# A mass rearing method to aliquot and hatch Aedes
mosquito eggs using capsules

F. Balestrino, A. Puggioli, W. Mamai, R. Bellini, Jérémy Bouyer

► To cite this version:

F. Balestrino, A. Puggioli, W. Mamai, R. Bellini, Jérémy Bouyer. A mass rearing method to aliquot and hatch Aedes mosquito eggs using capsules. Scientific Reports, 2024, 14 (1), pp.30276. 10.1038/s41598-024-81385-z. hal-05088634

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# A mass rearing method to aliquot and hatch *Aedes* mosquito eggs using capsules

F. Balestrino<fnref n="1" />✉, A. Puggioli², W. Mamai<fnref n="1" />, R. Bellini² & J. Bouyer<fnref n="1" />,³

The sterile insect technique (SIT) is a sustainable genetic control method to effectively suppress mosquito vector populations below sanitary and nuisance thresholds. The SIT requires a cost-effective production and release of radio sterilized insects of high and consistent quality and quantity. In this study, we report a new procedure to hatch, aliquot and introduce *Aedes albopictus* and *Aedes aegypti* larvae into larval mass rearing units using dissolvable vegetable capsules containing brushed eggs and larval diet. The results demonstrated that the egg brushing procedures, the opening of the hatching container and the presence of diet in the hatching solution increased *Aedes* eggs hatching success. The diet added during the hatching process decreased the dissolved oxygen (DO) concentration, which constituted the main stimulus for egg hatching success in both species. However, a DO reduction below 3 mg/l can effectively stimulate hatching in both species regardless of the deoxygenation method adopted. The capsule method produced similar hatching success without affecting larval survival and pupation rates in comparison with standard hatching procedures. The use of capsules eliminates intermediate laborious procedures for hatching, counting and transferring larvae into mass rearing trays with an expected improvement in production times and costs for *Aedes* SIT operational programs.

**Keywords** Sterile insect technique, Mosquito mass rearing, *Aedes albopictus*, *Aedes aegypti*, Dissolvable capsule, Hatching response

Vector-borne diseases are infectious diseases with high mortality and prevalence in the world. *Aedes* mosquitoes are among the most invasive vectors of disease capable to impose a substantial threat to global public health. Increased urbanization, mobility of goods and people and ongoing climate changes are favouring the active and passive distribution of these vectors with a concurrent increase of outbreaks and epidemic risks throughout the world<fnref n="1" />. In the absence of effective therapies and vaccines, vector control represents the main strategy to reduce the incidence of important diseases such as dengue, Zika and chikungunya. Moreover, current conventional mosquito control strategies are mainly based on the reactive and often ineffective chemical interventions during epidemics that are harmful to the environment and human health²,³. The large use of chemical molecules has also generated the selection of vector populations resistant to various insecticides, further decreasing the effectiveness of mosquito control activities⁴. New effective and sustainable control methods are therefore necessary to reduce the sanitary burden caused by *Aedes* mosquitoes around the world. Among the most recent and innovative control strategies proposed, genetic insect control techniques certainly represent the most promising alternative for the effective control of mosquito populations. These techniques aim to introduce sterility or disease refractoriness into target wild populations using self-limiting or self-sustaining insect release methods⁵. Among these, the sterile insect technique (SIT) represents a consolidated and successful self-limiting strategy for the control of key insects of agronomic, veterinary and sanitary importance⁶⁻⁸. The SIT technique is based on the continuous release of large quantities of radio-sterilized insects capable of mating with conspecific females, progressively introducing sterility into the target wild population. The SIT has already demonstrated its effectiveness against mosquitoes in small- and medium-scale field trials but to address the challenge of mosquito control, such techniques must be effectively tested and validated at a large operational scale. To support the SIT technique applied as a component of area-wide integrated pest management (AW-IPM) programs, it is therefore necessary to develop dedicated procedures and equipment for the mass production of the insect with consistent quantity, quality and sustainable costs⁹. The Insect Pest Control Laboratory of the Food and

<footnote n="1">¹Insect Pest Control Laboratory IPCL, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA, Seibersdorf 2444, Austria. ²Sanitary Entomology and Zoology Department, Centro Agricoltura Ambiente "G. Nicoli", Crevalcore 40014, Italy. ³ASTRE, CIRAD, INRAE, Plateforme Technologique CYROI, Sainte-Clotilde, Univ. Montpellier, La Réunion 97490, France. ⁴email: fabriziobalestrino@hotmail.com; f.balestrino@iaea.org</footnote>
Agriculture Organization / International Atomic Energy Agency (FAO/IAEA Joint Division) has been working for several years on the development and transfer of the mass rearing procedures and technology necessary to support the field application of the SIT technique against mosquito vectors. Beside developing dedicated mass rearing technologies and procedures¹⁰, methods to facilitate and optimise egg hatching and introduction of the young larval stages inside the larval rearing units are necessary. As existing genetic sexing strains for early male separation are not yet scalable or applicable for large-scale mosquito production and filed release¹¹,¹², male mosquito selection must be performed at the pupal or adult stage under mass rearing conditions. Considering the protandry and the typical pupal dimorphism detectable in *Aedes* mosquito species, the effectiveness of the dimensional sex separation procedures relies on a synchronous and homogeneous larval development to standardize and maximize the collection of male pupae. In addition to maintaining constant environmental rearing parameters and the use of effective larval rearing schedules and diets, it is therefore essential to introduce an adequate and homogeneous number of larvae of the same age into the larval rearing trays¹³. Current techniques for introducing an accurate number of first-instar larvae into the larval rearing trays require the hatching of an estimated quantities of eggs inside single sealed hatching containers containing deoxygenated water to stimulate eggs hatching effectively and simultaneously¹⁰,¹⁴,¹⁵. In the standard IAEA hatching method one hatching container for each larval tray needs to be prepared and conditioned overnight at controlled temperature. The larvae present in each container after hatching must therefore be manually transferred into the larval trays which are stacked inside the rack unit and already filled with water¹⁰. To facilitate this procedure and simplify the hatching operations, some projects adopt the simultaneous hatching of large quantity of eggs, subsequently taking homogeneous aliquots of larvae according to the previously estimated larval density¹⁶. The aliquots of larvae are taken from the hatching container while stirring to ensure thorough mixing, and subsequently transferred into the larval trays. To further reduce the workload and stress that might affect the larvae along this process, automatic hatching and optical larval counting devices have been proposed¹⁷. These optical devices designed to mechanize larval mass rearing process have counting inaccuracies of 5% and can process larvae at a maximum rate of about 1000–2000 larvae per minute. However, even with the use of numerous devices operating in parallel, it is still necessary to manually transfer the larval samples into the rearing trays at a great expense of time and manpower. To improve *Aedes albopictus* and *Aedes aegypti* mass rearing procedures and to reduce larval rearing management time and costs, the direct introduction of the eggs inside the larval trays was tested using dissolvable vegetable capsules containing eggs and larval diet powder. After embryonic maturation, the eggs of *Ae. aegypti* and *Ae. albopictus* can hatch at different times after their immersion in water. The hatching response of *Aedes* mosquito eggs is strongly affected by the depletion of the water dissolved oxygen in the water caused by microbial activity in the vicinity of the eggs¹⁸⁻²⁰. The current methods to reduce oxygen from hatching solution in the mass rearing facilities includes the use of boiled water¹⁶, the addition of yeast, larval diet²¹,²² and bacterial broth into the hatching media¹⁵,²³. This study reports the results of egg aliquoting and hatching obtained using dissolvable vegetable capsules on the mass rearing productivity of *Ae. albopictus* and *Ae. aegypti* in comparison with standard IAEA procedures. The method proposed for these species can significantly reduce the management times and costs of *Aedes* mass rearing facilities supporting large-scale SIT activities.

