INTRODUCTION

Aedes aegypti (L.) is recognized as one of the most invasive mosquito species worldwide (Gratz 2004, Lambrechts et al. 2010). It is widely distributed across tropical and subtropical regions and has highly adapted to urban environments (Wilke et al. 2020). This species poses a significant public health risk as a vector for pathogens that causes diseases such as dengue, chikungunya, yellow fever, and Zika (CDC 2016, Wilder-Smith et al. 2017). In the absence of effective treatments, controlling mosquito vector populations remains the primary strategy for preventing the transmission of these diseases to humans (Trewin et al. 2017, Roiz et al. 2018, Wilson et al. 2020). The species’ ability to develop resistance to insecticides (Moyes et al. 2017, Parker et al. 2020, Aryaprema et al. 2025), exploit a wide range of artificial and natural habitats (Wilke et al. 2019), utilize cryptic breeding and resting habitats (Perich et al. 2000), and survive in a desiccated egg state for extended periods (Kumar et al. 1995, Momen et al. 2019) has made population control using traditional methods particularly challenging (Cuervo-Parra et al. 2016). As such, there is an urgent need to develop new and effective strategies to complement the Integrated Vector Management toolbox for controlling this species.

The sterile insect technique (SIT) has emerged as a promising, species-specific, and environmentally friendly approach for suppressing mosquito populations. SIT was first applied in the 1950s to control New World Screwworm populations (Cochliomyia hominivorax (Coquerel) in the USA, Mexico, and Central America (Bushland et al. 1955, Dame et al. 2009, Klassen et al. 2021). Since then, the technique has been successfully used to manage multiple insect species (Miller et al. 1994, Hendrichs et al. 1995, Ant et al. 2012, Enkerlin et al. 2015, Kebede et al. 2015, Klassen et al. 2021). It was later extended to mosquitoes, with the first releases of sterile mosquitoes occurring in South Florida in 1959–1960, using Anopheles quadrimaculatus (Say) (Weidhaas et al. 1962), followed by releases of sterilized Ae. aegypti in Pensacola, FL, in 1960–1961 (Morlan et al. 1962). Despite early challenges due to poor quality of male mosquitoes (Benedict and Robinson 2003, Dame et al. 2009), successful applications of SIT began in the 1960s using Culex quinquefasciatus Say in India and Florida (Patterson et al. 1975, 1977). Since then, significant progress has been made, and SIT has proven successful in controlling mosquito populations in multiple locations worldwide (Bellini et al. 2013, Ernawan et al. 2019, Kittayapong et al. 2019, Zheng et al. 2019, Velo et al. 2022, Balatsos et al. 2024).

The SIT involves mass-rearing and releasing sterile males to inundate a target area. These sterile males mate with wild females, and through continued release of sterile males, the population is expected to decrease over time (Knipling 1985). The success of SIT primarily depends on the ability of released males to compete with wild males for mates (Pérez-Staples et al. 2013; Tejeda et al. 2017, Kapranas et al. 2022). Therefore, having strong quality control over production conditions at every stage of the process—from mass-rearing to release—is critical to the effectiveness of the technique. For SIT to be successful, mass-rearing must be scalable, meeting program demands without sacrificing efficiency, product quality, or cost effectiveness. A key factor in achieving this is identifying a larval diet for mass-rearing that produces high-quality sterile males (product quality control) while maintaining good levels of process quality control given the specific needs of the mass-rearing program, including available products for larval food, product certifications and quality control, efficiency of workflows given program equipment, and efficient use of rearing staff effort (Briegel and Timmermann 2001, Cohen 2003, Benedict et al. 2009b, Mamai et al. 2020, 2025). An appropriate larval diet promotes synchronized larval growth to adequate sizes both for pupal sex separation and for adult male performance. Insufficient nutrition can compromise the quality of the released mosquitoes, ultimately hindering the success of the SIT program (Joy et al. 2010, Lang et al. 2018). The International Atomic Energy Agency (IAEA) has recommended a standardized larval diet composed of tuna meal, bovine liver powder, and brewer’s yeast for Aedes rearing (Bond et al. 2017), However, this diet is not always feasible for large-scale mass-rearing in many countries due to importation restrictions. Consequently, several alternative diets have been tested in different mass-rearing programs to achieve similar or improved performance (Puggioli et al. 2013, Bond et al. 2017, Momen et al. 2019, Obra et al. 2022). The present study aimed to assess 3 candidate diets to identify an appropriate larval diet for scalable mass-rearing of Ae. aegypti at the Anastasia Mosquito Control District (AMCD) in St. Johns County, FL to support effective implementation of SIT.

