It is no surprise to people rearing mosquitoes that the basic utensils and methods of mosquito rearing are similar to those described 60 years ago [21]. Personal experience and inherited methods rather than controlled experiments have been the mainstay for developing mosquito rearing, and these methods are sufficient for laboratory use. In the following section, some of the practices are discussed that have been helpful to large-scale production efforts. Simple innovations are also suggested with potential to simplify and improve mass rearing.
Management
Effective management systems are absolutely critical for the success of production and distribution of millions of sterile mosquitoes. The statement [22] that, "...the failure of area-wide programmes is usually due to poor management and inadequate public support and not to poor technology." is a widely recognized truth. Good management requires methods similar to those used in business and industrial mass-production - disciplines in which few scientists who are charged with this responsibility have any training. Notably, the two largest release programmes quickly adopted such management systems and carefully controlled production levels and quality analysis. Most notably the World Health Organization/Indian Council of Medical Research (WHO/ICMR) programme attributed its successes to reliance on the Program Evaluation and Review Technique (PERT) [23]. This method is still widely used and provides a useful model for programme management. The production of An. albimanus in El Salvador required careful planning and analysis of production systems which have been well described [24]. However, no overall management system of that project has been described. Of great value to wider application of mosquito SIT will be development of a standard model of management system for the operation of facilities worldwide.
Production systems
Because previous mass-rearing efforts have essentially increased the scale of laboratory methods, integrated production systems are an area in which rapid dramatic improvements can be expected. Several infrastructure improvements could significantly improve the quality of rearing: circulating water systems with continuous quality monitoring and waste removal, continuous larval/pupal separation and sterilisation systems, measures to reduce and prevent the development of pathogens, and feeding systems that maintain optimal diet availability.
In spite of the demands and scale of mosquito release programmes, it is surprising that the infrastructure differed mainly in number from that used in laboratories for small scale rearing. While the WHO/ICMR used a centralized aquatic stage collection system, the El Salvador An. albimanus SIT project relied on individual rack-mounted trays. Both programmes utilized pupal separation methods and adult cages nearly identical to those used in research laboratories. Neither mechanized nor continuous methods for blood feeding, egg collection, larval feeding or pupal separation were implemented. Useful models exist however: on a small scale, a circulating system that included biological water conditioning and automatic feeding for rearing of Anopheles stephensi provides an example of how such systems can be created [25].
The economic considerations that limited the development of mechanized specialized systems decades ago are now a smaller obstacle because of the availability of inexpensive personal computers, software, control devices, and solid-state sensors. In the final analysis of the utility of SIT, economics of production will make developing these systems essential. The low cost of labour in disease endemic countries should not be used to justify a lack of technology development. Investing in the training and infrastructure of the persons working in the factory and the host country will be better served by transferring efficient advanced technical skills rather than instruction in menial labour. Furthermore, strikes, absenteeism, and inconsistent performance are less likely to interfere with a production system that does not depend excessively on numerous personnel. Finally, some programmes have found it difficult to decrease staff sizes when increased efficiencies and demand resulted in diminished labour needs. Starting with as small a staff as possible reduces these problems.
Larvae and pupae
Larval rearing conditions have a direct, and often irreversible, effect on adult traits therefore a clearly defined diet is a priority. Developing such a diet is not as straightforward as it might appear and is usually accomplished by trial and error. Because mass rearing has not been performed under aseptic conditions and mosquito larvae are opportunistic feeders, microbes constitute a large portion of their diet, and no truly defined diet for mosquitoes has been used in a large-scale system. Regardless of the fact that controlled conditions are desirable for mass production, and aseptic defined-diet rearing of mosquitoes would help accomplish this, it will likely not be practical on a large scale.
