Effects of a bloodless diet on fitness and malaria susceptibility in Anopheles mosquitoes from Burkina Faso

Abstract

Emerging resistance in malaria vectors and parasites has accelerated the development of novel control strategies such as the Sterile Insect Technique and Wolbachia-based approaches, which require large-scale mosquito rearing. These methods traditionally rely on vertebrate Blood, raising ethical and logistical concerns. This study evaluated the effects of a BLOODless™ on wing size, longevity, and Plasmodium falciparum susceptibility in Anopheles coluzzii and An. gambiae from Burkina Faso, and the Kisumu An. gambiae strain. Blood and Bloodless-reared mosquitoes were compared across seven generations. Wing size was unaffected by diet in the 3rd and 7th generations, though sex impacted size in the 3rd generation only. A diet-species interaction was detected in the 7th generation. Plasmodium falciparum infection rates were similar in the 3rd generation, but by the 6th, Blood-fed mosquitoes showed higher oocyst prevalence, though intensity remained unchanged. Infection outcomes were species-dependent only at this later stage. Longevity, initially comparable across groups, increased significantly in Bloodless-fed mosquitoes by the 7th generation. Species had no effect on survival. A Blood-free diet enables sustained mosquito rearing while preserving traits essential for malaria research, providing an ethical, effective alternative to vertebrate Blood in vector control and experimental programs.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-40458-x.

Background

Mosquito species can acquire and transmit pathogens responsible for human diseases such as malaria, dengue fever, yellow fever, chikungunya, Zika, West Nile virus, Japanese encephalitis and lymphatic filariasis¹. The highest incidence of these mosquito-borne diseases is in the tropical and subtropical regions of the world¹. Among these mosquito-borne diseases, malaria remains a major public health problem, despite numerous control efforts¹.

Malaria control have focused primarily on the use of antimalarial drugs and insecticides, such as long-lasting insecticide-treated nets and indoor residual spraying^2–4against mosquito vectors. While these interventions have reduced malaria transmission⁵, their widespread use has also contributed to the development of drug and insecticide resistance, which are a threat to ongoing control efforts^6,7.

To address the challenges posed by resistance, new control measures have been recommended, focusing mainly on the RTS/S, AS01 in October 2021⁸ and R21/Matrix-M in October 2023⁹ vaccines¹⁰, which have been approved by the World Health Organisation for use in malaria-endemic areas in 2024. In addition, new strategies have been developed in laboratories to support malaria control, including the Sterile Insect Technique (SIT), which uses radiation to sterilise male mosquitoes¹¹; the release of insects carrying a dominant lethal gene, in which male mosquitoes are genetically modified so that their mated offspring die before reaching adult stage¹²; and mosquitoes infected with Wolbachia to reduce disease transmission^13,14. A complementary approach is to disrupt the parasite development in mosquito vectors following Blood-feeding on a vaccinated or drug-treated patient, i.e. transmission-blocking drugs or vaccines¹⁵. Indeed, in anautogenous mosquitoes, the blood meal provides essential nutrients that initiate vitellogenesis and egg development, serving as a critical benchmark for assessing the performance of blood-free diets in mass-rearing systems¹⁶.

The implementation of these new vector-based control strategies relies heavily on the ability to rear large numbers of mosquitoes. For experimental purposes, mass rearing of mosquitoes in insectaries requires a reliable supply of Blood of sufficient quantity and quality. In most of the world’s insectaries, mosquito colonies are often maintained by a substantial supply of Blood from human donors, slaughterhouse Blood from various animal types, and sedated or restrained live animals^17,18. However, the use of live animals has several drawbacks, including high costs associated with their purchase and maintenance¹⁹. Additionally, ethical concerns, regulatory constraints, and oversight by animal protection authorities limit the feasibility of this approach in certain laboratories¹⁸.