# Methods

## Mosquito colonies and eggs hatching quality control

*Aedes albopictus* and *Aedes aegypti* strains used in this study were collected in the field in Emilia-Romagna (Italy) and Bahia (Brazil) and colonized respectively at the Centro Agricoltura Ambiente (CAA, Crevalcore, Italy) and Moscamed (Petrolina, Brazil), both IAEA collaborating centres on the development of SIT against mosquitoes, and then at the IPLC IAEA (Seibersdorf, Austria) following FAO/IAEA guidelines mass-rearing procedures¹⁰. Research carried out on invertebrates do not require a specific permit according to the directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. The blood used for routine mosquito blood-feeding was collected during routine slaughtering of pigs in authorized abattoirs at the highest possible standards strictly following EU laws and regulations. This study was carried out in accordance with ARRIVE guidelines (https://arriveguidelines.org). The eggs collected from adult colonies over oviposition paper substrates were dried (24–36 h at 24±2 °C; RH 70±5%) and maturated at higher humidity conditions (24±2 °C; RH 90±5%) for at least 14 days before undergoing hatching procedures.

The *Ae. albopictus* and *Ae. aegypti* eggs batches needed in these trials were preliminarily tested to assess their viability. The eggs were gently brushed off from the oviposition papers and separated from debris using a sieve of 300 µm mesh size. Three samples of about 300 brushed eggs were hatched inside a 50 ml Falcon tubes filled with 45 ml of a larval hatching solution obtained by adding 0.43 gr of IAEA larval diet mixture¹⁰ per litre of deionised water. The tubes were closed and incubated overnight (16–20 h) at larval rearing room conditions (28±2 °C; 85±10% RH; 14:10 L: D) followed by egg hatching rate determination under a stereoscopic microscope.

## *Experiment 1: effect of diet, egg brushing and closure of container on egg hatching success*

The efficacy of the new proposed hatching method is based on the possibility of achieving high and uniform hatching rate for *Aedes* eggs introduced together with larval diet inside dissolvable capsules that will be released inside larval mass rearing trays. The effect of egg brushing, addition of diet and closure of the hatching container on the hatching success was evaluated on *Ae. albopictus* and *Ae. aegypti* eggs. Paper strips of equal width with approximately 500 eggs each were collected from *Ae. albopictus* and *Ae. aegypti* oviposition papers and hatched at different conditions inside conical centrifuge tubes with screw cap (50 ml Falcon tubes) filled with 45 ml deionised water. The hatching success using (i) eggs brushed or left attached to their oviposition substrate, (ii) hatching containers left open or closed and (iii) hatching solutions supplemented or not with 20 mg of larval diet was evaluated on both species in eight treatments as reported below.
ECD : E - brushed eggs C - hatching container closed D - 20 mg larval diet added.

ECN : E - brushed eggs C - hatching container closed N - no larval diet added.

EOD : E - brushed eggs O - hatching container open D - 20 mg larval diet added.

EON : E - brushed eggs O - hatching container open N - no larval diet added.

PCD : P - eggs on paper C - hatching container closed D - 20 mg larval diet added.

PCN : P - eggs on paper C - hatching container closed N - no larval diet added.

POD : P - eggs on paper O - hatching container open D - 20 mg larval diet added.

PON : P - eggs on paper O - hatching container open N - no larval diet added.

All tubes were maintained overnight (16-20 h) at larval rearing room conditions (28 ± 2 °C; 85 ± 10% RH; 14:10 L : D). After incubation, the eggs hatching response from each treatment was estimated on a random sample of 200-300 eggs under a stereoscopic microscope and measurement of dissolved oxygen (DO) concentration in each hatching solution was recorded using a dissolved oxygen analyser (DO9100 YINMIK, Shandong, China). Two biological replicates, with at least five technical repetitions each, were performed for each treatment and species tested.

## Experiment 2: Capsule egg hatching method against standard egg hatching method

The results of the first experiment confirmed the possibility of effectively hatching *Aedes* brushed eggs when placed together with larval diet inside larval rearing trays. The capsule hatching method was compared to the IAEA standard hatching procedures to evaluate the hatching success and the pupal productivity of *Ae. aegypti* and *Ae. albopictus* under larval mass rearing conditions based on the IAEA guideline¹⁰ with a modified rearing schedule²⁴.