MATERIALS AND METHODS

Eggs from a local strain of Ae. aegypti, derived from a colony established with wild-type eggs collected in 2024, were provided by MosquitoMate (MosquitoMate, Lexington, KY), where they are continuously reared. Six thousand eggs (by weight) packaged into a cellulose capsule (size 00 vegetarian capsules; Herb Affair, Chicago, IL) were hatched in 3 groups in 4-liter reverse osmosis water in Wolbaki trays (70 cm × 60 cm × 3 cm, WBK-P0003-V2; Guangzhou Wolbaki Biotec Co., Guangzhou, China), with 4 trays per group. The estimated larval density in each tray was approximately 1.5 larvae/ml, which lies within the range of densities regularly used for mass-rearing of Ae. aegypti (Bargielowski et al. 2011, Mamai et al. 2025). Each group was fed 1 of 3 diets, TetraMin® Tropical Flakes (Tetra, Spectrum Brands Pet, Blacksburg, VA), Ziegler® Tropical Pro-Start45 Meal (Ziegler Bros., Gardners, PA), or bovine liver powder (MP Biomedicals, San Diego, CA) administered in cellulose capsules (size 00 vegetarian capsules), each capsule containing 500 mg of food (Dobson et al. 2023). Here onwards the 3 diets will be referred to as Tetramin, Ziegler, and liver powder, respectively. The macronutrient compositions, as reported by the manufacturers, are 1) Tetramin—minimum (min) 46% crude protein, min. 11% crude fat, maximum (max) 3% fiber, 2) Ziegler—min. 45% crude protein, min. 10% crude fat, max. 5% crude fiber, and 3) liver powder—75.6% protein, 14.6% fat. Each diet was administered according to a predetermined daily regimen (Table 1) based on established feeding programs at Lee County Mosquito Control District, FL for the Ziegler diet, MosquitoMate for the liver powder diet, and AMCD for the Tetramin diet (Bangonan et al. 2022, Dobson et al. 2023, Stenhouse et al. 2025). Diets were scaled to a density of 6,000 larvae per tray and tested with several pilot trials before the complete study to observe for excess food or water foulage and monitor any abnormal larval mortality or behavior. Daily food amounts were adjusted accordingly from pilot trials to minimize problems with water quality and to maintain healthy larvae during this study. Due to the low pupal count on Day 7, the Ziegler diet was provided with an additional day of development before collection (Table 1).

Table 1.Predetermined daily feeding regimen for Aedes aegypti larvae and the total food intake during development (mg/larva).¹

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Male pupae were separated from larvae and female pupae based on size differences on the second day of pupation (at the most significant portion of pupation before adult eclosion) using a modification of the Fay-Morlan glass plate approach as implemented by the Mosquito Pupae Sex Automatic Sorter (WBK-P0001-V2). Hatching and rearing were conducted in a climate-controlled insectary at 26 ± 2°C, 80 ± 10% relative humidity, and a photoperiod of 14:10 h (light:dark). Water temperature in the rearing trays was recorded daily from the day of egg hatching until pupation using HOBO MX2303 data loggers (Onset, Bourne, MA). The average water temperature in the trays during the rearing period was 24.29°C, ranging from 22.43°C to 26.30°C across replicates and trials.