To overcome the limitations of fresh animal Blood, Blood-free alternatives such as the Bloodless diet have been proposed as viable substitutes for mosquito rearing²⁰. The Bloodless diet offers feeding rates comparable to those achieved with vertebrate Blood and mimic the physiological effects of a conventional Blood meal²¹. This approach is cost-effective, more standardised, and reduces the use of animals for mosquito rearing. Additionally, it helps prevent the ingestion of contaminants such as microsporidia, which adversely impact mosquito colonies and compromise insectary operations¹⁹. Nevertheless, understanding the effects of Blood-free diets on key mosquito life-history traits is necessary before their large-scale implementation. Recent studies have shown that Bloodless does not negatively affect the fitness, genome, microbiota, or vector competence of Anopheles stephensi for Plasmodium berghei¹⁷. These previous studies, although innovative, were carried out in a non-malaria endemic area, using mosquito strains that had been established for over a decade in the laboratory, and used murine parasites. Another study, carried out in a malaria-endemic country with An. gambiae s.s., showed that the mean number of eggs per female fed on bovine Blood was higher than those fed on Bloodless, but that the hatching rate was higher in the Bloodless diet group, when compared to the bovine Blood group²². It is therefore important to carry out further studies in malaria-endemic areas, on additional malaria vector species, and to use natural isolates of P. falciparum to carry out experimental infections.

Despite recent advances in blood-free mosquito diets that support reproduction and long-term colony maintenance in laboratory settings (e.g., artificial BLOODless diets; Mosi et al. 2024; long-term blood-free rearing studies 2024), there remains a critical lack of data on the multi-generation effects of bloodless diets in malaria-endemic, field-derived mosquito populations evaluated with natural Plasmodium falciparum isolates.

The current study aimed to evaluate the effect of Bloodless diet on mosquito fitness and P. falciparum development in three key malaria vector strains: the highly insecticide-resistant Anopheles gambiae and An. coluzzii strains from Burkina Faso^23,24, and the pyrethroid-susceptible An. gambiae Kisumu strain, originally from Kenya.

Methods

Ethics approval and consent to participate

The study protocol was approved by the Institutional Ethics Committee of the Institut de Recherche en Sciences de la Santé (IRSS), Burkina Faso (Approval No. A 009-2023/CEIRES/IRSS; IRB registration #00004738; FWA #00007038). Rabbit blood was obtained from laboratory-reared rabbits maintained at the IRSS animal facility. Blood collection (approximately 2 mL from the ear vein into heparinized tubes) was performed by trained personnel under veterinary supervision, in accordance with institutional animal welfare regulations. All experiments involving vertebrate animals were conducted in accordance with relevant guidelines and regulations, and all methods are reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). For human participants, prior to inclusion, written informed consent was obtained from the parents or legal guardians of the child volunteers.

Mosquito strains

Laboratory-reared An. gambiae and An. coluzzii were obtained from outbred colonies established in 2023 from wild caught females collected in Soumousso, Burkina Faso (11°23′14′′N, 4°24′42′′W) and identified by SINE PCR²⁵. The An. gambiae Kisumu strain was established in 2019 from eggs obtained from the Institut de Recherche pour Développement in Montpellier (France). Mosquitoes were held in 30 × 30 × 30 cm mesh-covered cages under standard insectary conditions (12 h:12 h light: dark [L: D], 27 ± 2 °C, 80 ± 10% relative humidity). Approximately 2 mL of venous blood were collected from the rabbit’s ears into heparinized tubes. The females were then maintained on this blood using the direct membrane feeding assay (DMFA) system. Larvae were reared at a density of 350 first-instar larvae in 700 mL of water in plastic trays measuring 36 cm × 3.14 (≈ 113.04 cm), calculated circumference using the formula C = D*π and fed with Tetramin Baby Fish Food (Tetrawerke, Melle, Germany). Adult mosquitoes were maintained with a 10% glucose solution, prepared using D-(+)- glucose powder, (Sigma-Aldrich^®, country) and distilled water.