Based on the viability of the *Aedes* eggs preliminarily measured, the respective weight of eggs required to obtain 18,000 larvae of *Ae. albopictus* and *Ae. aegypti* per tray was calculated using a conversion table (adapted from¹⁴). All the eggs necessary for the tests were weighted over an analytical balance (ME204T, Mettler-Toledo, India). In the IAEA standard hatching method (STD) the necessary amount of eggs was transferred inside a glass jar filled with 700 ml of deionised water added with 0.3 gr of IAEA larval diet powder (0.4 gr/l) for both species²²,²⁵. The jar was sealed and incubated overnight at larval rearing room conditions (day 0). The following day (day 1) first instar larvae were poured inside a mass rearing tray²⁶ together with the hatching solution. To accommodate for this additional liquid volume, the mass rearing tray was filled with a reduced quantity of deionised water (about 4.3 L, total hardness 1 °F) since the day before to ensure the desired final larval density and the appropriate larval rearing temperature. Before the introduction of the larvae into the trays, a random sample of 200-300 eggs were collected using a pipette from the bottom of the jars to measure the hatching rate and estimate the number of larvae introduced in each tray.

In the proposed capsule method (CAP), the same amount of eggs used in the previous method was manually placed inside an dissolvable vegetable capsule (HPMC, size 0 Clear, Galeno, Italy) together with 0.2-0.3 gr of larval IAEA diet powder mixture. The overall amount of diet introduced was equivalent to the necessary quantity of powder needed to completely fill the capsule. We selected the cellulose-based HPCM capsule over the animal derived gelatin capsule, as it exhibits no cross-linking reactions with various excipients, is stable during production and storage at different temperature and humidity conditions, does not exhibit temperature-dependent disintegration or dissolution and is able to retain any filling material²⁷. As soon as assembled and at the same time when eggs were placed in the jar in the previous method (day 0), the capsules were directly introduced into the larval mass rearing trays. In this method the trays were filled the day before the introduction of the capsule with the final quantity of deionised water needed (about 5 L). The day after introduction of the capsule in the mass rearing tray, a random sample of 200-300 eggs were collected using a pipette from the bottom of mass rearing trays to measure the hatching rate and to estimate the number of larvae added to each tray.

Beside the different hatching method employed, the same larval rearing schedule was adopted in all mass rearing trays. A 4% IAEA larval liquid diet was provided to the trays on day 1 (300 ml), day 4 (300 ml), day 5 (200 ml) and day 6 (300 ml) and the larvae and pupae produced in each tray were harvested on day 7 at about 160 h from hatching. Larvae, male and female pupae from each tray were manually collected and separated by size using a Fay-Morlan glass plate separator²⁸ (Wolbaki Biotech Co. Ltd., Guangzhou, China). The number of larvae, male and female pupae collected from each tray were volumetrically estimated using a modified tube following standard operating procedures¹⁰. Pupal male and female recovery rates were calculated as the ratio of pupae collected on the estimated initial number of first instars larvae introduced in the tray assuming a balanced sex ratio. The survival rate in each tray was estimated as the ratio between the pupae and larvae present at the time of collection in relation to the initial number of first instars larvae estimated in the tray. Four and six biological replicates with at least three technical repetitions each were performed for each treatment for both *Ae. aegypti* and *Ae. albopictus*.

## Experiment 3: effect of diet and oxygen concentration on egg hatching

In this experiment, the presence of larval diet and its effect on oxygen depletion was uncoupled by exposing samples of about 500 brushed eggs of *Ae. albopictus* and *Ae. aegypti* at different combinations of oxygen concentration and diet in the hatching media. All eggs used were tested for viability as described above and only batches with high viability (hatching rate HR > 0.8) were used. The day before the trials, four litres of deionised water were treated as follows: two litres were boiled for two hours and left to cool inside a sealed glass bottle overnight at larval room conditions. This prolonged boiling procedure generated a hatching solution with low dissolved oxygen value (DO = 2.5 mg/l) in the absence of nutrients. Additionally, two litres were added with IAEA larval diet (0.4 gr/l) and left overnight at larval rearing room conditions. This fermented water generated a hatching media with a low dissolved oxygen value (DO = 2.0 mg/l) with the presence of nutrients and other
fermentation products. These two different hatching solutions were used in two trials to evaluate the effect of oxygen concentration and diet using deionised boiled water or fermented water on the hatching success of brushed *Ae. albopictus* and *Ae. aegypti* eggs.

In the first trial, the effect of diet and hydrogen peroxide (H2O2, 3% concentration) was evaluated using deionised boiled water as hatching solution. The larval diet and the H2O2 were employed to decrease and increase the DO concentration in the hatching solution, respectively.

Hatching solutions consisting of 45 ml of boiled deionised water supplemented or not with (i) 20 mg of larval diet and (ii) 0.25 ml of 3% H2O2 were tested on both species using the following four treatments.

**HD**: H – 0.25 ml H2O2 added D – 20 mg larval diet added.

**HN**: H – 0.25 ml H2O2 added N – no larval diet added.

**OD**: O – no H2O2 added D – 20 mg larval diet added.

**ON**: O – no H2O2 added N – no larval diet added.

In the second trial, the effect of H2O2 on the egg hatching success was evaluated using fermented deionised water as hatching solution. The two different hatching solutions tested consisted of 45 ml of fermented deionised water added (H) or not (O) with 0.25 ml of H2O2. Hatching trials were performed using 50 ml Falcon tubes maintained closed at larval rearing room conditions overnight.

After incubation, the eggs hatching response was estimated on a random sample of 200–300 eggs under a stereoscopic microscope and measurement of dissolved oxygen (DO) concentration in each hatching solution was recorded using a dissolved oxygen analyser. At the end of these trials, all the eggs that did not hatch were gently dried overnight and hatched again the following day in Falcon tubes using IAEA standard hatching procedures to confirm eggs viability. Two biological replicates with three technical repetitions each were performed for each treatment and species tested.