Performance of the 3 diets was evaluated only for males using the following quality control parameters: 1) dry weight per pupa, 2) estimated total number of pupae produced, 3) pupal cephalothoracic length, 4) percent pupae eclosing to adulthood, 5) adult wing length, and 6) adult male longevity. To estimate the dry weight per pupa, the mass of male pupae (after sex separation) was run through a sieve, aliquoted, and spread out on paper towels (WypAll®; Kimberly-Clark Global Sales, Roswell, GA) to remove most of the excess water. The mass of male pupae was then transferred to coffee filters, and additional water was removed using a Nesco food and jerky dehydrator (Metal Ware Corporation, Two Rivers, WI) at 35°C for 7 min, following Carvalho et al. (2022). A group of 30 pupae per replicate was randomly selected and weighed to the nearest 0.1 mg using an analytical balance (MR304; Mettler-Toledo, Leicester, UK). The weight of the group was divided by 30 to obtain the average dry weight per pupa for that replicate, which served as the conversion factor to estimate pupal production. The total dry pupal mass from each tray was then divided by the corresponding conversion factor to estimate the total number of pupae. While weighing multiple groups per replicate could further reduce sampling error, pupae were reared under uniform conditions and exhibited minimal visible size, thus 1 group per replicate (3 groups per diet) was considered representative. Cephalothoracic length was measured as the distance between the anterior point of the median keel and the ventral tip of the pupal wing sheath (Koenraadt 2008). Thirty pupae were haphazardly selected from each replicate of each diet were measured using a Keyence digital microscope (VHX-7000N; Keyence Corporation, Osaka, Japan). An image of the pupa was captured directly on the microscope screen, and measurements were taken on-screen at 50× magnification using the microscope’s internal scale for conversion to millimeters. Measurements were validated using a microscale (1 div = 0.1 mm; Minitool Inc., Redding, CA).

The percent pupae eclosing to adulthood was estimated by placing 30 pupae from each replicate into small plastic cups (473 ml, ECO16DELI-PP; Ecosystems Holdings, Jefferson City, MO) containing RO water. The cups were then placed in 30 × 30 × 30 cm mesh cages (BugDorm-4E3030; MegaView, Taichung, Taiwan) and monitored until all pupae had either emerged or died.

Wing length was measured from the distal edge of the alula to the end of the radius vein, excluding fringe scales (Packer and Corbet 1989). Thirty adults from each replicate were haphazardly selected and immobilized by placing them in a refrigerator at 4–5°C for ∼10–20 min. The right wing of each individual was then detached using an entomological pin and a pair of forceps, then placed on a Petri dish for measurement. Wing lengths were measured using the Keyence digital microscope at 50× magnification, as described above.

Adult longevity and daily percent survival were assessed by placing 30 adult males per replicate into 30 × 30 × 30 cm mesh cages (BugDorm-4E3030). Mosquitoes were provided access to a 10% sucrose solution ad libitum, which was changed out weekly (Benedict et al. 2009a). Mortality was recorded daily, except on a few unavoidable days, by counting and removing the dead individuals from each replicate. Mosquito longevity was expressed as the median lifespan, which is defined as the age at which 50% of the mosquitoes in each replicate had died. All experiments were repeated 3 times, with each replicate of the 3 diet treatments coming from different cohorts of eggs.

RESULTS

Because rearing density can affect pupal and adult size, each tray was prepared with the same number of eggs as outlined above. We were able to harvest approximately 1,500 male pupae from each tray with no significant difference in the number of male pupae per tray across our 3 diets (Fig. 1A, GLM, df = 2, χ² = 0.76, P > 0.05). Over 90% of pupae successfully eclosed as adults from each tray across all 3 diets (Fig. 1B, GLM, df = 2, χ² = 0.39, P > 0.05).

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Overall, pupae reared on the Ziegler diet were 5.2% and 5.6% heavier than those reared on Tetramin and liver powder diets, respectively, which did not differ significantly from each other (Fig. 1C, ANOVA, F_2,30 = 3.60, P < 0.05). Percent differences are based on mean pupal weight (mean ± SE: Ziegler 2.63 ± 0.1 mg, Tetramin 2.5 ± 0.1 mg and liver powder 2.49 ± 0.1 mg) and may differ from median values shown in Fig. 1C. Similarly, the Ziegler diet produced pupae with a significantly greater cephalothoracic length than Tetramin or liver powder, which did not differ significantly from each other (Fig. 1D, ANOVA, F_2,1062 = 3.60, P < 0.05). However, the actual magnitude of the size difference was relatively small, representing a 0.03–0.04 mm difference in length between Ziegler and the other diets (mean ± SE: Ziegler 2.01 ± 0.004 mm, Tetramin 1.97 ± 0.003 mm, liver powder 1.98 ± 0.004 mm). Similarly, the wings of adults reared on the Ziegler diet as larvae were significantly longer than those of adults that had been reared on either Tetramin or liver powder as larvae, which did not differ from each other (Fig. 1E, ANOVA, F_2,1062 = 46.9, P < 0.05). Again, while statistically significant, the wing-length differences were modest with adults that fed on the Ziegler diet as larvae having 1.8% longer wings than adults reared on the liver powder diet as larvae, and Ziegler-reared adults had 2.2% longer wings than adults reared on Tetramin as larvae (mean ± SE: Ziegler 2.30 ± 0.004 mm, Tetramin 2.25 ± 0.005 mm, liver powder 2.26 ± 0.004 mm).

