We tested the hypothesis that bioclimatic conditions affect the species richness, abundance, and diversity of sand flies in neighboring physiographic regions with endemic transmission of cutaneous leishmaniasis. A sampling program was performed in southeastern Mexico in the High Plateau of Chiapas and the Eastern Mountains physiographic regions, between April 2009 and March 2011. CDC light traps were placed in five randomly selected houses, four CDC traps in four transects in the surrounding vegetation zones, and a modified Magoon trap baited with one protected person was installed at the periphery of the villages at each study site. A total of 12,349 sand flies were identified as species, of which 98.2

Mass-production of Aedes aegypti and Ae. albopictus for use in vector control programs involving the sterile insect technique (SIT) requires the standardization of rearing techniques, including the production of eggs. Following published protocols, egg quantification for both species was performed based on the weight and volume characteristics of batches of 1,000 to 27,000 eggs from insect colonies that originated from natural populations in Chiapas, Mexico. The findings were validated by direct measurement of egg dimensions. On average, Ae. aegypti eggs were similar to 40% heavier and occupied similar to 25% larger volume than those of Ae. albopictus (p < 0.001). Egg numbers were readily predicted by linear correlation with the weight and volume of eggs of both species (p < 0.001). Volumetric measures were significantly more consistent among replicates than the weight measurements (p < 0.02). Direct measurements revealed that the eggs of Ae. aegypti were similar to 10% longer (p < 0.001), similar to 13% wider (p < 0.001), and also more variable in size than those of Ae. albopictus. These species also differed significantly in egg length:width ratios (p < 0.001). We conclude that proxy indicators of egg numbers, such as weight and volume, should greatly assist in standardizing larval rearing procedures.

In the sterile insect technique, it is important to measure the impact of mass-rearing and handling of sterile males to allow a successful control of the target wild population. This study evaluates the effect of pre-release chilling on the survival, escape ability, and sexual competitiveness of male Aedes aegypti . To determine survival and escape ability, mosquitoes were chilled at 4 °C using four different treatments of either one exposure (25 min) or two consecutive exposures (25 + 25 min, 25 + 50 min, 25 + 100 min). For sexual competitiveness, two different treatments were evaluated, chilling for 25 min once and twice. Results showed that the longest exposure to chilling caused a significant reduction of survival time, from 67 to 54 days. Escape ability was reduced by the first chilling from 25 to 7% and with the second chilling, it was reduced from 30 to 24% in the control to 4.9, 2.0 and 0.5% for 25, 50 and 100 min, respectively. Sexual competitiveness index was reduced from 1.16 in the control, to 0.32 and − 0.11 for treatments involving one and two chilling periods, respectively. It is recommended to increase the chilling temperature and reduce the exposure time to reduce the harmful effects on sterile males.

Worldwide, vector-transmitted diseases account for more than 17% of all infectious diseases and cause more than 700,000 deaths annually. The primary strategy to control the transmission of dengue, chikungunya and Zika focuses on the control of their vectors. Vector control programs depend mainly on the use of insecticides. However, these measures have failed to yield the desired results because the lack of proper control decreases their effectiveness over time. Also, the effect of insecticide use on non-target organisms, environmental contamination, and the development of insecticide resistance impose continuous challenges for insecticide-dependant control programs. The sterile insect technique, on the other hand, is a non-polluting and environment-friendly technique. The use of sterile insects is generally well established in agriculture, but human health interventions often require a consensus that combines social perceptions with scientific evidence. Factors associated with acceptance of the releases of sterile male Aedes aegypti mosquitoes were assessed with logistic regression models for two communities (Rio Florido and Hidalgo) in Tapachula, Chiapas, Mexico. The favourable opinion of the respondents about the project workers, and the fact that the releases of sterile mosquitoes would be managed by the Secretaría de Salud, were found to be factors that supported the acceptance of sterile mosquito releases. Factors associated with disapproval were community (Rio Florido and Hidalgo) and the misbelief that sterile male mosquitoes can bite. The differences observed between the communities included in this study might be the result of experiences with the releases, of social and community organisation, and of leadership by social actors.

