Effects of a blood-free mosquito diet on fitness and gonotrophic cycle parameters of laboratory reared Anopheles gambiae sensu stricto – Parasites & Vectors

This study aimed to compare the effectiveness of a previously formulated blood-free diet (BLOODless) and bovine blood on rearing Anopheles gambiae s.s. in a laboratory in Tanzania. We hypothesized that there would be no evidence of a significant difference on the impact of feeding rates, fecundity (number of stage V eggs in ovaries or eggs laid per female), fertility (egg hatching rates), larvae pupation rates, longevity and wing length between mosquitoes reared on the BLOODless diet and those fed on bovine blood. Our results showed that the mosquitoes exclusively fed on BLOODless presented similar behaviour in terms of pupation rates, longevity and wing length compared with mosquitoes fed on bovine blood, while the hatching rate and the number of laid eggs per female were higher in the blood-fed colony.

We observed that female mosquitoes fed on BLOODless diet showed feeding rates that were not significantly different to those fed on blood; Marques et al., reported higher engorgement rates in various Anopheles species fed on this blood-free diet, suggesting that the BLOODless diet may be more attractive to female mosquitoes than mice blood [13, 15]. A possible reason is that mosquitoes frequently rely on sugar as their initial and primary energy source for flight and, the diet consists of a sugary solution enriched with amino acids and phagostimulant, that may attract the mosquitoes more towards the diet [8, 16]. Feeding choice experiments to determine whether mosquitoes will prefer the diet over vertebrate blood are required.

The survival of adult female mosquitoes 48 h post-feeding was similar for both the blood-fed and BLOODless-fed mosquitoes, showing that there was no evidence that the BLOODless causes preliminary death of mosquitoes, in accordance to what was previously observed on An. coluzzii [13, 15].

In terms of fecundity (number of laid eggs per female), we observed that blood-fed An. gambiae s.s laid more eggs than the BLOODless-fed females. Similar findings were observed when a different artificial diet was used in An. darlingi [17]. However, a previous study performed on An. coluzzii using the same BLOODless diet found that there was no significant difference between the number of eggs produced by the diet-fed females and the blood-fed mosquitoes [13, 15]. The differences could be owing to different larvae-diet composition, settings, species and study methodology used. In our study, the number of eggs were recorded in each generation from F₁ to F₈ and then analysed, while on previous publications the data were generated from three independent experiments. In addition, in previous studies [13, 15] BLOODless was prepared freshly before each feeding experiment, in the present study BLOODless was stored on its lyophilized form until use, which might impact the final results.

Fertility was affected by the BLOODless diet since fewer eggs produced by mosquitoes hatched. It has been reported that eggs from mosquitoes fed with BSA-based diets, such as the one used in this study, had a lower hatching rate compared with vertebrate blood. This fact suggests that supplementing the diet with additional nutrients to facilitate embryo development and survival of the eggs could be beneficial [4, 10, 11, 17, 18]. There is evidence that adding commercially available hemoglobin or iron (III) chloride as iron sources, respectively, to artificial diets would increase eggs viability and hatching rates [4, 19, 20]. Although these studies were performed on Aedes aegypti, the mechanism for oogenesis is similar in all female mosquitoes, thus adding such supplements to the current BLOODless diet might improve egg viability and hatching rates of An. gambiae s.s.

Our results showed no difference in pupation rates of larvae from the BLOODless-fed mosquitoes even though there is a higher concentration of isoleucine in the diet that is hypothesized to improve larvae pupation [21]. The findings differ from those of An. coluzzi, where larvae from BLOODless-fed mosquitoes had lower larval mortality compared with mice blood [13, 15].

The life expectancy and wing length of adult BLOODless-fed mosquitoes were similar to the bovine blood-fed mosquitoes for both male and female mosquitoes. However, we found that males lived longer than females for both colonies. These results differ from a previous study that tested the same diet and that showed that females lived longer than males [15]. The reason why males had a longer life span in our study is yet unknown.

According to Gonzales et al. [11], for an artificial diet to successfully replace vertebrate blood, it should meet the following criteria: (i) it must be able to attract females to fully engorge in substantial quantities, (ii) it should facilitate vitellogenesis (the process of egg development), (iii) it should allow for the production of large batches of eggs, (iv) the offspring competitiveness (such as blood seeking behaviour, mating, flight capacity) should be similar to that of wild mosquitoes, and finally, and more importantly, v) the meal should not adversely affect mosquito behaviour and immunity.

Findings from our study indicate that BLOODless can promote the healthy and successful development of viable eggs in female An. gambiae s.s., their hatching and growth from larvae to pupae and into adults, which is essential for sustaining mosquitoes in the laboratory. It has been observed that different vertebrate sources of a bloodmeal impact mosquito gonotrophic parameters and fitness [10, 12, 20]. Artificial diets like the one tested in this study offer the opportunity of creating balanced and stable meals for female mosquitoes containing all the essential nutrients for the development and maturation of eggs, as well as the aquatic stages to emergence onto adults. Furthermore, BLOODless serves as an ethical substitute for the utilization of vertebrate blood [20].

The BLOODless diet offers several advantages. First, it boosts chemical stability in comparison with vertebrate blood, which contains anticoagulants to prolong shelf life but may affect fecundity and fertility [4, 11]. Secondly, its usage aligns with animal welfare regulations by promoting the 3 R’s (replacement, reduction, and refinement), thus encouraging the utilization of non-animal protocols to minimize laboratory animal use. Despite initial higher costs for BLOODless ingredients, it can ultimately reduce operational expenses associated with animal husbandry. The cost per millilitre depends on the amount of bovine serum albumin (BSA) purchased and ranges from US$0.17 to US$0.34 [4]. On the other hand, whole blood’s pricing varies on the basis of animal source and blood treatment method. For instance, defibrinated blood from Haemostat Laboratories may cost: bovine, UD$0.14–0.60, sheep, US$0.05–0.38, and rabbit, US$0.39–0.73 [4]. Consequently, the unit price of whole blood is comparable to that of a BSA-based artificial diet.

We recommend further studies to validate the effectiveness of BLOODless for rearing Anopheles funestus – a competent malaria vector that is highly resistant to pyrethroids and other public insecticides – and Anopheles arabiensis – a secondary vector that is also highly resistant to public health insecticides – among other laboratory strains and populations collected from the field. Other tests, such as host-seeking behaviour in females as well as mating success, flight capacity and mating competitiveness of males, should also be conducted before implementation in insectaries worldwide.