### Experiment 4. Effect of diet on water dissolved oxygen (DO) concentration in mass rearing tray

The previous experiments demonstrated that a strong reduction in the DO concentration produced by the presence of the diet in the hatching media provided an effective stimulus for hatching in *Ae. albopictus* and *Ae. aegypti* eggs, regardless its concentration. A trial was therefore conducted to estimate the relationships between the diet concentration introduced into mass rearing trays and the relative water deoxygenation produced as a function of time. Mass rearing trays were stacked inside a rack unit and filled with 5 L of deionised water at larval rearing room conditions. The trays were provided with 5 different diet concentrations: 0, 50, 100, 200 and 400 mg/l, by spreading over the entire water surface 0, 250, 500, 1000 and 2000 mg of IAEA larval diet, respectively. The lower diet concentration tested (50 mg/l) was equivalent to the diet quantity contained in a dissolvable capsule (250 mg) while the larger dose was equivalent to the diet concentration used in IAEA standard hatching procedures either in jars or Falcon tubes (400 mg/l). The water DO concentration was recorded on each tray at 0, 6, 24 and 36 h from diet introduction. Nine repetitions were performed for each of the five treatments by measuring the DO values in the water 4 times consecutively.

### Experiment 5. Effect of diet on capsule hatching method under mass rearing conditions

A final test was conducted on *Ae. albopictus* and *Ae. aegypti* encapsulated brushed eggs to evaluate, under deionised fermented and not fermented water conditions, the hatching rate on eggs introduced without diet (CAP_E) or together with diet in the same (CAP_T) or separate capsules (CAP_E and CAP_D) placed in the opposite side of the tray. Capsule containing only eggs (CAP_E; 120 mg eggs), only diet (CAP_D; 250 mg IAEA larval diet) and eggs with diet (CAP_T; 120 mg eggs plus 250 mg IAEA larval diet) were therefore prepared and introduced in different combinations into the mass rearing trays. The trays were filled with 5 L of deionised water and stacked inside a rack under larval rearing room conditions 24 h before the introduction of the capsules. The same combinations of capsules were also prepared and introduced into different mass rearing trays filled with 5 L of water added with diet (400 mg/l larval diet) 24 h before the introduction of the capsules. The same amount of egg used to fill the capsule was also transferred inside a glass jar filled with 700 ml of deionised water added with 0.3 gr of IAEA larval diet powder for both species in order to maintain a comparison with the IAEA standard hatching method (STD). The day after introduction of the capsule in the mass rearing tray, a random sample of 200–300 eggs were collected using a pipette from the bottom of mass rearing trays and from the jars to estimate the hatching rates obtained with the different treatments performed. Two and four biological replicates with three technical repetitions each were performed for each treatment for *Ae. aegypti* and *Ae. albopictus* respectively.

## Statistical analysis

Statistical analyses were carried out with R version 4.1.0²⁹ using RStudio³⁰ and with IBM SPSS Statistic v.23 (IBM Corporation, Armonk, NY) with a significance level (a) of 5%. Generalized binomial linear mixed-effects models (GLMM) fit by maximum likelihood (Laplace approximation) with a logit link, was used to analyse the hatching success, the larval survival and the pupal productivity of *Ae. albopictus* and *Ae. aegypti*, considering these parameters as response variables, treatments and their interaction as fixed effects and the replicates nested with technical repetition as a random effect. After initial investigations using the specie as a fix factor, the databases were partitioned and analysed separately for each species tested. A binomial GLMM fit by maximum likelihood were also used to analyse the relationships between the water dissolved oxygen measured (DO) and the species tested on the eggs hatching success considering the egg hatch success (HR > 0.80) as response variable, the DO, the specie and their interaction as fixed effects and the replicates as a random effect for each species tested. For validation, the full models were checked for overdispersion (using Bolker's function; package bblme)³¹,³² and for normality and homogeneity of variances on the residuals³³. The stepwise removal of terms followed by likelihood ratio tests (LRTs) or based on the lowest value of Akaike's Information Criterion (AICc)
was used for model simplification. ANOVA function was used to compare the fits of models. The minimal adequate model retained only factors that significantly reduced explanatory power (*p* < 0.05) when removed³⁴. Repeated-measures analysis of variance (ANOVA) was used to analyse the data obtained between treatments. Mauchly's test with Greenhouse-Geisser and Huynh-Feldt adjustments were used to test sphericity assumptions and multiple comparisons between treatments were assessed using Tukey's honestly significant difference (HSD) post hoc test.

## Results

### Experiment 1: effect of diet, egg brushing and closure of container on egg hatching success

The diet added in the hatching media was one of the main factors affecting the hatching rate of eggs in both *Ae. albopictus* and *Ae. aegypti* (*z* = 23.38, *p* < 0.001). However, the hatching rate was different in the two species (*z* = -27.62, *p* < 0.001) varying according to the presence or absence of the diet in the hatching media (*z* = 16.87; *p* < 0.001). While in absence of diet the mean (Estimated Marginal Mean ± SE) hatching rate of *Ae. aegypti* (55.60 ± 1.50%) was significantly higher than *Ae. albopictus* (15.83 ± 1.81%), in presence of diet the two species had similar egg hatching successes (91.73 ± 0.79 and 91.67 ± 0.69%, respectively) (Fig. 1A and B).

The addition of diet (*z* = 45.24, *p* < 0.001) the brushing of the eggs (*z* = 12.72, *p* < 0.001) and the closure of the container (*z* = -3.25, *p* < 0.01) were all parameters that interact and significantly affected the egg hatching success in *Ae. albopictus* (Fig. 1A). The brushing of the eggs increased the eggs hatching success only when hatching was performed using open containers in absence of diet (*z* = 5.37, *p* < 0.001). The presence of diet in the hatching media was the main parameter affecting the eggs hatching success in *Ae. aegypti* (*z* = 32.66, *p* < 0.001) with a significant increase in hatching rate of brushed eggs discriminable only in presence of diet (*z* = 2.30, *p* < 0.05) (Fig. 1B).

The analysis performed indicated that dissolved oxygen measurement (*z* = -4.54, *p* < 0.001) and the species considered (*z* = -2.25, *p* < 0.05) were both significant statistic predictors for the eggs hatching success. According to the model obtained, *Ae. albopictus* has a significant reduced probability (B = -2.22, Exp (B) = 0.11) of hatching success in comparison with *Ae. aegypti* and for each unit increase in DO in the hatching media we expect a ~30-fold reduction (B = -3.46, Exp (B) = 0.03) in the likelihood of eggs hatching success. The mean (± SE) DO values measured in the hatching media for *Ae. albopictus* and *Ae. aegypti* after successful hatching procedure (HR > 0.8; Fig. 1) were equal to 2.36 ± 0.18 mg/l and 2.79 ± 0.25 mg/l while treatments with unsatisfactory hatching (HR < 0.8) showed higher (all *p* < 0.001) DO values for both *Ae. albopictus* (3.82 ± 0.37) and *Ae. aegypti* (and 4.33 ± 0.26).

