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Egg number quantification for mass-rearing of Aedes aegypti and Aedes albopictus: validation by direct measurement
Abstract
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 ∼40% heavier and occupied ∼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 ∼10% longer (p < 0.001), ∼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.
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Background
The diseases for which Aedes aegypti is a vector are worrisome. The high vector competence of this mosquito, as well as its anthropophilia and ability to adapt to the urban environment, allows it to exploit many habitats, making its prevention an arduous task. Despite current disease control measures focused on the mosquito, the effectiveness in containing its dispersion still requires improvement; thus greater knowledge about this insect is fundamental.
Methods
Aedes aegypti egg morphology and embryonic development were analyzed from eggs of the insectary of the Institute of Biomedical Sciences of the University of São Paulo. Optical (light and confocal) and electronic (transmission and scanning) microscopy were used to analyze the morphological and ultrastructural features of the eggs. Embryos were observed in the initial (0–20.5 h after egg-laying), intermediate (20.6–40.1 h after egg-laying), and final (40.2–61.6 h) stages of development, and kept at a temperature of 28 °C ± 1 °C until collection for processing.
Results
Eggs of Ae. aegypti were whitish at the time of oviposition, and then quickly became black. The egg length was 581.45 ± 39.73 μm and the width was 175.36 ± 11.59. Access to the embryo was difficult due to the egg morphology, point of embryonic development, and difficult permeability of the exochorion (mainly in fixation). Only about 5% of the collected eggs were successfully processed. In the initial stage of embryonic development, characteristics suggestive of intense cellular activity were found. In the intermediate stage, the beginning of the segmentation process was evident. In the final phase, it was possible to differentiate the cephalic region and the thoracic and abdominal segments.
Conclusion
The chorion was found to be an important protective barrier and a limiting factor for the evaluation of the embryos and mosquito embryonic cells, indicating that further studies need to be carried out to identify the reason that this occurs.
Graphical abstract
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.
BACKGROUND:Important arboviral diseases, such as dengue, chikungunya, and Zika virus infections, are transmitted mainly by the Aedes aegypti vector. So far, controlling this vector species with current tools and strategies has not demonstrated sustainable and significant impacts. Our main objective was to evaluate whether open field release of sterile males, produced from combining the sterile insect technique using radiation with the insect incompatible technique through Wolbachia-induced incompatibility (SIT/IIT), could suppress natural populations of Ae. aegypti in semi-rural village settings in Thailand. METHODOLOGY/PRINCIPAL FINDINGS:Irradiated Wolbachia-infected Aedes aegypti males produced by the SIT/IIT approach were completely sterile and were able to compete with the wild fertile ones. Open field release of these sterile males was conducted in an ecologically isolated village in Chachoengsao Province, eastern Thailand. House-to-house visit and media reports resulted in community acceptance and public awareness of the technology. During intervention, approximately 100-200 sterile males were released weekly in each household. After 6 months of sterile male release, a significant reduction (p
Background
Aedes aegypti mosquitoes are vectors of a variety of emerging viral pathogens, including yellow fever, dengue, chikungunya, and Zika virus. This species has established endemic populations in all cities across southern New Mexico sampled to date. Presently, control of Aedes-borne viruses relies on deployment of insecticides to suppress mosquito populations, but the evolution of insecticide resistance threatens the success of vector control programs. While insecticide resistance is quite common in Ae. aegypti field populations across much of the U.S., the resistance status of this species in populations from New Mexico has not previously been assessed.
Results
First, we collected information on pesticide use in cities in southern New Mexico and found that the most commonly used active ingredients were pyrethroids. The use of insecticides with the same mode-of-action over multiple years is likely to promote the evolution of resistance. To determine if there was evidence of resistance in some cities in southern New Mexico, we collected Ae. aegypti from the same cities and established laboratory strains to assess resistance to pyrethroid insecticides and, for a subset of populations, to organophosphate insecticides. F2 or F4 generation mosquitoes were assessed for insecticide resistance using bottle test bioassays. The majority of the populations from New Mexico that we analyzed were resistant to the pyrethroids permethrin and deltamethrin. A notable exception to this trend were mosquitoes from Alamogordo, a city that did not report using pyrethroid insecticides for vector control. We screened individuals from each population for known knock down resistance (kdr) mutations via PCR and found a strong association between the presences of the F1534C kdr mutation in the para gene of Ae. aegypti (homologue to F1534C in Musca domestica L.) and pyrethroid resistance.