Origin of bloodless diet and standard blood source

The lyophilized Bloodless diet was provided by the Global Health and Tropical Medicine/Instituto de Higiene e Medicina Tropical-Universidade Nova de Lisboa (GHTM/IHMT-NOVA), Portugal²⁰. Joana Marques and Henrique Silveira are inventors of the patented Bloodless diet (PCT/IB2019/052967, WO/2019/198013 and US2021030025A1), owned by Universidade Nova de Lisboa and Universidade do Algarve¹⁷. Standard Blood was obtained by collecting venous Blood from one of the ears of rabbits reared at the Institut de Recherche en Sciences de la Santé.

Bloodless diet and blood direct membrane feeding assays

The artificial BLOODless diet was prepared by dissolving 0.3 g of BLOODless powder in 1 mL of distilled water. Two parallel mosquito colonies were maintained from F1 to F7: one fed the BLOODless diet and the other fed vertebrate blood. All comparisons were conducted using mosquitoes derived from the same founder population but maintained on these parallel diets. Larval feeding was identical across generations and treatments, eliminating potential confounding effects of larval nutrition. For adult feeding, direct membrane feeding assays were performed: feeding systems were maintained at 38 °C, and 500 µL of BLOODless or blood was offered to female mosquitoes in paper cups with separate feeding cells for 50–60 min. Three independent experiments were performed to assess the effects of the BLOODless diet on wing size, longevity, and susceptibility to Plasmodium falciparum in laboratory-reared Anopheles coluzzii and Anopheles gambiae mosquitoes.

Effects of bloodless on wing size

A total of 35 males and 35 females five-day-old from each species and experimental group (Bloodless diet and Blood) of the generations F3 and F7 were anaesthetised at -20 °C for 5 min to evaluate their wing size¹⁷. For males, it is important to note that measurements reflect the progeny of females maintained on either BLOODless or blood diets, rather than males fed directly on these diets. Under a binocular magnifying glass, the two wings of each mosquito were carefully dissected using N^o5 Dumont forceps while gently holding the thorax. The dissected wings were then placed on a slide containing a drop of 70% alcohol to ensure proper spreading. To secure them, a transparent coverslip was carefully cut and gradually lowered onto the wings, sealing them against the slide. Wing size was measured under the magnifying glass and photographed using a specially adapted LASX camera (Leica Microsystems, Switzerland Ltd. CH-9435 Heerbrugg, image resolution: 1024 × 768; actual field of view: 4.77 mm × 3.58 mm).

Effects of bloodless on susceptibility to Plasmodium falciparum

Female An. gambiae, An. coluzzii and An. gambiae Kisumu mosquitoes were fed with Blood drawn from naturally P. falciparum gametocyte-infected patients recruited among 5–15-year-old school children in villages surrounding Bobo-Dioulasso, Burkina Faso. Briefly, thick Blood smears were taken from each volunteer, air-dried, Giemsa-stained and examined by microscopy for the presence of P. falciparum at the IRSS laboratory in Bobo-Dioulasso. Asexual trophozoite parasite stages were counted against 200 leucocytes, while infectious gametocyte stages were counted against 1000 leukocytes. Children with asexual parasitaemia of > 1000 parasites per microlitre (estimated based on an average of 8000 leucocytes ml − ¹) were treated in accordance with national guidelines. Asymptomatic P. falciparum gametocyte-positive children were recruited for the study. Blood was collected by venipuncture in heparinized tubes. Two distinct parasite from 2 donors named hereafter A and B, with respective gametocytaemia of 440 and 144 gametocytes µl^− 1 of Blood, were used for the experimental infections using direct membrane feeding assays (DMFA) as previously described²⁶. DMFA was performed using whole donor Blood (i.e. no serum replacement)²⁶.

Four-five-day-old female mosquitoes from two generations (generation 3 - F3 and generation 6 - F6) of An. gambiae, An. coluzzii, and An. gambiae Kisumu were used to assess susceptibility to P. falciparum. Mosquitoes were held in the paper cups (see above) and allowed to feed on infective Blood for 1 h. Unfed or partially fed females were removed and discarded. The remaining fully engorged mosquitoes were placed into 30 × 30 × 30-cm mesh-covered cages and kept in a biosafety room under the same standard conditions (12 h:12 h L: D, 27 ± 2 °C, 70 ± 5% relative humidity). The mosquitoes were given a 10% glucose solution on cotton wool thereafter.