Sterile males of Aedes aegypti were released once a week for 8 weeks to evaluate the dispersal efficiency of ground and aerial drone release methods in a rural village of 26 Ha in southern Mexico. Indoor and outdoor BG-Sentinel traps were placed in 13–16 houses distributed throughout the village. The BG traps were activated 48 h after the release of the sterile males and functioned for a 24 h period following each release. Over the 8-week period of simultaneous ground and aerial releases, an average of 85,117 ± 6457 sterile males/week were released at ground level and 86,724 ± 6474 sterile males/week were released using an aerial drone. The ground release method resulted in higher numbers of captured males (mean = 5.1 ± 1.4, range 1.1–15.7 sterile males/trap) compared with the aerial release method (mean = 2.6 ± 0.8, range 0.5–7.3 sterile males/trap) (p < 0.05). Similarly, the prevalence of traps that captured at least one sterile male was significantly higher for ground release compared to the aerial release method (p < 0.01). The lower numbers of sterile males captured in the aerial release method could be due to mortality or physical injury caused by the chilling process for immobilization, or the compaction of these insects during transport and release. However, aerial releases by a two-person team distributed insects over the entire village in just 20 min, compared to ~90 min of work for a five-person team during the ground release method. Ground release also resulted in higher aggregations of males and some villagers reported feeling discomfort from the presence of large numbers of mosquitoes in and around their houses. We conclude that modifications to the handling and transport of sterile males and the design of containers used to store males are required to avoid injury and to improve the efficiency of aerial releases for area-wide SIT-based population suppression programs targeted at mosquito vectors of human disease.

Dengue and other Aedes-borne diseases have dramatically increased over the last decades. The Sterile Insect Technique (SIT) has been successfully used as part of integrated pest strategies to control populations of insect-plant and livestock pests and is currently being tested as a potential method to reduce mosquito populations in an environmentally friendly approach. However, during the mass rearing steps needed to produce millions of mosquitoes, egg storage and preservation are essential for a certain amount of time. Eggs of Aedes aegypti have a chorionic pad that functions as a sticky substance to glue them onto the inner walls of larval breeding sites. The chorionic pad is chemically made of hyaluronic acid, a hygroscopic compound, responsible to protect them from desiccation over time. Two commercial products with hygroscopic properties, hydrolyzed collagen, and Hyalurosmooth®, both were tested to assess their ability to prolong egg life storage for A. aegypti and A. albopictus. Results showed that 85–95% of Ae. aegypti eggs were able to hatch up to week 8 after being treated with both hydrophilic compounds, compared with the control 66.3%. These two substances showed promising effects for keeping Ae. aegypti eggs viable during prolonged storage in mass rearing insect production focused on vector control SIT programs.

The sterile insect technique may prove useful for the suppression of mosquito vectors of medical importance in regions where arboviruses pose a serious public health threat. In the present study, we examined the effects of sterilizing irradiation doses across different ratios of fertile:irradiated males on the mating competitiveness of Ae. aegypti and Ae. albopictus under laboratory and field-cage conditions. For both species, the percentage of females inseminated and the number of eggs laid over two gonotrophic cycles varied significantly in mating treatments involving 1:1, 1:5, and 1:10 fertile:irradiated males compared to controls of entirely fertile or entirely irradiated males but was not generally affected by the irradiation dose. Egg hatching was negatively affected in females exposed to increasing proportions of irradiated males in both laboratory and field cages. Male competitiveness (Fried’s index) values varied from 0.19 to 0.58 in the laboratory and were between 0.09 and 1.0 in field cages, depending on th species. Competitiveness values were negatively affected by th eirradiation dose in both species under field-cage conditions, whereas in the laboratory, Ae. albopictus was sensitive to the dose but Ae. aegypti was not. In general, male competitiveness was similar across all mating regimes. Most importantly, induced egg sterility was positively correlated with the proportion of irradiated males present in the mating treatments, reaching a maximum of 88% under field-cage conditions for both Ae. aegypti and Ae. albopictus males treated with 50 and 40 Gy irradiation, respectively. These results indicate that sterile males produced at our facility are suitable and competitive enough for field pilot SIT projects and provide guidance to decide the optimal sterile:fertile ratios.