Adult mosquitoes that were reared on liver powder as larvae had a significantly higher daily mortality rate than Tetramin or Ziegler-reared mosquitoes, which did not differ from each other (Fig. 2A, GLM, df = 2, χ² = 334.47, P < 0.01). However, more than 90% of mosquitoes from all 3 diets survived beyond 18 days, indicating comparable early survival rates. Survival of liver powder-reared mosquitoes declined steadily after approximately 20 days, whereas Tetramin- and Ziegler-reared mosquitoes maintained more than 80% survival up to 30 days. Further analysis showed that adult mosquitoes reared on the Ziegler diet as larvae had a median lifespan that was twice as long as mosquitoes that fed on liver powder as larvae, and adult mosquitoes from larvae reared on the Tetramin diet had a median lifespan nearly twice as long as mosquitoes fed on liver powder as larvae (Fig. 2B, ANOVA, F_2,33 = 27.44, P < 0.01).

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Although not quantitatively measured, our observations revealed that the average development time from first-instar to peak pupation, before the onset of eclosion, was approximately 7 days for larvae fed Tetramin or liver powder, and approximately one day longer for those larvae fed Ziegler.

DISCUSSION

Choosing the appropriate larval diet for mass-rearing programs requires balancing mosquito growth and performance with the practicality and scalability of the rearing process to ensure the efficiency and success of SIT programs. A high-quality diet should support rapid and uniform larval development, consistent larval and pupal survival, synchronized pupation, and uniform pupal sizes, ultimately producing high-quality adult male mosquitoes with substantial longevity, flight ability, and mating competitiveness (Bellini et al. 2007).