Susceptibility was evaluated based on two parameters: infection rate (the proportion of mosquitoes harboring at least one oocyst in their midgut following an infectious Blood meal) and infection intensity (the number of oocysts per infected female). For the experimental infections, two P. falciparum isolates (n = 2: isolates A and B) were used. On day 7 post-Blood meal (dpbm), 300 females from each generation were dissected. This included 150 mosquitoes fed on Bloodless (50 An. gambiae, 50 An. coluzzii, and 50 An. gambiae Kisumu) and 150 mosquitoes fed on Blood (50 An. gambiae, 50 An. coluzzii, and 50 An. gambiae Kisumu). The midguts of these mosquitoes were dissected and stained with 1% mercurochrome to microscopically assess the presence and number of oocysts at 400× magnification.

Effect of bloodless on survival

To determine how Bloodless influences mosquito longevity, 35 females and 35 males of each experimental group were kept in 25 × 25 × 25 cm cages, provided with a 10% glucose solution continuously every day, according to mosquito species. The experiment was performed at generations F3 and F7, with three replicates per group. Dead mosquitoes were removed and counted in each cage every morning at 08:00 am, until all the mosquitoes in the cages had died.

Statistical analysis

All analyses were performed with R software (version 4.3.1). We used a Generalized Linear Model (GLM, glm function, stats package in R) with a “Gaussian” distribution was used to analyse the effect of mosquito feeding treatment (2 levels: Bloodless diet and Blood), mosquito species (3 levels: An. coluzzii, An. gambiae and An. gambiae Kisumu), mosquito sex (2 levels: Male and Female), and their interactions on mosquito wing size for each mosquito generation (F3 and F7). Analysis of mosquito wing size was carried out separately for each mosquito generation because of the non-normal distribution of the data. Indeed, the mean wing size of F7 mosquitoes was 1.5 times larger than that of F3 ones, resulting in a bimodal distribution of data. Therefore, we analysed mosquito wing size separately for each mosquito generation to fit the Gaussian distribution of data. We used a binomial GLM to analyse the effect of mosquito feeding treatment, species, generation, and their interactions on oocyst prevalence (number of females harboring oocysts in their stomach out of the total number of females exposed to the infectious Blood meal). We used a GLM with a Poisson distribution to analyse the effect of mosquito feeding treatment, species, generation, and their interactions on oocyst intensity (number of oocysts in the stomachs of infected females). We use a Cox proportional hazards regression model (coxph function, survival package in R) to analyse the effect of mosquito feeding treatment, sex, species, generation, and their interactions on mosquito longevity.

For model selection, we used the stepwise removal of terms, followed by likelihood ratio tests. Term removals that significantly reduced explanatory power (p < 0.05) were retained in the minimal adequate model²⁷. Analyses for normality and homogeneity of variances were performed with a Shapiro and Fligner test, respectively (shapiro.test and fligner.test functions, stats package). If necessary, data were transformed using the Box-Cox power transformation method (powerTransform function, car package in R). When needed, multiple comparison tests were used to compare the levels of explanatory variable or explore significant interactions (emmeans function, emmeans package in R).

Results

Impact of bloodless on mosquito wing size

A total of 840 mosquitoes (both males and females) were dissected for wing measurements, comprising 420 mosquitoes from each generation (F3 and F7). This included 140 mosquitoes per species per generation (An. coluzzii, An. gambiae and An. gambiae Kisumu).

F3 generation. There was a significant feeding × sex interaction on mosquito wing size (=10.38, p=0.001; Fig. 1a). The wing size of male mosquitoes fed on Bloodless diet was similar to that of male mosquitoes fed on Blood, while the wing size of female mosquitoes fed on Bloodless diet was significantly higher than that of female mosquitoes fed on Blood (Fig. 1a). There was a significant feeding × species interaction on mosquito wing size (=22.66, p<0.001; Fig. 1b). In both An. coluzzii and An. gambiae, the wing size of mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood; while in An. gambiae Kisumu, the wing size of mosquitoes fed on Bloodless diet was significantly higher than that of mosquitoes fed on Blood (Fig. 1b). There was no significant feeding × sex × species interaction on mosquito wing size (=2.44, p=0.295; Fig. S1a).