A chemically defined diet was used to successfully rear several culicines [26], but was not successful for Anopheles freeborni [27], though small-scale sterile anopheline rearing is possible [28]. In an interesting example of the hunter becoming the hunted, Munderloh et al. [29] were able to rear An. stephensi larvae on cultured cells of a mosquito that itself preys on mosquito larvae, Toxorhynchites. An ideal diet would provide adequate nutritional components that positively influence rearing conditions and insect quality, is inexpensive, globally available, and of consistent quality. Those that are used for smaller scale culture such as TetraMin™ Flake Food are of high quality but of high cost and would not be desirable for mass-rearing. As an indicator of the amount of diet required - and the significance of cost - we have estimated that approximately 2-3 kg of larval diet per day will be required for production of one million males per day. Mass rearing diets have consisted of inexpensive ground dried animal foods (sometimes defatted to prevent 'scumming'), cereals, yeast, beef liver powder - and of course the happenstance unidentified microorganisms. Conversely, cat and monkey chow are often used, but are neither designed from mosquito larvae nor can they be obtained in the same form globally. Successful combinations were determined empirically, but when used under septic conditions, it is impossible to say to what extent the diets were useful because either they fed the larvae directly, supported the microorganisms on which the larvae fed or both. In response to these concerns, the IAEA mosquito SIT programme is currently testing various ingredients that we feel meet the necessary requirements described above e.g. beef liver powder, yeast, fish meal and squid liver meal (unpublished data).
Key issues for colonisation and production
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What genetic factors control eurygamy/stenogamy?
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What is the relationship between stenogamy and field mating competitiveness?
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What are the genetic and mating consequences on colonised mosquitoes of cage introductions of field-collected individuals, and what method best accomplishes genetic introgression of wild genotypes into colonies?
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Does genetic heterogeneity affect laboratory vigour and mating competitiveness?
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What is the relationship of dispersal capacity - as measured in the laboratory using behavioural and biochemical tests - to actual field dispersal behaviour?
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Do tactile interactions of mosquito larvae affect development independently of related factors such as food availability and waste accumulation?
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Do various geographic strains of a species have different levels of competitiveness for females of a target population?
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Can artificial selection increase male competitiveness? If so, what characters are altered in the process?
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What is the relative importance of dispersal behaviour to program effectiveness under various population density and distribution scenarios?
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What do male age, size, and longevity contribute to mating competitiveness?
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Do specific dietary components not reflected in gross energy reserves affect competitiveness?
Experimental guidance exists regarding dietary components that affect competitiveness. A rare, clear example exists where a specific dietary component was elegantly linked to field performance of mosquitoes and has influenced our choice of test diets described above. During a series of release experiments in California, a dramatic reduction in first generation Cx. tarsalis mating competitiveness was observed [30]. Because these males were genetically identical to field males, laboratory rearing factors were suspected. (n.b.: This should be considered as an excellent routine method to determine the effect of factory production alone on competitiveness independently of genetic effects.) A marked reduction in fatty acids in laboratory reared mosquitoes had previously been observed relative to wild type [31] and linked to flight ability [32]. A series of experiments identified several fatty acids that were reduced in laboratory mosquitoes reared on a defined diet and that were necessary for flight and competitive mating. As a result, the authors recommended supplementing the diet with fish oil or other sources of eicosapentanoic acid, a consideration not heeded by many laboratories that have carefully avoided fatty diets for mosquito larvae. More recent experiments conducted by Huho et al. [33] demonstrated that total lipids were less abundant in laboratory reared mosquitoes relative to wild type though no attempt was made to analyse specific fatty acids. Both of these series of observations should focus attention on the importance of controlling the supply of fatty acids in the factory independently of total weight, lipid content and growth rates.
Spatial and tactile interactions in the larval environment are also poorly understood, due largely to the lack of appropriate experimental equipment that isolates the effects of larval and diet density, total diet amount and availability and waste accumulation. Timmermann and Briegel [34] clearly demonstrated that increasing water depth adversely affects anophelines but not Aedes, and inhibitory tactile interactions have been detected in Culex sitiens [35], but the absence of circulating/exchanging water systems and a constant diet concentration and amount makes it difficult to separate interacting variables.
A practical compromise between an undefined and defined aseptic rearing system is possible and has not received significant attention: numerous species of microorganisms (e.g. Tetrahymena, Euglena, Chlamydomonas) can be produced in large amounts, are of an appropriate size, and would provide a complex food for larvae. Advantages of many such protists are that they scavenge unwanted bacteria and fungi and can be aseptically cultured in simple medium in large amounts. Moreover, many of these are motile and accumulate at the water surface where most larval feeding occurs. These might provide a controlled complex diet that would be compatible with sanitation, mass-production, cost requirements, automation, and importantly, anopheline feeding behaviour.
