Background
The urgent need to better control mosquito numbers and interrupt disease transmission has guided much mosquito research in laboratories worldwide. Such research including the study of the biology, physiology, anatomy, genetics, taxonomy and ecology usually use individual or quantity rearings of mosquitoes. The goal of insect rearing is to provide reliable, affordable sources of high-quality insects. For most of these purposes or for the routine colony maintenance,
Anopheles mosquitoes were reared in standard laboratory rearing trays containing water that held less than 500 larvae [
1,
2]. Pupae, individually picked, were transferred into a small bowl that is placed inside a small rearing cage (30 × 30 × 30 cm) for emergence and maintenance. However, rearing for large-scale needs requires a variety of improvement in methodology and equipment. The use of the sterile insect technique (SIT) for the control of pest insects as part of an integrated, area-wide approach is widely accepted. This technique utilizes radiation-sterilized individuals, which are released into the field and the wild population of the pest is then suppressed by the occurrence of sterile mating [
3‐
5].
In order to reach a sustainable field population reduction, one of the key challenges when applying SIT is the production of sufficient mosquitoes to achieve the target production level of males to be released and for colony replacement [
4,
6]. For this reason, it is necessary to continually produce large numbers of eggs (millions of eggs/day) to fill several tray-rack larval rearing units [
7,
8] in order to reach a daily operational level able to sustain continuous large scale operation activities. With revived interest in recent years for the use of sterile male release for mosquito control [
9‐
15], there is a requirement for the development of more efficient and economical methods to produce large numbers of sterile male mosquitoes. Therefore, in the production facility, pressure is directed towards parameters which are important for high egg productivity.
Since 2005, the Insect Pest Control Laboratory (IPCL) of the joint Food and Agriculture Organization/International Atomic Energy Agency (FAO/IAEA) division of Nuclear Techniques in Food and Agriculture has developed dedicated technology and procedures [
16] to support mosquito vector control programmes in Member States that use the SIT as a component of area-wide integrated pest management (AW-IPM). The mass rearing of mosquitoes is a key element of the application of the SIT, and so custom equipment has been designed for both larval and adult components of
Anopheles arabiensis rearing [
7,
17,
18]. A prototype of mass production cage (MPC) has been developed [
18], and designed to minimize handling and the opening of the cages during operations such as blood feeding and egg collection. This cage exists in two different sizes, a large cage with dimensions length 200 cm × width 20 cm × height 100 cm (400-litre volume) and a small cage length 200 cm × width 10 cm × height 100 cm (200-litre volume). Both cages include: (i) an external blood feeding system (modified Hemotek system, Discovery Workshops, Lancashire, UK), (ii) a built-in sugar feeding system, and (iii) an oviposition system located at the bottom of the cage. Although the cages have been transferred to Sudan and South Africa for validation under local conditions, and improvements in equipment and techniques have been made as a result, there is still a need to fully evaluate the productivity of such equipment and to quantify how operational parameters affect egg productivity.
The objective of this study was to optimize the rearing method to ensure high egg productivity of An. arabiensis in the mass rearing cage prototypes (Anopheles MPC, large and small). Experiments were conducted to determine the impact of four parameters on eggs production: (i) cage volume, (ii) blood meal source, (iii) total number of pupae introduced into the cages, and (iv) loading cages with all pupae on cage set-up compared to cages topped up with a daily addition of further pupae. A greater understanding of the effects of these factors would allow to define the conditions under which An. arabiensis adults should be maintained in order to maximize the effectiveness of the rearing process.
Discussion
Pilot studies prior to SIT application against
An. arabiensis mosquitoes are currently being undertaken in two endemic countries, Sudan and South Africa [
4,
12,
27]. Maximizing egg production is important for the mass-rearing of any insect species, and the optimization work described here provides important insights into the development of effective standard operating procedures (SOPs) for producing large numbers of eggs in a mass-rearing setting. A number of factors were evaluated: blood meal source, total number of pupae introduced into the cages, cage loading strategy and cage volume.