The 3 diets tested in this study performed similarly in terms of pupal and adult production numbers, indicating comparable larval and pupal survival and overall adult production. However, the Ziegler diet resulted in the largest size and extended longevity. At the same time, liver powder and Tetramin yielded pupae of similar sizes, but with mosquitoes fed liver powder living significantly shorter than those fed either the Ziegler or Tetramin diets. The larger pupae produced by the Ziegler diet, as evidenced by greater weight and cephalothoracic length, may reflect the extra day of larval development and the nutritional status achieved during larval development. As expected from the pupal size, the Ziegler diet also produced mosquitoes with longer wing lengths, indicative of larger adult male body size (Packer and Corbet 1989), which is primarily determined during the larval stage by the quality and quantity of larval nutrition (Koenraadt 2008, Bond et al. 2017, Schoor et al. 2020). Larger mosquitoes may exhibit greater overall performance, including enhanced longevity (Maciel-De-Freitas et al. 2007, Gutiérrez et al. 2020). The Ziegler diet produced larger mosquitoes and longer-lived adults suggesting that it not only supports larger size but also facilitates the accumulation of nutrient reserves or other beneficial physiological traits that contribute to adult longevity. Interestingly, the Tetramin diet produced slightly smaller mosquitoes with longevity comparable to those reared on the Ziegler diet. This suggests that, despite their smaller size, these mosquitoes may have accumulated sufficient nutritional reserves and other physiological benefits to support adult longevity. Mosquitoes reared on liver powder were comparable in size to those from the Tetramin diet but exhibited a significant reduction in longevity. This likely reflects a poorer nutrient balance, or deficiency in specific nutrients, in the liver powder diet that are critical for adult energy storage and metabolic functions compared to our other two diets. Previous studies have demonstrated the importance of key nutrient constituents in larval diets, such as carbohydrates, because they significantly influence adult male size, longevity, and flight ability (van Handel 1988, Maciel-de-Freitas et al. 2007, Souza-Neto et al. 2007, Joy et al. 2010, Yee et al. 2012, Puggioli et al. 2013, Bond et al. 2017, Schoor et al. 2020). Despite differences in adult longevity, mosquitoes reared on all 3 diets demonstrated high and comparable early survival rates under laboratory conditions. This suggests that all diet treatments have the potential to support adequate survival through the critical early period required for successful mating activity in the field. This is particularly relevant in the context of SIT programs, where early survival is more critical than extended adult lifespan, given that the majority of successful matings typically occur within the first few days following release (Oliva et al. 2012, Damiens et al. 2016). A practical and resilient SIT program in Florida reported an average lifespan of only 2.46 days for released Ae. aegypti males in the field, demonstrating that short postrelease survival is sufficient for program success and highlighting the critical importance of early mating activity (Carvalho et al. 2022). If early survival remains high, potential reductions in performance traits, such as flight ability or mating competitiveness, may be at least partially compensated for by the high release ratios of sterile males relative to wild males typical of SIT programs. SIT releases are designed to inundate wild populations and ensure sufficient encounters between sterile males and females, thereby helping to maintain program efficacy even when some sterile male performance parameters are diminished compared to those of wild males (Dame et al. 2009). However, it is important to note that our study did not include direct comparisons of mating competitiveness either between diet treatments or against wild males. Therefore, the high early survival observed across all 3 diets is promising, but competitiveness between diets or against wild males requires further investigation. The extended development time required by Ziegler may indicate differences in daily nutrient availability that influence developmental timing or that larger body size simply took longer to achieve. Given that shorter development time improves scalability, efficiency, and reduces operational costs, this delay could be a limiting factor in mass-rearing programs. While all 3 diets produced comparable numbers of pupae and adults, the delayed development associated with Ziegler may affect rearing turnover and production efficiency in large-scale operations. Notably, a separate mass-rearing program reported a shorter development time (6 days) using a different Ziegler-based diet with a different feeding regimen (Stenhouse et al. 2025). This suggests that further optimization of the daily feeding regimen of the Ziegler diet could help reduce development time.

Additionally, preparation and handling requirements varied notably among the diets. The liver powder diet was provided in premeasured capsules, making it the easiest to handle and dispense. In contrast, the Tetramin diet required weighing, dissolving in water to make a slurry, and measuring a slurry for distribution, which is more labor- and time-intensive than simply delivering capsules. The Ziegler diet was moderate in preparation and handling effort. These practical aspects, including human resource availability, ease of daily preparation, and workflow integration, are crucial considerations when selecting a diet for scalable mass rearing.

Although diet cost was not formally assessed in this study, it remains a key factor for mass-rearing programs where economic sustainability plays a crucial role in long-term operational success. Based on manufacturer-reported information, the ingredient cost over the development period for approximately 1,500 male mosquitoes is substantially higher for liver powder compared to the Ziegler and Tetramin diets (approximately 24 times and 4 times, respectively), highlighting a significant economic consideration. The cost of Tetramin is approximately 7 times higher than that of Ziegler.

Considering all these factors, selecting the most appropriate diet among the 3 tested for the scalable mass rearing of Ae. aegypti in the SIT facility at Anastasia Mosquito Control District, St. Augustine, FL, requires balancing multiple goals, including program objectives, resource availability, operational efficiency, and cost. Although the differences in growth traits among the 3 diets were statistically significant, all 3 diets can be considered viable candidates, with each offering strengths that can align with different program priorities. If the primary goal is to maximize average mosquito quality at the lowest cost, Ziegler stands out as the best choice. The Ziegler diet produced larger and longer-lived adults and is also the least expensive in terms of ingredient cost. For programs that prioritize operational efficiency while maintaining acceptable performance, liver powder may be a more practical option, despite its higher price. Tetramin represents a viable intermediate option, offering moderate cost and performance, for programs with flexible operational priorities. However, its trade-offs in performance and preparation effort must be considered when assessing long-term scalability. Ultimately, all 3 diets can be used to meet specific program needs. With further refinement of feeding regimens, the Ziegler diet has the greatest potential to offer the best overall balance of biological performance, scalability, and affordability. These findings provide a framework for selecting larval diets for scalable mass-rearing in SIT programs and highlight the importance of aligning diet choice with both biological performance and operational feasibility.