### Experiment 2: Capsule egg hatching method against standard egg hatching method

The trials performed under mass rearing conditions indicated that the hatching rate of *Ae. albopictus* eggs was affected by both the hatching method (*z* = 3.78, *p* < 0.001) and the initial viability of the eggs tested (*z* = 3.78, *p* < 0.001) with a significant interaction between factors (*z* = -0.24, *p* < 0.01). Using low-viable eggs (HR < 0.8), the capsule method induced a lower (*z* = -3.78, *p* < 0.001) mean (± SE) hatching rate (0.667 ± 0.043) in comparison with the standard method (0.711 ± 0.039) while, using high-viable eggs (HR > 0.8) the capsule

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Egg hatching rate measured in *Ae. albopictus* and *Ae. aegypti* in the different treatments performed with eggs brushed or maintained on paper (E vs. P), using container open or closed (O vs. C) during hatching procedures performed in presence or absence of larval diet (D vs. N).</figcaption></figure>
method (0.743 ± 0.036) and the standard method (0.736 ± 0.038) equally stimulated hatching (z = 0.548, p > 0.05) (Fig. 2A).

The survival rate of *Ae. albopictus* larvae obtained with the hatching capsule method (0.713 ± 0.015) was higher (z=-13.33, p < 0.001) than the standard method (0.700 ± 0.015) with different intensity according to the viability of the eggs used (z=2.35, p < 0.05). However, the use of low viable eggs significantly increased (z=-19.81, p < 0.001) the larval survival rate of *Ae. albopictus* (0.722 ± 0.016) in comparison with data collected on high-viable eggs batches (0.691 ± 0.017) (Fig. 2B).

The capsule and standard methods showed similar (z=2.24, p > 0.05) male pupal production rate (0.371 ± 0.011 and 0.367 ± 0.011, respectively) when low-viable eggs were tested, while the use of high-viable eggs improved (z=6.91, p < 0.001) the number of *Ae. albopictus* male pupae collected on the initial number of larvae reared with the capsule method (0.368 ± 0.011) in comparison with the standard method (0.357 ± 0.010). The hatching method used did not affect the *Ae. albopictus* female pupal production rates (z=-0.62, p > 0.05). The female production rates obtained with low-viable eggs with the capsule and the standard methods (0.201 ± 0.012 and 0.200 ± 0.011, respectively) were higher (z=-15.29, p < 0.001) than the production rates registered with the same methods using high-viable eggs (0.173 ± 0.010 and 0.193 ± 0.011). The increase in the number of larvae initially introduced into the mass rearing trays (L1 range: 14,749–19,281) reduced ($t_{1,56}$ = -3.34; p < 0.01) the larval survival rate (Fig. 3A) and did not produce any increase ($t_{1,56}$ = 1.81; p > 0.05) in the number of *Ae. albopictus* male pupae collected 160 h after hatching (Fig. 3C).

All trials performed on *Ae. aegypti* were carried out with high-viable eggs (HR > 0.8) and the hatching rate observed with the capsule method (0.806 ± 0.024) was not significantly different (z=-1.798, p > 0.05) than

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Egg hatching rates (A) and survival rates of the immature stage (B) measured on low (HR < 0.8) and high (HR > 0.8) viable *Ae. albopictus* eggs using the capsule (CAP) and the standard (STD) hatching methods under mass rearing conditions.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Scatter plots and regression lines describing the relationships between the number of larvae L1 introduced in the mass rearing trays and the survival rates (A and B) and the male pupae productivity (C and D) registered at 160 h from larval introduction in *Ae. albopictus* and *Ae. aegypti*.</figcaption></figure>

with the standard method (0.786 ± 0.026). The survival rate of *Ae. aegypti* larvae produced with the hatching capsule method (0.848 ± 0.027) was significantly lower (z = 19.92, *p* < 0.001) than with the standard method (0.870 ± 0.024). The male pupal production in *Ae. aegypti* was not different under the two hatching methods tested (z = 1.903, *p* > 0.05) while the female pupal production rate was higher (z = -21.05, *p* < 0.001) with the capsule methods (0.228 ± 0.010) in comparison with the standard method (0.204 ± 0.009). The increase in the number of larvae initially introduced into the mass rearing trays (L1 range: 13,634–18,391) significantly reduced (t<sub>1,22</sub> = -3.13; *p* < 0.01) the larval survival rate (Fig. 3B) but produced a significant increase (t<sub>1,22</sub> = 4.99; *p* < 0.001) in the number of *Ae. aegypti* male pupae collected 160 h after hatching (Fig. 3D).

**Experiment 3: effect of diet and oxygen concentration on egg hatching**

In the first trial conducted (Fig. 4A, C, E and G), the species used (z = -7.65, *p* < 0.001), the addition of larval diet (z = 3.45, *p* < 0.001) and H2O2 (z = -29.04, *p* < 0.001) in the boiled water used as hatching medium (DO = 2.5 mg/l) were all factors that significantly influenced the egg hatching rate with significant mutual interactions (all *p* < 0.001). When H2O2 was added to the water, the hatching rate of *Ae. albopictus* eggs with (0.025 ± 0.004) and without diet (0.035 ± 0.005) were equally (z = 1.52, *p* > 0.05) strongly inhibited. In the absence of H2O2, the addition of larval diet in the hatching solution increased (z = -5.42, *p* < 0.001) hatching success (0.868 ± 0.010) as compared with eggs immersed exclusively in deoxygenated water (0.785 ± 0.012) (Fig. 4A). The DO values measured in the hatching solution for *Ae. albopictus* were significantly affected by the presence of larval diet (t<sub>3,20</sub> = -6.87; *p* < 0.001), H2O2 (t<sub>3,20</sub> = 70.61; *p* < 0.001) and their interaction (t<sub>3,20</sub> = 5.35; *p* < 0.001). In the absence of H2O2, the addition of larval diet in the hatching solution decreased (t<sub>3,20</sub> = 6.87; *p* < 0.001) the DO values (1.07 ± 0.11) as compared with eggs immersed exclusively in deoxygenated boiled water (2.23 ± 0.12). In presence of H2O2 the values of the DO measured in the hatching media in presence (14.33 ± 0.12) or absence (14.23 ± 0.12) of diet were not significantly different (t<sub>3,20</sub> = -0.69; *p* > 0.05; Fig. 4E).