To produce offspring, a female must take a blood meal, develop eggs, and lay her eggs at a suitable oviposition site, and the blood meal provides essential nutrients for eggs production and reproductive fitness. Past research has shown a variation in the feeding preferences between
Anopheline species, ranging from those that feed on a wide range of mammals and birds, to those that feed on just one species [
28‐
30]. Several haematological properties, including biochemical composition and red cell density, vary between vertebrate species [
31,
32] and could influence its nutritional value and subsequent reproductive fitness of mosquitoes that imbibe it [
28,
32]. When rearing mosquito vectors in the laboratory, it is important that a blood source is selected that will facilitate predictably high egg production, both for routine colony maintenance and for experimentation, and finding the best blood source is challenging [
33]. Results showed that whether a blood meal from a pig or from a cow was used there was no effect on egg productivity in colonized
An. arabiensis under conditions of mass-rearing. It is important to note that the bovine blood tested had first been frozen and defrosted for use, while the porcine blood was always used fresh or after refrigerated storage. This is particularly important in the context of mass-rearing as fresh blood may not always be available, for example from the local slaughterhouse, on the day it is needed; it has been shown that blood can be stored under refrigeration for weeks or for several months if frozen, and still facilitate good levels of egg production.
Aedes spp. have been shown to feed poorly or not at all on blood that had been previously frozen [
34], but
An. arabiensis is known for its more zoophilic proclivities [
35,
36] and often show plastic responses in host feeding patterns, readily diverting to feeding on the most common or most amenable host(s). The explanation for the lack of influence of blood origin may alternatively be either these two blood meals are similar in quality in terms of amino acid composition in supporting egg development and ultimately egg production, or that blood source and quality are irrelevant for reproductive fitness in
An. arabiensis which expresses only weak host preference. In contrast, in the tsetse fly,
Glossina morsitans, blood source has a strong impact on fecundity [
37], with those feeding on pig blood producing more offspring than those feeding on cow blood. However, remarkably, no relationship between the preferred host and optimum reproductive output is reported. In the context of the development of a system for mass-rearing insects, where efficiency, economics and availability of blood source are of the utmost importance [
38], this result is interesting, as bovine blood and porcine blood could be used without any effect on egg production.
As insect rearing conditions become more crowded, their survival and fecundity usually decrease [
39,
40], and indeed in this study high adult density adversely affected egg production in
An. arabiensis. The decline in egg production with density might be related to competition among females for blood. Although demonstrating intraspecific competition in mass-rearing cages in hematophagous insects is extremely difficult, physical access to blood feeders was probably limited due to increased stocking density, based on authors’ observations. Kelly et al. [
41] demonstrated that increasing rearing densities of female sandflies are associated with smaller blood meals in female
Lutzomyia longipalpis. It is also likely that high density conditions constitute a form of stress which could negatively affect the performance and reproduction of the adults. Reproductive output has been shown to decline with increasing population density in many populations, as a result of direct competition for limited resources, elevated stress levels from intraspecific interactions [
42‐
46]. In the early experiments with 10,000 pupae added per cage, egg production was lower than when 15,000 pupae were added, but later experiments found that 30,000 pupae/cage resulted in a dramatically reduced egg production. Thus, density is clearly an important parameter and must be optimized to maximize egg production.
In these experiments, neither egg production/cage nor female fecundity were affected by the volume of the cage. It was expected that increasing the volume would provide more space for mating and would result in increased inseminated females. Although the original hypothesis was not supported by the data, this observation does not mean that in all cases the volume of the cage cannot affect egg productivity. It may depend on the value of the resting surface, as a 1.8 density-resting surface value is generally reported to promote suitable adult mosquito-rearing conditions [
1]. In the conditions of the present study, the resting surface area in both cages were almost the same and above the value of 2. In the Mediterranean fruit fly (Diptera: Tephritidae) and
Anastrepha oblique (Diptera:Tephritidae), Liedo et al. [
47] and Orozco-Davila et al. [
48], respectively, demonstrated that an increase in the surface resting area within adult cages of the mother colony, as well as the use of low adult cage density during rearing resulted in strains with higher mating competitiveness. Thus, a role of the cage volume cannot be conclusively ruled out, and specifically internal surface area, on egg productivity. Further studies will be carried out to determine the effect of different resting surface areas on egg productivity. However, the MPC should be of adequate size for easy handling and mating.
One purpose of the present study was to look for a suitable rearing method from the standpoint of developing space-efficient cages with potential for saving time and reducing associated costs of mass-rearing of An. arabiensis. Cages loaded with an initial 15,000 pupae with 1000 additional pupae added daily gave acceptable results in term of egg production and offer an advantage over cages with only an initial load of 15,000 pupae. In the latter case twice as many cages would need to be maintained to produce the same number of eggs, considerably increasing the rearing costs, and making blood feeding, for example, more time consuming. For mass rearing purposes, this is desirable and necessary in order to produce large numbers of insects in an efficient manner, keeping costs below a threshold acceptable for an operational SIT programme.