Indoor and outdoor ovitraps were placed in 15 randomly selected houses in two rural villages in Chiapas, southern Mexico. In addition, ovitraps were placed in five transects surrounding each village, with three traps per transect, one at the edge, one at 50 m, and another at 100 m from the edge of the village. All traps were inspected weekly. A transect with eight traps along a road between the two villages was also included. Population fluctuations of Aedes aegypti and Ae. albopictus were examined during 2016–2018 by counting egg numbers. A higher number of Aedes spp. eggs was recorded at Hidalgo village with 257,712 eggs (60.9%), of which 58.1% were present in outdoor ovitraps and 41.9% in indoor ovitraps, compared with 165,623 eggs (39.1%) collected in the village of Río Florido, 49.0% in outdoor and 51.0% in indoor ovitraps. A total of 84,047 eggs was collected from ovitraps placed along transects around Río Florido, compared to 67,542 eggs recorded from transects around Hidalgo. Fluctuations in egg counts were associated with annual variation in precipitation, with 2.3 to 3.2-fold more eggs collected from ovitraps placed in houses and 4.8 to 5.1-fold more eggs in ovitraps from the surrounding transects during the rainy season than in the dry season, respectively. Aedes aegypti was the dominant species during the dry season and at the start of the rainy season in both villages. Aedes albopictus populations were lower for most of the dry season, but increased during the rainy season and predominated at the end of the rainy season in both villages. Aedes albopictus was also the dominant species in the zones surrounding both villages. The numbers of eggs collected from intradomiciliary ovitraps were strongly correlated with the numbers of eggs in peridomiciliary ovitraps in both Río Florido (R2adj = 0.92) and Hidalgo (R2adj = 0.94), suggesting that peridomiciliary sampling could provide an accurate estimate of intradomiciliary oviposition by Aedes spp. in future studies in these villages. We conclude that the feasibility of sterile insect technique (SIT)-based program of vector control could be evaluated in the isolated Ae. aegypti populations in the rural villages of our baseline study.

Objective. To compare the efficacy of three modern larvicides with the organophosphate temephos for control of Aedes aegypti in water tanks in Chiapas. Materials and methods. Trials were performed to compare the efficacy of pyriproxyfen, novaluron, two formulations of spinosad (granules and tablets) and temephos in oviposition traps and domestic water tanks. Results. Pyriproxyfen and te­mephos provided 2-3 weeks of complete control of larvae in oviposition traps, whereas spinosad granules and novaluron provided 7-12 weeks of control. Treatment of water tanks resulted in a significant reduction in oviposition by Ae. aegypti in houses (p<0.001). Higher numbers of larvae were present in temephos and pyriproxyfen-treated water tanks compared to novaluron and spinosad tablet treatments during most of the study. Conclusion. Spinosad formulations and novaluron were effective larvicides in this region. The poor performance of temephos may be indicative of reduced susceptibility in Ae. aegypti populations in Chiapas

Objective.To determine the time of oogenic development and the length of the gonotrophic cycle of Ae. aegypti and Ae. albopictus in laboratory. Materials and methods. Bloodfed females of Ae. aegypti and Ae. albopictus were dissected every 4 h to determine the development status of the follicles according to the Christophers' stages. Results. The minimum time of oocyte maturation in Ae. aegypti and Ae. albopictus was 64-82 h and 52-64 h post-feeding, respectively. We found that the gonotrophic cyde of Ae. aegypti (3.7-4.2 d) is longer than that of Ae. albopictus (3.2-3.7 d).The follicle length showed significant differences between species at Christophers' stages 2" and 5, whereas follide amplitude was different between the two mosquitoes at stages 2", 3 and 4. Conclusions. The study provided new evidence on the reproductive strategies of Ae. albopictus and Ae. aegypti females that coexist in the Neotropical region of Mexico.

The Sterile Insect Technique (SIT) is a pest control method where large numbers of sterile males are released to induce sterility in wild populations. Since a successful SIT application depends on the released sterile males being competitive with wild males, standard quality control tests are a necessary component of any SIT program. Flight ability (ability to fly out from a device) is a reliable indicator of insect quality. Based on previous studies, we developed four new tubular devices constructed with locally available materials to explore their potential as flight test devices for Aedes aegypti (L.) mass-reared males. Males were allowed to fly upwards through a vertical tube, the ones that flew out were considered successful. The effect of male age (0 to 21 d old), test time interval (30 min to 24 h), and the design of the device (40 and 80 cm height and 2 and 3.5 cm diameter) were evaluated. Our devices determined differences in the flight ability of Ae. aegypti males of different ages. During the first minutes, more old males escaped than young males in three out of four types of devices. However, young males reached higher rates of escape in all cases after 24 h. For standard quality control tests, we recommend testing 2- to 3-d-old sexually mature males in the high and narrow device (80 × 2 cm). Further observations for time intervals between 1 and 5 h might be performed to decide the shortest and more representative interval to use.