Conclusion
High-level pyrethroid resistance is common in Ae. aegypti from New Mexico and geographic variation in such resistance is likely associated with variation in usage of pyrethroids for vector control. Resistance monitoring and management is recommended in light of the potential for arbovirus outbreaks in this state. Also, alternative approaches to mosquito control that do not involve insecticides should be explored.
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.
Aedes aegypti represents the principal vector of many arthropod-borne diseases in tropical areas worldwide. Since mosquito control strategies are mainly based on use of insecticides, resistance development can be expected to occur in frequently exposed Ae. aegypti populations. Surveillance on resistance development as well as testing of insecticide susceptibility is therefore mandatory and needs further attention by national/international public health authorities. In accordance, we here conducted a study on Ae. aegypti resistance development towards several often used insecticides, i.e., malathion, deltamethrin, permethrin, λ -cyhalothrin, bendiocarb, and cyfluthrin, in the periurban area of Banjarmasin city, Kalimantan, Indonesia. Our results clearly showed resistance development of Ae. aegypti populations against tested insecticides. Mortalities of Ae. aegypti were less than 90% with the highest resistance observed against 0.75% permethrin. Collected mosquitoes from Banjarmasin also presented high level of resistance development to 0.1% bendiocarb. Molecular analysis of voltage-gated sodium channel ( Vgsc ) gene showed significant association of V1016G gene point mutation in resistance Ae. aegypti phenotypes against 0.75% permethrin. However, F1534C gene point mutation did not correlate to Ae. aegypti insecticide resistance to 0.75% permethrin. Irrespective of periurban areas in Kalimantan considered as less densed island of Indonesia, Ae. aegypti -derived resistance to different routinely applied insecticides occurred. Our findings evidence that Ae. aegypti insecticide resistance is most likely spreading into less populated areas and thus needs further surveillance in order to delay Ae. aegypti resistance development.
Aedes aegypti is a major vector of arboviruses that may be controlled on an area-wide basis using the sterile insect technique (SIT). Larval diet is a major factor in mass-rearing for SIT programs. We compared dietary effects on immature development and adult fitness-related characteristics for an International Atomic Energy Agency (IAEA) diet, developed for rearing Ae. albopictus, and a standardized laboratory rodent diet (LRD), under a 14:10 h (light:dark) photoperiod ("light" treatment) or continuous darkness during larval rearing. Larval development was generally fastest in the IAEA diet, likely reflecting the high protein and lipid content of this diet. The proportion of larvae that survived to pupation or to adult emergence did not differ significantly between diets or light treatments. Insects from the LRD-dark treatment produced the highest proportion of male pupae (93% at 24 h after the beginning of pupation) whereas adult sex ratio from the IAEA diet tended to be more male-biased than that of the LRD diet. Adult longevity did not differ significantly with larval diet or light conditions, irrespective of sex. In other aspects the LRD diet generally performed best. Adult males from the LRD diet were significantly larger than those from the IAEA diet, irrespective of light treatment. Females from the LRD diet had ~25% higher fecundity and ~8% higher egg fertility compared to those from the IAEA diet. Adult flight ability did not differ between larval diets, and males had a similar number of copulations with wild females, irrespective of larval diet. The LRD diet had lower protein and fat content but a higher carbohydrate and energetic content than the IAEA diet. We conclude that the LRD diet is a low-cost standardized diet that is likely to be suitable for mass-rearing of Ae. aegypti for area-wide SIT-based vector control.
Dengue and chikungunya are increasing global public health concerns due to their rapid geographical spread and increasing disease burden. Knowledge of the contemporary distribution of their shared vectors, Aedes aegypti and Aedes albopictus remains incomplete and is complicated by an ongoing range expansion fuelled by increased global trade and travel. Mapping the global distribution of these vectors and the geographical determinants of their ranges is essential for public health planning. Here we compile the largest contemporary database for both species and pair it with relevant environmental variables predicting their global distribution. We show Aedes distributions to be the widest ever recorded; now extensive in all continents, including North America and Europe. These maps will help define the spatial limits of current autochthonous transmission of dengue and chikungunya viruses. It is only with this kind of rigorous entomological baseline that we can hope to project future health impacts of these viruses.