Similar to what was observed in *Ae. albopictus*, the hatching rate of *Ae. aegypti* eggs was affected by the presence of diet (z = 3.44, *p* < 0.001) and H2O2 (z = -29.05, *p* < 0.001). The presence of diet increased *Ae. aegypti* egg hatching rate both in presence (6.11 ± 0.68 vs. 2.62 ± 0.45; z = -4.17, *p* < 0.001) and absence of H2O2 (93.65 ± 0.69 vs. 89.84 ± 0.85; z = -3.45, *p* < 0.01; Fig. 4C). The DO values measured in the hatching solution for *Ae. aegypti* were significantly affected by the presence of larval diet (t<sub>3,20</sub> = -2.62; *p* < 0.05), H2O2 (t<sub>3,20</sub> = 32.17; *p* < 0.001) and their interaction (t<sub>3,20</sub> = 2.14; *p* < 0.05). In the presence of H2O2, the values of the DO measured in the hatching media in presence (13.98 ± 0.24) or absence (13.85 ± 0.24) of diet were not significantly
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Egg hatching rates (A, B, C and D) and the related DO values (E, F, G and H) measured in the different hatching treatments performed in Falcon tubes with brushed eggs of *Ae. albopictus* and *Ae. aegypti* using boiled deionised water (Experiment 3, trial 1; DO = 2.5 mg/l) and fermented deionised water (Experiment 3, trial 2; DO = 2.0 mg/l) as hatching solutions.</figcaption></figure>

different ($t_{3,20} = -0.40; p > 0.05$). However, in the absence of H2O2, the addition of the larval diet only marginally reduced ($t_{3,20} = -0.40; p = 0.071$) the DO values ($2.13 \pm 0.24$) as compared with eggs immersed exclusively in deoxygenated boiled water ($3.02 \pm 0.24$) (Fig. 4G).

In the second trial (Fig. 4B, D, F and H), the species used ($z = -6.15, p < 0.001$) and the addition of H2O2 ($z = -26.11, p < 0.001$) in the diet fermented water used as hatching media (DO = 2.0 mg/l) were all factors that influenced the egg hatching rate, with significant interactions ($z = 5.45, p < 0.001$). When H2O2 was added to the fermented water, the hatching rates of *Ae. albopictus* ($0.052 \pm 0.007$) and *Ae. aegypti* ($0.026 \pm 0.005$) were reduced ($z = -28.01, p < 0.001$ and $z = -26.01, p < 0.001$, respectively) in comparison with their untreated control groups ($81.04 \pm 0.011$ and $90.43 \pm 0.009$, respectively) (Fig. 4B and D). The DO values measured in the hatching solution for *Ae. albopictus* and *Ae. aegypti* increased with the presence of H2O2 ($t_{1,10} = -23.48; p < 0.001$ and $t_{1,10} = -98.03; p < 0.001$, respectively), up to $13.53 (\pm 0.35)$ and $14.35 (\pm 0.35)$ mg/l, respectively (Fig. 4F and H).

**Experiment 4. Effect of diet on water dissolved oxygen (DO) concentration in mass rearing tray**

In the mass rearing trays, the DO concentration varied over time ($F_{2,19,40} = 2136.05, p < 0.001$) and as a function of the diet concentration used ($F_{8,77,40} = 83.26, p < 0.001$) (Fig. 5). Diet concentration impacted the average DO scores across time ($F_{4,40} = 163.29, p < 0.001$). The diet introduced in the larval mass rearing trays at a concentration of 50 mg/l reduced ($p < 0.001$) the water DO concentration measured after 6 ($4.90 \pm 0.05$ mg/l) and 24 h ($3.47 \pm 0.09$ mg/l) in comparison with the untreated water trays. The introduction of larval diet in the trays at concentration of 400 mg/l induced a higher DO concentration reduction ($p < 0.001$) at 6 ($4.18 \pm 0.05$ mg/l) and 24 h ($2.21 \pm 0.03$ mg/l) after treatment in comparison with the value registered at the same time in untreated trays ($4.9 \pm 0.04$ mg/l and $4.46 \pm 0.03$ mg/l).

**Experiment 5. Effect of diet on capsule hatching method under mass rearing conditions**

The eggs of *Ae. albopictus* (Fig. 6A) and *Ae. aegypti* (Fig. 6B) introduced together with larval diet in capsules have a significant higher ($p < 0.001$) hatching success than the eggs introduced alone (CAP_E) or away from the capsule containing larval diet (CAP_E + CAP_F) when submerged in trays filled with deionised water. The addition of diet in the larval tray 24 h before eggs introduction significantly increase the hatching success of eggs introduced alone or away from larval diet in both species. The hatching rate of *Ae. albopictus* and *Ae. aegypti* eggs introduced alone (CAP_E) or positioned away from the larval diet (CAP_E + CAP_F) are satisfactory only when performed inside trays containing water previously combined with diet within 24 h.