Fig. 1.

Mosquito feeding treatment × sex interaction, and feeding treatment × species interaction on mosquito wing size for (a,b) F3 generation, and (b,c) F7 generation; respectively. The box plots indicate the median (wide horizontal bars), the 25th and 75th percentiles (squares) and the minimum and maximum values (whiskers). The black dots represent the mean values. Box plots with different letters indicate a statistical difference at P < 0.05.

F7 generation. There was no significant feeding × sex interaction on mosquito wing size (=0.98, p=0.321; Fig. 1c). The wing size of mosquitoes fed on Bloodless diet was significantly higher that of mosquitoes fed on Blood, regardless of mosquito sex (Fig. 1c). There was a significant feeding × species interaction on mosquito wing size (=16.35, p<0.001; Fig. 1d). In An. coluzzii, the wing size of mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood; while in both An. gambiae and An. gambiae Kisumu, the wing size of mosquitoes fed on Bloodless diet was significantly higher than that of mosquitoes fed on Blood (Fig. 1d). There was a significant feeding × sex × species interaction on mosquito wing size (=42.16, p<0.001; Fig. S1b).

Effect of bloodless on oocyst prevalence

A total of 600 female mosquitoes, including 300 individuals per generation F3 and F6), were experimentally infected with Blood containing P. falciparum gametocytes with a gametocytemia of 440 gam/µl for F3 generation and 144 gam/µl for F6 generation.

Oocyst prevalence. There was no significant feeding × species interaction on oocyst prevalence (=1.31, p=0.518; Fig. 2a). The oocyst prevalence in mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood, regardless of mosquito species (Fig. 2a). However, there was a significant feeding × generation interaction on oocyst prevalence (=10.85, p = 0.001; Fig. 2b). In the F3 generation, the oocyst prevalence in mosquitoes fed on Bloodless diet was similar to that in mosquitoes fed on Blood, while in F6 generation, the oocyst prevalence in mosquitoes fed on Bloodless diet was significantly higher than that in mosquitoes fed on Blood (Fig. 2b). There was no significant feeding × species × generation interaction on oocyst prevalence (=2.08, p=0.360; Fig. S2a).

Fig. 2.

(a) Mosquito feeding treatment × species interaction, and (b) feeding treatment × generation interaction on oocyst prevalence. (c) Mosquito feeding treatment × species interaction, and (d) feeding treatment × generation interaction on oocyst intensity. The box plots indicate the median (wide horizontal bars), the 25th and 75th percentiles (squares) and the minimum and maximum values (whiskers). The black dots represent the mean values. Bars or box plots with different letters indicate a statistical difference at P < 0.05.

Oocyst intensity. There was no significant feeding × species interaction (=4.32, p=0.115; Fig. S2c), and no significant feeding × generation interaction (=2.48, p = 0.115; Fig. S2d) on oocyst intensity. The oocyst intensity in mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood, regardless of mosquito species or generation (Fig. 2c and d, respectively). There was no significant feeding × species × generation interaction on oocyst intensity (= 1.09, p=0.578; Fig. S2b).

Mosquito longevity

There was a significant feeding × species interaction on mosquito longevity (=13.49, p=0.001; Fig. 3a). In An. coluzzii, mosquitoes fed on Blood lived significantly shorter than mosquitoes fed on Bloodless diet; while in both An. gambiae and An. gambiae Kisumu, the longevity of mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood (Fig. 3a). There was no feeding × sex interaction on mosquito longevity (= 0.12, p = 0.721; Fig. 3b). The longevity of mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood, regardless of the sex (Fig. 3b). There was a significant feeding × generation interaction on mosquito longevity (=9.13, p = 0.002; Fig. 3c). In F3 generation, the longevity of mosquitoes fed on Bloodless diet was similar to that of mosquitoes fed on Blood; while in F7 generation, mosquitoes fed on Blood lived shorter than mosquitoes fed on Bloodless diet (Fig. 3c). There was no feeding × sex × species interaction on mosquito longevity (=2.92, p=0.231; Fig. S3).