OBJECTIVE:To determine the species distribution, abundance, and diversity of culicids in the Yucatan Peninsula (YP); their potential distribution, using ecological niche modeling (ENM), and the risk of contact with urban and rural populations.MATERIALS AND METHODS:A cross-sectional study was carried out through the YP. The diversity of species was determined with the Shannon index. The potential distribution of the culicids was determined through the ENM, as well as the risk of urban and rural populations through contact with vectors.RESULTS:A total of 10 699 specimens, 15 genera and 52 species were registered. Campeche and the Biosphere Reserve of Calakmul exhibited the highest diversity.CONCLUSIONS:The ENM predict a high suitability in all the YP of Ae. aegypti, An. albimanus, An. pseudopuntipennis, Cx. coronator, and Cx. quinquefasciatus. The vector species that exhibited the highest risk of contact in the YP were Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus.

The sterile insect technique (SIT) may offer a means to control the transmission of mosquito borne diseases. SIT involves the release of male insects that have been sterilized by exposure to ionizing radiation. We determined the effects of different doses of radiation on the survival and reproductive capacity of local strains of Aedes aegypti and Ae. albopictus in southern Mexico. The survival of irradiated pupae was invariably greater than 90% and did not differ significantly in either sex for either species. Irradiation had no significant adverse effects on the flight ability (capacity to fly out of a test device) of male mosquitoes, which consistently exceeded 91% in Ae. aegypti and 96% in Ae. albopictus. The average number of eggs laid per female was significantly reduced in Ae. aegypti at doses of 15 and 30 Gy and no eggs were laid by females that had been exposed to 50 Gy. Similarly, in Ae. albopictus, egg production was reduced at doses of 15 and 25 Gy and was eliminated at 35 Gy. In Ae. aegypti, fertility in males was eliminated at 70 Gy and was eliminated at 30 Gy in females, whereas in Ae. albopictus, the fertility of males that mated with untreated females was almost zero (0.1%) in the 50 Gy treatment and female fertility was eliminated at 35 Gy. Irradiation treatments resulted in reduced ovary length and fewer follicles in both species. The adult median survival time of both species was reduced by irradiation in a dose-dependent manner. However, sterilizing doses of 35 Gy and 50 Gy resulted in little reduction in survival times of males of Ae. albopictus and Ae. aegypti, respectively, indicating that these doses should be suitable for future evaluations of SIT-based control of these species. The results of the present study will be applied to studies of male sexual competitiveness and to stepwise evaluations of the sterile insect technique for population suppression of these vectors in Mexico.

Collections of mosquitoes were conducted for the surveillance of species of medical importance in the state of Quintana Roo, Mexico, in June of 2017. Species collected included Psorophora (Psorophora) cilipes and Culex (Melanoconion) theobaldi, both new records for the state. Psorophora cilipes was previously recorded in Mexico State and Cx. theobaldi in Chiapas State. With the addition of these species, the mosquito fauna of Quintana Roo State now includes 81 species. Quintana Roo and Tamaulipas State with 82 species are the most mosquito-rich states in Mexico, based on currently available records. The specimens collected in this study were deposited in the Culicidae collection of the Universidad Autónoma Agraria Antonio Narro Unidad Laguna.

The invasive mosquito Aedes albopictus is currently distributed in most of the southern Mexican region. Since the species was first recorded in the state of Tamaulipas, in northeastern Mexico in 1988, it has expanded its distribution throughout the Sierra Madre Oriental and Gulf of Mexico to the Neotropical region of the country. Currently the species occurs in the states of Tamaulipas, Coahuila, Nuevo Leon, Veracruz, Chiapas, Morelos, Quintana Roo, Sinaloa, San Luis Potosi, and Hidalgo. This is the first report of the mosquito in the states of Tabasco and Yucatan and the confirmation of its presence in Quintana Roo state. Aedes albopictus has been incriminated as a secondary vector of diseases such as those caused by dengue, chikungunya, and Zika viruses, which have caused epidemic outbreaks in most tropical and subtropical regions of Mexico; therefore, surveillance for the detection of Ae. albopictus is paramount so that targeted control strategies can be implemented for its control throughout Mexico.