Due to the absence of a perfect method for mosquito sex separation, the combination of the sterile insect technique and the incompatible insect technique is now being considered as a potentially effective method to control Aedes albopictus. In this present study first we examine the minimum pupal irradiation dose required to induce complete sterility in Wolbachia triple infected (HC), double-infected (GUA) and uninfected (GT) female Ae. albopictus. The HC line is a candidate for Ae. albopictus population suppression programmes, but due to the risk of population replacement which characterizes this triple infected line, the individuals to be released need to be additionally irradiated. After determining the minimum irradiation dose required for complete female sterility, we test whether sterilization is sufficient to prevent invasion of the triple infection from the HC females into double-infected (GUA) populations. Our results indicate that irradiated Ae. albopictus HC, GUA and GT strain females have decreased fecundity and egg hatch rate when irradiated, inversely proportional to the dose, and the complete sterilization of females can be acquired by pupal irradiation with doses above 28 Gy. PCR-based analysis of F 1 and F 2 progeny indicate that the irradiated HC females, cannot spread the new Wolbachia wPip strain into a small cage GUA population , released at a 1:5 ratio. Considering the above results, we conclude that irradiation can be used to reduce the risk of population replacement caused by an unintentional release of Wolbachia triple-infected Ae. albopictus HC strain females during male release for population suppression.
With the global burden of mosquito-borne diseases increasing, and some conventional vector control tools losing effectiveness, the sterile insect technique (SIT) is a potential new tool in the arsenal. Equipment and protocols have been developed and validated for efficient mass-rearing, irradiation and release of Aedines and Anophelines that could be useful for several control approaches. Assessment of male quality is becoming more sophisticated, and several groups are well advanced in pilot site selection and population surveillance. It will not be long before SIT feasibility has been evaluated in various settings. Until perfect sexing mechanisms exist, combination of Wolbachia-induced phenotypes, such as cytoplasmic incompatibility and pathogen interference, and irradiation may prove to be the safest solution for population suppression.
Background
Quantification of eggs prior to rearing the immature stages of mosquitoes is an essential step in establishing a standardized mass rearing system. To develop a simple and accurate method of egg quantification for Aedes aegypti and Aedes albopictus, the relationship between egg number and weight, as well as egg number and volume, were studied.Methods
Known quantities of eggs (1,000, 3,000, 6,000, 12,000, 15,000, 18,000, 21,000 and 27,000) were counted and subsequently their weight and volume were measured. Best-fit curves and regression equations were used to describe relationships between Aedes egg number and both weight and volume.ResultsEighteen thousand Ae. aegypti eggs weighed 159.8 mg and had a volume of 277.4 ¿l, compared to measurements of 131.5 mg and 230.3 ¿l for Ae. albopictus. The eggs of Ae. aegypti were thus larger and heavier than those of Ae. albopictus. The use of weight and volume to quantify egg number was validated by counting volumes and weights of eggs expected to correspond to 3,000 and 18,000 eggs of each species; significant correlations were found in all cases except in the case of 3,000 Ae. albopictus eggs measured by volume.Conclusion
Methods for egg quantification were validated and shown to be a consistent and practical means to achieve uniform distribution of Aedes larvae between rearing trays, important for optimal mass rearing of the immature stages of Aedes mosquitoes.
About this Book The sterile insect technique (SIT) is an environment-friendly method of pest control that integrates well into area-wide integrated pest management (AW-IPM) programmes. A first of its kind, this book takes a generic, thematic, comprehensive, and global approach in describing the principles and practice of the SIT. The strengths and weaknesses, and successes and failures, of the SIT are evaluated openly and fairly from a scientific perspective. The SIT is applicable to some major pests of plant, animal and human health importance, and criteria are provided to guide in the selection of pests appropriate for the SIT. This technology, using radiation to sterilize insects, was first developed in the USA, and is currently applied on six continents. For four decades it has been a major subject for research and development in the Joint FAO/IAEA Programme on Nuclear Techniques in Food and Agriculture, involving both research and the transfer of this technology to Member States so that they can benefit from improved plant, animal and human health, cleaner environments, increased production of plants and animals in agricultural systems, and accelerated economic development. A great variety of subjects are covered, from the history of the SIT to improved prospects for its future application. The major chapters discuss the principles, technical components, and application of sterile insects. The four main strategic options in using the SIT — suppression, containment, prevention, and eradication — with examples of each option, are described in detail. Other chapters deal with supportive technologies, economic, environmental, and management considerations, and the socio-economic impact of AW-IPM programmes that integrate the SIT. The 28 chapters were all peer reviewed before final editing.