In conclusion, until studies based on clear and relevant adult male mating performance outcomes are completed, a conservative approach is to choose a complex diet containing animal fats, even if it is undefined. As basic as this element is to the success of SIT, diet development is a high priority for efforts to mass-rear mosquitoes, and adequacy of diets cannot be determined merely by one's ability to consistently rear mosquitoes in the laboratory.
Pupa collection
In some laboratories pupae are not separated from larvae but rather adults are collected directly from larval trays, a method that will likely not be practical for mass rearing. However, unless rearing methods are devised that result in synchronous pupation of a large proportion of larvae in a single day or two, we assume that in mass-rearing systems, pupae will be separated from larvae prior to adult emergence. Fortunately, size, behaviour, and buoyancy differences between larvae and pupae allow selection by means that are readily amenable to mass production and mechanization. To stimulate thinking toward this goal, a conceptual separator is shown that could operate continuously rather than in a batch-wise manner (Figure 1). The basis of the separation is the greater diameter of pupae relative to larvae: since female pupae are larger than males in some species, it might also be suitable for sex separation. This device would have several advantages over previous methods: larvae and pupae could be automatically counted in the stream for redistribution to rearing containers and release, partial or complete water and food changes could be performed automatically, and separations could be performed several times daily from a cohort to provide precisely staged pupae for irradiation.
Adults
Sucrose, glucose and fructose solutions are common adult diets, though fruits are sometimes provided, such as raisins. The use of anti-oxidants in diets to increase longevity and fecundity e.g. [36, 37] should be considered and implemented as a routine measure. One report described a complex vitamin mixture which was offered to mosquitoes during colonisation, but no information was provided about its effectiveness [38]. The simplicity of designing and conducting survival and fecundity experiments to test various supplements should lead to greatly improved adult diets. A common food preservative for human diets, methylparaben, has been shown to increase adult longevity of anophelines [39] and was adopted by that programme for routine use. For mass production though, additional information on fecundity would be needed prior to implementation.
For blood-feeding a large number of female mosquitoes, defibrinated blood or blood treated with an anti-coagulant, is provided via a membrane feeding system. Fecundity is reduced in these systems, and the absence of human pathogens in the blood meal must be assured. An ideal blood meal would be totally synthetic, but continued reliance on treated natural blood should not prevent production of competitive adults. For relatively small amounts (e.g. several litres per week), blood can be collected sterile in veterinary bags from cattle and used fresh or after refrigeration. Larger volumes would be problematic as measures to sterilise the blood after collection in an abattoir might be necessary. If this is required, methods for preparation and storage of bovine blood have been developed and tested for tsetse rearing [40, 41]. Commercial companies will supply rather large volumes of aseptically collected animal blood but at a relatively high cost.
Adult rearing usually consists of a larger number and size of cages than those used in numerous research laboratories with little consideration of adult biology. A multitude of concepts should be reconsidered for the next generation of adult rearing cages and tested by experimentation. These are the needs for mating arenas, sanitation, egg collection, blood-feeding, and adult resting behaviour. A concept for such a cage is shown in Figure 2. Several features of such a cage might meet these requirements and be consistent with large-scale production. Similar considerations to those that resulted in this concept cage should be applied to other prototypes that are capable of holding large numbers yet require little maintenance. Preliminary testing and modification of this cage has indeed demonstrated mating at the artificial horizon (Figure 3), but we have found (unpublished data) that extensive modification to the egg collection device is needed. The removable paper bottom also functions well to prevent waste accumulation, and we have retained this feature in a second prototype, which is stocked with 40,000 adults. Like the first version, it retains extensive adult daytime-resting sites, which An. arabiensis prefer.
Controlling disease in the factory
Interventions for disease control have consisted largely of routine hygiene, bleaching trays and equipment, use of disposable items etc. No air and water filtration sufficient to prevent contamination has been used. If selection for greater competitiveness or ease of production in the factory occurs at the expense of immuno-competence, this will become of greater concern.