Many countries in Latin America have recently experienced outbreaks of Zika and chikungunya fever, in additional to the usual burden imposed by dengue, all of which are transmitted by Aedes aegypti in this region. To identify potential larvicides, we determined the toxicity of eight modern insecticides to A. aegypti larvae from a colony that originated from field-collected insects in southern Mexico. The most toxic compounds were pyriproxyfen (which prevented adult emergence) and λ-cyhalothrin, followed by spinetoram, imidacloprid, thiamethoxam, and acetamiprid, with chlorantraniliprole and spiromesifen the least toxic products. Field trails performed in an urban cemetery during a chikungunya epidemic revealed that insecticide-treated ovitraps were completely protected from the presence of Aedes larvae and pupae for 6 and 7 weeks in spinosad (Natular G30) and λ-cyhalothrin-treated traps in both seasons, respectively, compared to 5–6 weeks for temephos granule-treated ovitraps, but was variable for pyriproxyfen-treated ovitraps with and 1 and 5 weeks of absolute control in the dry and rainy seasons, respectively. Insecticide treatments influenced the mean numbers of Aedes larvae + pupae in each ovitrap, mean numbers of eggs laid, and percentage of egg hatch over time in both trials. The dominant species was A. aegypti in both seasons, although the invasive vector Aedes albopictus was more prevalent in the rainy season (26.7%) compared to the dry season (10.2%). We conclude that the granular formulation of spinosad (Natular G30) and a suspension concentrate formulation of λ-cyhalothrin proved highly effective against Aedes spp. in both the dry and rainy seasons in the cemetery habitat in this region.

The arrival of chikungunya fever (CHIKF) in Latin American countries has been expected to trigger epidemics and challenge health systems. Historically considered as dengue-endemic countries, abundant Aedes aegypti populations make this region highly vulnerable to chikungunya virus (CHIKV) circulation. This review describes the current dengue and CHIKF epidemiological situations, as well as the role of uncontrolled Ae. aegypti and Aedes albopictus vectors in spreading the emerging CHIKV. Comments are included relating to the vector competence of both species and failures of surveillance and vector control measures. Dengue endemicity is a reflection of these abundant and persistent Aedes populations that are now spreading CHIKV in the Americas. This article forms part of a symposium in Antiviral Research on "Chikungunya discovers the New World."

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

The abundance, richness and diversity of mosquitoes and aquatic insects associated with their oviposition sites were surveyed along eight states of the Pacific coast of Mexico. Diversity was estimated using the Shannon index (H’), similarity measures and cluster analysis.

Background The larvicidal efficacy of the naturally derived insecticide spinosad, for control of immature stages of Anopheles albimanus and associated culicids, was compared to that of synthetic and biological larvicides. Effects on non-target insects were also determined. Methods A field trial was performed in replicated temporary pools during the rainy season, in southern Mexico. Pools were treated with 10 ppm a.i. spinosad (Tracer 480SC), Bti granules applied at 2 kg/ha (VectoBac WDG, ABG-6511), and 100 ml/ha temephos (50 EC), or an untreated control. Numbers of immature mosquitoes, and aquatic insects in pools were monitored for 20 weeks. Results Samples of immature mosquitoes comprised approximately 10% An. albimanus , 70% Culex spp. (mostly Cx. melanoconion and Cx. coronator ) and 20% Uranotaenia lowii . The most effective larvicides were spinosad and temephos that eliminated An. albimanus in 16 out of 20 post-treatment samples, or 9 weeks of continuous control of immature stages, respectively. These larvicides resulted in 15 and 5 weeks of elimination of Culex spp., respectively, or 20 and 4 weeks of continuous elimination of U. lowii , respectively. Bti treatment provided little consistent control. Aquatic insects were recorded comprising 3 orders, 20 families, 40 genera and 44 species. Shannon diversity index values (H’) for aquatic insects were highest in the control (0.997) and Bti (0.974) treatments, intermediate in the spinosad treatment (0.638) and lowest in the temephos treatment (0.520). Severely affected non-target insects in the spinosad and temephos treated pools were predatory Coleoptera, Hemiptera and Odonata, which in the case of spinosad was likely due to the high concentration applied. Bti had little effect on aquatic insects. Conclusions The spinosad treatment retained larvicidal activity for markedly longer than expected. Spinosad is likely to be an effective tool for control of anopheline and other pool-breeding mosquitoes in tropical regions. Non-target effects of spinosad on aquatic insects merit further study, but were likely related to the concentration of the product used.