Mating competitiveness trials have been conducted in large net-screened enclosures (8 by 5 by 2.8 m) built in a natural shaded environment, in the summers of 2006, 2007, 2008, and 2009 in northern Italy. Aedes albopictus (Skuse) males were radio-sterilized by applying gamma radiations at doses in the range 30-60 Gy. Gamma radiation was administered to aged pupae at the rate of 2.3 Gy/min. Reared radiated males (originally collected in Rimini, Forli, Bologna, Matera, Pinerolo) and hybrid radiated males were tested against wild fertile males (originated from eggs collected in Rimini and Cesena) and reared fertile males, in multiple comparisons for mating competitiveness with reared or wild females. The ratio was kept constant at 100-100_100 (fertile males-radiated males_virgin females). Mating competitiveness was estimated through the calculation of the hatching rate of the eggs laid in oviposition traps positioned inside enclosures. No clear effect of the strains tested (reared, wild, or hybrid) was found. Results demonstrated that reducing the radiation dose from 60 to 30 Gy increases males' competitiveness. Laboratory investigations conducted after controversial results in the 2006 preliminary trials, showed that radiation induces precociousness in adult male emergence.
Effective vector control, and more specifically mosquito control, is a complex and difficult problem, as illustrated by the continuing prevalence (and spread) of mosquito-transmitted diseases. The sterile insect technique and similar methods control certain agricultural insect pest populations in a species-specific, environmentally sound, and effective manner; there is increased interest in applying this approach to vector control. Such an approach, like all others in use and development, is not a one-size-fits-all solution, and will be more appropriate in some situations than others. In addition, the proposed release of pest insects, and more so genetically modified pest insects, is bound to raise questions in the general public and the scientific community as to such a method's efficacy, safety, and sustainability. This article attempts to address these concerns and indicate where sterile-insect methods are likely to be useful for vector control.
Monitoring reproductive rates in experiments involving aedine mosquitoes is tedious and time demanding. Here, we demonstrate a protocol for rapid estimation of aedine mosquito egg number. The protocol uses ImageJ, a publicly available image analysis program developed at the U.S. National Institutes of Health. The method relies upon the oviposition behavior of Aedes (i.e., ovipositing on a moist substrate instead of water surface) and upon the contrast between dark-colored aedine eggs and a light, uniformly colored paper that is used as an oviposition substrate. The results for 3 Aedes species show that, following the generation of separate calibration curves for each species, the protocol allows for the accurate and repeatable estimation of samples containing hundreds of aedine eggs. We discuss the use of the protocol for monitoring immature aedine populations in both laboratory and field applications.
Aedes notoscriptus and Aedes aegypti are both peri-domestic, invasive container-breeding mosquitoes. While the two potential arboviral vectors are bionomically similar, their sympatric distribution in Australia is limited. In this study, analyses of Ae. aegypti and Ae. notoscriptus eggs were enabled using scanning electron microscopy. Significant variations in egg length to width ratio and outer chorionic cell field morphology between Ae. aegypti and Ae. notoscriptus enabled distinction of the two species. Intraspecific variations in cell field morphology also enabled differentiation of the separate populations of both species, highlighting regional and global variation. Our study provides a comprehensive comparative analysis of inter- and intraspecific egg morphological and morphometric variation between two invasive container-breeding mosquitoes. The results indicate a high degree of intraspecific variation in Ae. notoscriptus egg morphology when compared to the eggs of Ae. aegypti. Comparative morphological analyses of Ae. aegypti and Ae. notoscriptus egg attributes using SEM allows differentiation of the species and may be helpful in understanding egg biology in relation to biotope of origin.
This chapter describes sterile insect techniques (SIT). It is a biologically based method for the control of key insect pests. Wild female insects inseminated by released, radiation-sterilized males do not reproduce, and repeated releases of the sterilized insects lead to a reduction in pest population numbers. Effective control using sterile insects is achieved when they are used systematically as part of area-wide integrated pest management (AW-IPM) programs. SIT is species-specific, nonpolluting, and resistance-free. Since the original concept was developed in the United States in the 1940s, SIT has been used successfully for screwworm flies, tsetse flies, fruit flies, and moths. Technical progress in behavioral ecology, mass rearing, strain improvement, global information, positioning and monitoring systems, and aerial release, combined with economies of scale and a growing demand for pest-free and low-pesticide agricultural products in local and international trade, have increased the use of SIT in AW-IPM programs. These programs, by decreasing insecticide use, have also facilitated the use of biological control agents against secondary insect pests.