The addition of diet in the deionised water as a pre-treatment increased the hatching rate ($z = -3.084, p < 0.05$) observed in the capsule containing *Ae. albopictus* eggs and diet ($0.762 \pm 0.010$ vs. $0.716 \pm 0.011$, Fig. 6A) while
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Trends in the dissolved oxygen concentration (DO) measured in mass rearing trays filled with 5 L of deionised water (T=25 °C) at different larval diet concentration (0, 50, 10, 200 and 400 mg/l) as a function of time (hr). Mean values with 95% confidence interval error bars are reported. Different letters indicate significant difference (*p* < 0.05) between treatment (Tukey's test).</figcaption></figure>

in *Ae. aegypti*, the capsules containing eggs and diet had a similar (*p* > 0.05) hatching rate irrespective of the addition of diet in the larval tray 24 h before eggs introduction (0.916 ± 0.008 vs. 0.923 ± 0.008). However, the eggs introduced with diet in capsules in deionised and fermented water had similar (*p* > 0.05) hatching success in comparison with eggs hatched using standard hatching procedures in both species (Fig. 6A). The addition of diet in the larval tray 24 h before the introduction of *Aedes* eggs decreased (*p* < 0.001) the mean (± SE) DO value in the water (2.29 ± 0.05 mg/l) in comparison with trays filled with deionised water only (4.32 ± 0.05 mg/l).

Discussion

A new method for *Ae. albopictus* and *Ae. aegypti* egg hatching based on the direct introduction of dissolvable capsules filled with eggs and larval diet into the mass rearing trays has been proposed and successfully tested. This system aims to increase the cost-efficiency of the *Aedes* mass rearing process by eliminating intermediate procedures for hatching, counting and transferring the young fragile larvae into the mass rearing trays.

In the first experiment, possible hatching conditions found within an open larval rearing tray were simulated using 50 ml capacity Falcon tubes. These tests showed that brushing eggs from oviposition papers increases the egg hatching success in both species. Physical stimuli such as vibrations or water agitation³⁵⁻³⁷ have previously been reported to effectively increase egg hatching rate in some *Aedes* species, probably because capable to simulate the typical vibrational pattern produced during rainfall. However, as previously observed³⁶, the chemical hatching stimuli produced by the presence of diet were stronger than the physical cues produced by egg brushing and the closure of the container in both species in our study. The closure of the containers decreases the eggs hatching success in both species but with statistical significance only for *Ae. albopictus*. However, containers which were exposed to air were previously observed to produce the most efficient hatching also on *Ae. aegypti*³⁸. Currently, the containers used for *Ae. albopictus* and *Ae. aegypti* hatching procedures are generally kept closed¹⁰,¹⁵ but the results observed in this study indicate that open containers can equally be used to achieve successful hatching in these species. The larval diet added in the hatching media provides the main hatching stimulus for both species. The presence of diet produces a significant reduction in the amount of dissolved oxygen concentration which has a significant inverse relationship with the egg hatching success in both species. The reduction of the dissolved oxygen concentration in the hatching media has been often identified as the main stimulus for *Aedes* eggs hatching regardless of the deoxygenation method adopted¹⁸.

In our study, we confirmed that the DO concentration reduction in the hatching media is the main stimulus for egg hatching in the tested *Aedes* species. The use of hatching solutions characterized by deionised water subjected to prolonged boiling time or previously added with larval diet generate equally anoxic conditions (DO < 2.5 mg/l) capable of effectively stimulating egg hatching in both species. However, the addition of H2O2 in all hatching solutions increases DO values, leading to a complete inhibition of the hatching stimuli in both species regardless of the presence of diet or fermentation agents or products that may be present in the hatching media. Eggs subjected to H2O2 do not lose viability, showing high hatching success when subjected to standard hatching procedures 24 h after the exposure to hydrogen peroxide. Our results confirm how deoxygenated and
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Egg hatching rates of *Ae. albopictus* (A) and *Ae. aegypti* (B) brushed eggs introduced alone (CAP_E) or together with diet in the same (CAP_T) or separate capsules (CAP_E and CAP_D) placed in the opposite side of mass rearing trays filled 24 h before with 5 L of deionised water in presence (FERMENTED DEIONISED WATER) or absence (DEIONISED WATER) of larval diet (400 mg/l). Parallel eggs hatching procedures were also carried out on both species to have a comparison with current standard hatching procedures (STD).</figcaption></figure>

sterilized water obtained with prolonged boiling time can strongly stimulate hatching in both species tested. However, the use of boiled water is not generally considered an efficient hatching method for *Ae. albopictus* eggs¹⁵ probably due to the short boiling time adopted and the concomitant poor deoxygenation obtained (DO > 3 mg/l). All tests conducted confirmed the lower DO reduction required by *Ae. aegypti* in comparison with *Ae. albopictus* to achieve effective hatching rates under all the different hatching conditions tested. As previously observed in other *Aedes* species¹⁸, a reduction of the dissolved oxygen below 3 mg/l in the hatching medium has been showed to cause effective stimulation of egg hatching in *Ae. albopictus* and *Ae. aegypti*.

The capsule method proposed in this study to aliquot and hatch *Ae. albopictus* and *Ae. aegypti* eggs directly into mass rearing trays proved equally efficient as the IAEA standard hatching procedures. However, the use of eggs with initial low viability (HR < 0.8) reduced the hatching capacity of the capsule method in comparison with the standard hatching method. This different hatching rate could also be related to a smaller quantity of diet (0.2 mg vs. 0.3 mg) that can be introduced into the selected capsules (size 0) due to the different quantity of eggs needed to maintain the same larval density into the mosquito rearing trays (18,000 larvae per tray) using low and high viable eggs (0.25 mg and 0.15 mg, respectively). This condition was observed only in *Ae. albopictus* probably because this species suffers a greater decline in egg viability during embryonation and storage as compared with *Ae. aegypti*³⁹,⁴⁰. Newly deposited *Aedes* eggs require gradual maturation of the embryo and subsequent storage at high and constant relative humidity (90 ± 5%) avoiding strong ventilation and excessive temperature variations (28 ± 2 °C). As previously reported, even if the hatch rate decreases proportionally with the duration of eggs storage in both species¹⁵, *Ae. aegypti* has greater resistance to desiccation and greater tolerance to higher temperature in comparison with *Ae. albopictus*, resulting in longer eggs viability under different storage conditions³⁸⁻⁴⁰. However, hatching rates greater than or equal to 80% indicate a good viability of eggs in both species and can be considered a threshold value to assess egg quality under mass rearing conditions¹⁵.
The use of the different hatching methods on high-viable eggs in both species produced similar egg hatching success without affecting larval survival and pupation rates under mass rearing conditions. The larval survival rate of both species decreases proportionally with the increase of the larval density above 2.5-3 larvae/ml irrespectively from the eggs hatching method used. However, while in *Ae. aegypti* the male pupae collected 160 h after hatching increase proportionally with the number of larvae reared, in *Ae. albopictus* the production of male pupae remains unchanged by the increase in larval density. The negative effect of overcrowding on *Ae. albopictus* male pupal productivity was already observed during mass rearing validation procedures⁴¹ (max larval density 2-3 larvae per ml) while the use of higher larval density is adopted (3-4 larvae per ml) in mass rearing procedures supporting the release of *Ae. aegypti* sterile males⁴².