Fig. 3.

(a) Mosquito feeding × species interaction, (b) feeding treatment × sex interaction and (c) feeding treatment × generation interaction on mosquito longevity. P-value from multiple comparison is provided.

Discussion

In this study, laboratory assays were conducted to evaluate the impact of the Bloodless diet on mosquitoes by assessing (i) wing size (ii) susceptibility of An. gambiae, An. coluzzii and An. gambiae Kisumu females to natural isolates of P. falciparum, and (iii) survival of both male and female An. gambiae, An. coluzzii and An. gambiae Kisumu mosquitoes reared on the Bloodless diet, compared to those reared on Blood. Overall, our results demonstrated that the mosquitoes fed on Bloodless presented larger wing sizes than those fed on Blood, regardless of sex or species. Oocyst prevalence and intensity were similar between mosquitoes of the same species fed on either diet type, but differed between mosquito species. These results show that Bloodless does not affect mosquito susceptibility to P. falciparum and that the differences observed correspond to mosquito species-specific traits. However, we noticed significant difference between pooled Bloodless and b fed of generation F6. This finding could be explained by the fact that the Bloodless (artificial blood) probably influence the mosquito microbiota after generation F3 and this hypothesis should be verified in our next studies. Globally, our findings suggest that, under laboratory conditions, Bloodless performs comparably to Blood in supporting key Anopheles spp. traits. In addition, a recent work with An. stephensi and P. berghei reported comparable oocyst prevalence and intensity between the offspring of females fed on Bloodless and those fed on Blood¹⁷. In the third generation, oocyst prevalence was similar across the three mosquito species. However, the oocyst intensity of An. coluzzii was higher than that of An. gambiae which was also higher than that of An. gambiae Kisumu for both colonies. Experimental infection studies carried out with An. gambiae and An. coluzzii from larvae collected in Burkina Faso showed no differences in oocyst prevalence or intensity between the two species²⁸, while studies on the third generation of An. gambiae s.l. showed that An. gambiae harbored more oocysts than An. coluzzii²⁹. Ayo et al., 2024 reported that a newly established An gambiae s.s. and the Kisumu strain exhibited similar oocyst prevalence, although oocyst intensity was slightly higher in An. gambiae s.s. compared to Kisumu³⁰. Our results may be explained by differences in genetic background or bacterial microbiota among species, which could make An. coluzzii more permissive to P. falciparum infection than An. gambiae, and An. gambiae more permissive than the An. gambiae Kisumu strain. In the same way, our results for the sixth generation could be influenced by the composition of the bacterial microbiota. Previous research has shown that certain bacteria in the mosquito intestinal flora promote infection to Plasmodium^31,32. For the future work, it would be useful to characterise the bacterial communities of the three mosquito species over several generations in order to understand the variations observed in the oocyst prevalence and intensity between mosquito species. This is particularly relevant given that the microbial community in the mosquito midgut plays a crucial role in Plasmodium infection. In most cases, the introduction of certain bacteria inhibits Plasmodium development³³, whereas the absence of this microbial community following antibiotic treatment has been shown to increase the susceptibility of Anopheles mosquitoes to Plasmodium infection^34–38. In addition to the microbiota, mosquitoes can harbour other infectious organisms such as microsporidia, fungi, and filarial worms, which may reduce Plasmodium infections^39–41.

The male mosquitoes body size is a key parameter for the mosquito mating in swarms. It has been reported that larger males of An. gambiae s.s. mated more easily than smaller males in swarms^42,43. Our results show that regardless of sex and species, mosquitoes from the Bloodless diet group were slightly larger than those from the Blood group. The Bloodless diet contains bovine serum albumin, cholesterol, and amino acids, which may contribute to increased mosquito size by supporting key physiological processes. Although Blood also contains these components, their concentrations can vary significantly between batches and donors. In contrast, Bloodless provides these nutrients in defined and consistent amounts. Bovine serum albumin, acquired through the blood meal, serves as a reliable protein source, supplying amino acids essential for muscle development and other metabolic functions. Cholesterol, which mosquitoes must acquire through their diet, is a critical component of cell membranes and is involved in hormone biosynthesis necessary for growth and development²².