Egg size variation among female mosquitoes (Aedes aegypti) is documented. Females hatching from large eggs grow faster, attain a larger adult size, take larger blood meals, lay more and larger eggs than females hatching from small eggs. This cycle is self-perpetuating; offspring of large females have a high probability of attaining a large size. Three factors oppose selection for large egg size by this apparently deterministic cycle: (1) Smaller and slower-growing females may also produce large eggs under some circumstances; (2) genetic differences among sibships are more important than differences in egg size; (3) there is no effect of body size on the reproductive success of males, so males pass on genetic material which has not been size-selected. It does not appear that there is an optimal egg size within the limits of observed egg sizes.
Pupal mass is significantly correlated with the fecundity of female Aedes aegypti (Diptera: Culicidae); larger females mature more eggs than smaller females. Large pupal mass ameliorates the effects of starvation on adults of both sexes. Mass has no effect on the probability of being inseminated (for adult females) or inseminating (for adult males). Thus, larval growth directly affects the fecundity survival of individuals as adults. Also, it is likely that females will maximize their mass at pupation, whereas males will minimize age at pupation and maximize mass secondarily.
The purpose of this paper is to consider the possibility of controlling insects by releasing sexually sterile males among the existing natural population. The principles involved will be described and the potentialities as well as the limitations of the method as we know them at present, will be discussed.
Area-wide integrated pest management (AW-IPM) focuses on the preventive management of pest populations throughout the ecosystem.
It seeks to treat all habitats of the pest population so that none produces migrants to re-establish significant infestations
in areas of concern. In contrast, the conventional strategy focuses narrowly on defending the valued entity (crop, livestock,
people, buildings, etc.) from direct attack by pests. AW-IPM requires multiyear planning, and an organization dedicated exclusively
to its implementation, whereas conventional pest management involves minimal forward planning, tends to be reactive, and is
implemented independently by individual producers, businesses, or households. AWIPM tends to utilize advanced technologies,
whereas the conventional strategy tends to rely on traditional tactics and tools. The sterile insect technique (SIT) is a
species-specific form of birth control imposed on the pest population. It is a powerful tool for “mopping up” sparse pest
populations, and is most efficient when applied as a tactic in a system deployed on an area-wide basis. On environmental,
economic and biological grounds, the case for the SIT is compelling.
Aedes aegypti and Aedes albopictus are potential arboviral vectors leading to high human fatality worldwide. Efforts in the present study were made to differentiate the eggs of A. aegypti and A. albopictus morphologically and morphometrically using scanning electron microscopy (SEM). Morphometrically, these species' eggs were 48.48% significantly different of the 33 attributes including egg dimensions, micropylar apparatus, dimensions and density of outer chorionic cells (OCCs), tubercles and width of exochorionic network. In comparison to A. aegypti eggs, A. albopictus eggs were significantly smaller and more tapered at the posterior end; however, the micropylar disc of A. aegypti was wider and had incomplete circular sectors whereas it was a narrower polygon without sectors in A. albopictus. These species were also significantly different with regards to OCC which enclose both large central and small peripheral tubercles. Specifically, the exochorionic networks in A. aegypti were interwoven, reticulated and extensively wide whereas they were narrow, prominent and solid-wall-like in A. albopictus. This feature may strengthen A. albopictus eggs against desiccation, when they are laid in containers. The morphometrical and morphological analysis of the egg's attributes of A. aegypti and A. albopictus may be helpful in understanding egg biology as well as in species confirmation.
Complete descriptions, including details of the micropylar apparatus and outer chorion at the anterior and posterior poles and on the dorsal surface, are given for the eggs of Aedes (Stegomyia) albopictus Skuse and Ae. (S.) aegypti (L.). The egg of Ae. (Howardina) bahamensis Berlin, the first of this subgenus to be examined with the electron microscope, is described for the first time. Certain characters may be useful for differentiating eggs of these three species under a stereomicroscope. The substantially greater egg length in Ae. bahamensis alone separates it from the other two species, at least in the laboratory populations studied. The micropylar collar in Ae. aegypti clearly differentiates it from the other two. Further examination of mixed groups of eggs is needed to determine whether fine structural differences in the outer chorion can be correlated with consistent differences in the stereomicroscopic image.
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