Although the reduction of water DO concentration produced by diet fermentation resulted the main egg hatching stimulus, the quantity of diet introduced with eggs in the capsules (approximately 250 mg) was not sufficient to effectively reduce the water DO concentration in the mass rearing trays below the critical threshold for both species. A suitable reduction of the DO parameter can instead be achieved by adding 0.4 g of larval diet per litre of rearing water, 24 h before the introduction of the eggs in the mass rearing trays. Tests conducted by separating the eggs from the larval diet in trays containing only deionised water indicated that only the introduction of eggs and diet in the same capsule is capable to effectively stimulate hatching in both species. The eggs introduced with diet in the capsule could therefore be exposed to an effective hatching stimulus because released near the diet which would be capable to deoxygenate only a small volume of water in the immediate proximity. If the water in the rearing tray is previously added with 0.4 g of larval diet per litre, the reduction in dissolved oxygen concentration produced can effectively stimulate the egg hatching of both species even when introduced in absence of any additional larval diet.

The proposed capsule method and the IAEA standard hatching procedures are equally capable to stimulate eggs hatching in both species while the presence of fermented water in the mass rearing trays before capsule introduction produces a small but significant increase in eggs hatching success only in *Ae. albopictus*. The use of the capsule hatching method for the mass rearing of *Ae. albopictus* has been tested in parallel at the CAA in Italy using comparable mass rearing procedures and technologies. While the capsules containing eggs and diet used in the laboratory tests were filled manually with a total preparation time of approximately 20 capsules per hour, the capsules tested under mass rearing conditions at the CAA were created using a pharmaceutical manual capsule filling machine (MC2, Multigel, Florence, Italy) with a processing capacity of approximately 1,200 capsules per hour. Whitin six months of continuous mass production, a mean weekly production of about 500,000 males was achieved with a mean pupae male production rate of 31% and sex separation accuracy of about 99.0% using the Wolbaki automatic sorting device<sup>43,44</sup>.

The results obtained in this study demonstrate that adding larval diet when filling the trays 24 h before the introduction of the capsules in the *Ae. albopictus* larval mass rearing procedures could enhance the hatching success thus reducing the amount of eggs used. Moreover, considering that this species does not show an increase in pupae productivity as larval density increases above 2.5 larvae/ml, an accurate evaluation of the larval density employed is recommended. The effect of larval density in *Ae. albopictus* should be further investigated to maximize the male pupae productivity under mass rearing conditions. In any case, the use of the capsule method appears capable of generating a substantial improvement in the management of larval mass rearing procedures with a reduction in working times and staff employed.

With current standard Aedes mass rearing procedures, the preparation and overnight incubation of the eggs hatching jars and the subsequent manual transfer of the larvae into the rearing trays are necessary. The preparation of a rack with 50 larval trays therefore requires approximately 120 min, which includes the movement and transfer of the hatched larvae and requires the availability of approximately 0.7 sqm of climate-controlled rearing area for overnight incubation. Since a 50-tray rack unit can produce on average 100,000 to 150,000 male Aedes pupae per week, a production of 10 million males in a bisexual mosquito strain mass-rearing facility<sup>45</sup> would require the use of approximately 70 to 100 racks per week with larval rearing preparation times of approximately 140-200 h and the availability of 50 to 70 sqm of climatic controlled area for hatching procedures.

With the proposed capsule method, 300 capsules with egg and diet (necessary to fill 6 mass rearing racks) can be prepared in about 15 min using available manual capsule fillers. No additional rearing space would be necessary for the hatching procedures and the manual insertion of the capsules into the mass rearing trays would take place in approximately 2 min per rack and overall times of 6 to 8 h to set up a larval rearing to produce 10 million males per week.

As already experienced with *Ae. albopictus*, the eggs can be easily and safely brushed under mass rearing conditions and homogeneously mixed with larval diet to achieve the correct volume of eggs to be introduced into the capsules. The presence of diet in the capsules is necessary to promote effective hatching and to correctly calibrate the volume of eggs to be introduced in the selected capsule size according to the initial egg's viability level. Available manual or automatic capsule filling machines can be used to easily create capsules with eggs and diet with the possibility to store the capsules for at least two weeks without losing eggs viability (preliminary unpublish data). Prolonged mixing procedures between the diet and the eggs can further favour the hatchability of the eggs increasing the vibration received. With these new mass rearing procedures, a significant improvement in production times and costs for *Aedes* species is expected with a higher cost-efficiency of the SIT and all the different genetic control programs that relies on mass production and area-wide release of mosquitoes.

## Data availability

Data generated or analysed during this study is provided within the manuscript. Direct access to source data is available in the supplementary information files.

The raw data for each experiment conducted can be obtained in the dedicated attached files.

Source data experiment 1 (Supplementary Material 1)
Source data experiment 2 (Supplementary Material 2)

Source data experiment 3.1 (Supplementary Material 3)

Source data experiment 3.2 (Supplementary Material 4)

Source data experiment 4 (Supplementary Material 5)

Source data experiment 5 (Supplementary Material 6)

Received: 24 July 2024; Accepted: 26 November 2024

Published online: 04 December 2024

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**Author contributions**

F.B. and A.P. designed the experiments and conceived the project. F.B., A.P and W.M. carried out experiments. J.B., and F.B. analysed and interpreted the data. J.B. and R.B. supervised the work. All authors drafted, reviewed, and approved the final manuscript.

**Declarations**

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1038/s41598-024-81385-z.

**Correspondence** and requests for materials should be addressed to F.B.

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