These results are supported by recent studies showing that dietary amino acids, proteins, and lipids acquired during blood feeding and larval nutrition critically influence physiological processes such as egg maturation, resource allocation, and adult morphological traits, including body size and nutrient reserves (e.g., temporal amino acid dynamics coordinating egg maturation in mosquitoes; impacts of dietary nutrition on larval growth and adult composition; influence of larval diet on development and vector competence)^44,45.

Mosquito lifespan is a key determinant of vectorial capacity, with longer-lived vectors having a greater potential to transmit pathogens⁴⁶. It is therefore an important parameter in mosquito population dynamics and disease transmission²⁰. Recent studies on An. stephensi males and females reared on Bloodless and standard Blood diets showed that, regardless of the feeding regime, females lived longer than males¹⁷. In contrast, another study using An. gambiae mosquitoes reared on Bloodless and bovine Blood diets showed no significant difference in lifespan between sexes across both colonies²². In our study, we observed no difference in lifespan between mosquitoes reared on the Blood-free diet and those reared on Blood in the third generation. However, we observed that, regardless of mosquito species, females had a longer lifespan than males. Unlike the results observed in the 3rd generation, mosquitoes reared on the Bloodless diet exhibited a longer lifespan than those reared on Blood in the 7th generation. This increased longevity may be attributed to specific components of the Bloodless diet, such as amino acids and vitamins or to the greater stability of its artificial composition compared to mammalian Blood²². The Bloodless diet could therefore represent a promising alternative for supporting the production of mosquitoes required for novel vector control strategies, such as the sterile insect technique, transmission-blocking methods, and approaches based on the use of Wolbachia.

Conclusion

This study shows that Bloodless enhances body size and maintains susceptibility to P. falciparum infection of mosquitoes over at least 7 generations. While no differences in lifespan were observed in early generations, mosquitoes fed the Bloodless diet had a longer lifespan in later generations. These findings highlight the potential of Bloodless as an alternative to animal Blood in mosquito rearing, with implications for vector control and research. Assessment under semi-field conditions can serve as the next step prior to operational deployment. To ensure feasibility for large-scale implementation, acceptability studies and stakeholder engagement, particularly involving input from vector control practitioners and policymakers, will be essential.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

H.S., J.M., R.V., I.S., E.B and D.F.S.H conceived and designed the present study; J.M. and R.V. produced the diet. D.F.S.H, A.M.A.P.S, and I.S. conducted the experiments, with E.B. providing supervision and overseeing the study; B.S.P. and D.F.S.H carried out the data analysis; D.F.S.H drafted the manuscript; E.B., I.S., H.S., J.M., A.D and R.V. provided critical revisions to the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

The research received financial support from Fundação para a Ciência e Tecnologia and Aga-Khan Development Network (MosqDiet_FCT AGAKHAN/541725581/2019), and IHI (Training and Capacity Building Unit). J.M. and H.S. are supported by the Fundação para a Ciência e a Tecnologia (FCT Portugal) by funds to GHTM/IHMT-NOVA (GHTM-UID GHTM UID/04413/2020). J.M. is recipient of a contract by FCT (10.54499/CEECIND/00450/2017/CP1415/CT0001.

Data availability

The data generated in this study are available as Additional file 1.

Declarations

Ethics approval and consent to participate

The protocol of the study was approved by the Institut de Recherche en Sciences de la Santé, ethics committee: A 009-2023/CEIRES/IRSS. Prior to inclusion, informed consent was obtained from the parents or legal guardian of the volunteers.

Consent for publication

All authors concur with the submission presented by the corresponding authors.

Competing interests

The authors declare no competing interests.