technology Directory, Department of Homeland Security; Fogarty International Center, National Institutes of Health; Pasteur Institute – Cenci Bolognetti Foundation; Foundation for the National Institutes of Health through the Grand Challenges in Global Health (GCGH) initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare no conflict of interest. * E-mail: aajames@uci.edu PLOS Neglected Tropical Diseases | www.plosntds.org March 2014 | Volume 8 | Issue 3 | e2623

Development of a Semi-Field System for Contained Field Trials with Aedes aegypti in Southern Mexico

Abstract. Development of new genetic approaches to either interfere with the ability of mosquitoes to transmit dengue virus or to reduce vector population density requires progressive evaluation from the laboratory to contained field trials, before open field release. Trials in contained outdoor facilities are an important part of this process because they can be used to evaluate the effectiveness and reliability of modified strains in settings that include natural environmental variations without releasing mosquitoes into the open field. We describe a simple and cost-effective semi-field system designed to study Aedes aegypti carrying a dominant lethal gene (fsRIDL) in semi-field conditions. We provide a protocol for establishing, maintaining, and monitoring stable Ae. aegypti population densities inside field cages.

Dispersal of Male Aedes aegypti in a Coastal Village in Southern Mexico

Most Aedes aegypti dispersal studies have focused on females because of their central role in dengue virus transmission. Only a few mark-release-recapture (MRR) studies provided insights into male Ae. aegypti dispersal. To fill this knowledge gap, we conducted five male Ae. aegypti MRR experiments in a coastal village in southern Mexico. Small and large male cohorts were marked with fluorescent dusts, released outside buildings, and recaptures were carried out by using backpack aspirators. Recapture rates ranged between 0.35% and 6.55% and median distance traveled was 12-166 meters. A statistically significant difference in median distance traveled with large males dispersing farther than small ones was detected only in one experiment (MRR5: U = 3.5, P < 0.01). Male dispersal data will be useful for constructing and estimating parameter values and validating models that will be used to plan the most effective release strategies for genetically modified male Ae. aegypti.

Distribution of mosquito species in areas with high and low incidence of classic Kaposi’s sarcoma and seroprevalence for HHV‐8

Abstract The ‘promoter‐arthropod’ hypothesis, which postulates that exposure to the bites of certain species of haematophagous arthropods is an environmental risk cofactor linked to human herpes virus 8 (HHV‐8) and Kaposi’s sarcoma, was investigated in the Po River valley, northern Italy. The presence and density of adult female mosquitoes (Diptera: Culicidae) was determined by CDC light trap catches in two adjacent districts, at variance with respect to Kaposi’s sarcoma incidence and HHV‐8 seroprevalence. A total of 3910 specimens belonging to 11 species was collected in 34 rural sites (six municipalities) representative of the two districts. Five of these species are considered to be possible ‘promoters’ because of the irritation their bites cause humans: Aedes vexans (Meigen) and Ae. caspius (Pallas) (87% of sampled promoters), Culex modestus Ficalbi, Culiseta annulata (Schrank) and Coquillettidia richiardii (Ficalbi). Six are probable ‘non‐promoters’: Cx. pipiens s.l., Cx. martinii Medschid, Anopheles claviger (Meigen), An. maculipennis s.l., An. plumbeus Stephens and Uranotaenia unguiculata Edwards. The density of promoters by site was correlated with the incidence rates of Kaposi’s sarcoma at the district level (Pearson’s r= 0.33, P= 0.06) and at the municipal level (r= 0.50, P < 0.01). Similar correlations emerged for non‐promoters (r= 0.48, P < 0.01 and r = 0.42, P= 0.01, respectively). The density of promoters was higher than that of non‐promoters in sites with livestock (odds ratio, OR = 2.8, 95% CI 2.2–3.6) and in municipalities with Kaposi’s sarcoma cases (OR = 2.5, 95% CI 1.7–3.5). The study provides additional evidence of the association between the density of some mosquito species and Kaposi’s sarcoma.

Regulation of Aedes aegypti Population Dynamics in Field Systems: Quantifying Direct and Delayed Density Dependence

Transgenic strains of Aedes aegypti have been engineered to help control transmission of dengue virus. Although resources have been invested in developing the strains, we lack data on the ecology of mosquitoes that could impact the success of this approach. Although studies of intra-specific competition have been conducted using Ae. aegypti larvae, none of these studies examine mixed age cohorts at densities that occur in the field, with natural nutrient levels. Experiments were conducted in Mexico to determine the impact of direct and delayed density dependence on Ae. aegypti populations. Natural water, food, and larval densities were used to estimate the impacts of density dependence on larval survival, development, and adult body size. Direct and delayed density-dependent factors had a significant impact on larval survival, larval development, and adult body size. These results indicate that control methods attempting to reduce mosquito populations may be counteracted by density-dependent population regulation.

Mathematical Models as Aids for Design and Development of Experiments: The Case of Transgenic Mosquitoes

ABSTRACT We demonstrate the utility of models as aids in the design and development of experiments aimed at measuring the effects of proposed vector population control strategies. We describe the exploration of a stochastic, age-structured model that simulates field cage experiments that test the ability of a female-killing strain of the mosquito Aedes aegypti (L.) to suppress a wild-type population. Model output predicts that choices of release ratio and population size can impact mean extinction time and variability in extinction time among experiments. We find that unless fitness costs are >80% they will not be detectable in experiments with high release ratios. At lower release ratios, the predicted length of the experiment increases significantly for fitness costs >20%. Experiments with small populations may more accurately reflect field conditions, but extinction can occur even in the absence of a functional female-killing construct because of stochastic effects. We illustrate how the model can be used to explore experimental designs that aim to study the impact of density dependence and immigration; predictions indicate that cage population eradication may not always be obtainable in an operationally realistic time frame. We propose a method to predict the extinction time of a cage population based on the rate of population reduction with the goal of shortening the duration of the experiment. We discuss the model as a tool for exploring and assessing the utility of a wider range of scenarios than would be feasible to test experimentally because of financial and temporal restraints.

Comparison of model predictions and laboratory observations of transgene frequencies in continuously-breeding mosquito populations

The persistence of transgenes in the environment is a consideration in risk assessments of transgenic organisms. Combining mathematical models that predict the frequency of transgenes and experimental demonstrations can validate the model predictions, or can detect significant biological deviations that were neither apparent nor included as model parameters. In order to assess the correlation between predictions and observations, models were constructed to estimate the frequency of a transgene causing male sexual sterility in simulated populations of a malaria mosquito Anopheles gambiae that were seeded with transgenic females at various proportions. Concurrently, overlapping-generation laboratory populations similar to those being modeled were initialized with various starting transgene proportions, and the subsequent proportions of transgenic individuals in populations were determined weekly until the transgene disappeared. The specific transgene being tested contained a homing endonuclease gene expressed in testes, I-PpoI, that cleaves the ribosomal DNA and results in complete male sexual sterility with no effect on female fertility. The transgene was observed to disappear more rapidly than the model predicted in all cases. The period before ovipositions that contained no transgenic progeny ranged from as little as three weeks after cage initiation to as long as 11 weeks.