In previous mosquito release programmes, measures for disease control have been conducted without knowledge of potential pathogens in the colony. Given the paucity of information, past experiences with pathogens in mosquito mass-rearing systems are encouraging. For instance, mass rearing of An. albimanus in the early stages in El Salvador was fraught with a high number of "bad trays" (10%-16%) from which few or no pupae were harvested. Often these trays exhibited lack of synchrony in development and foul-smelling water suggesting that improper amounts of diet were provided. Later, when the rearing was increased to one million male pupae per day, losses were reduced to around 1%. In both circumstances, eggs were cleaned simply by washing with water, which apparently sufficiently reduced Nosema and/or other surface contamination.
As genes related to growth and reproduction may be independent of genes related to the immune system, the routine mass-rearing process in artificial conditions could lead to a decrease of immune fitness. In shrimp aquaculture, where disease resistance is a crucial component of productivity and consistency, domestication and breeding programmes consider the immune effectors as markers of individual selection. The selection of individuals with the highest immune capacity is based on real-time RT-PCR or microarray analysis of mRNA of the main immune genes such as penaeidins, hemocyanin, phenoloxidase, superoxide dismutase, LPS-binding protein and β-glucan-binding protein. Such a strategy aims not only at avoiding the decrease of immune response but also at increasing the general non-specific resistance against various types of pathogens.
In mosquitoes, several types of viruses have been described which are related to the baculoviruses [42, 43], iridoviruses [44–46], and parvoviruses particularly in Anopheles and Aedes spp. [47]. It must be considered that founder field-collected mosquito specimens may be infected - yet healthy - carriers of mosquito pathogens, some of which might be vertically transmitted. As in shrimp aquaculture, the founders could be analysed through e.g. nested-PCR based on highly conserved sequences in order to eliminate asymptomatic carriers. Routine analysis could also be performed in order to confirm that the production continues to be virus-free.
In Anopheles specifically, several intracellular pathogens have been described, such as Rickettsia [48] and Spiroplasma [49]. Recent information in shrimp aquaculture has shown that such pathogens are efficiently transmitted vertically and horizontally, and when these occur in high prevalence can result in mortality and severe losses of fecundity and vigour. Consequently, control of intracellular bacteria is a priority.
Performance standards in other insect SIT programmes
Large size of individuals is a well-proven indicator of general nutritional status including energy reserves available for dispersion. Adult male size has also been correlated with successful mating in Cochliomyia hominivorax, being even more important in one study than strain type [50]. Evidence is inconclusive to support its prediction of mating success in mosquitoes. Large females of An. gambiae have been observed to be preferred mates [51], but it has not been demonstrated whether females preferentially mate larger males: observations for An. gambiae suggest male size is irrelevant [52]. Until the general observation that male size is relevant in Diptera is discounted for anophelines, production methods must consider that not merely consistent size, but a particular size itself may be a valuable goal.
For fruit flies standard quality control protocols have been established for mass-reared flies e.g. for pupal size and weight, emergence, flight ability, longevity, sex ratio, and mating propensity [19, 53, 54]. Comparison of results of the same tests from different mass-rearing facilities has allowed the establishment of standards for each of the parameters for several fruit fly species with, and without, irradiation. Though such quality control tests do not directly measure the competitiveness of the insect, they do provide standard indicators of the consistency of the production process. When linked to male releases that are suppressing populations, this is a valuable quality control measure.
Experimental approaches
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Experimental field cages should be constructed at potential release sites in which field-collected mosquito mating behaviour occurs and tests of release material can be conducted. These facilities should be capable of measuring longevity and competition effects with varied numbers of wild males present.
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An experimental system must be developed in which the individual effects of larval density, tactile interactions, food density and type and waste accumulation can be incontrovertibly and individually tested.
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Rapid, extensive and inexpensive genotyping methods are needed to determine the effectiveness of many different colonisation and introgression methods.
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Laboratory systems for automation and control of production must be developed.
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Colonisation cages must be developed and their effects on founder population sizes compared. This characteristic reasonably reflects the probable long-term level of selection.
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Calculation methods should be developed that allow factory management of production characteristics (numbers, size, age etc.) to predict population control effects rather than merely numerical production outcomes.
To measure competitiveness, a complementary set of data is required from tests in which sterile males compete sexually in a semi-natural environment with the feral population - the mating performance field test [55]. Sterility induced in open field tests can be obtained after sterile insects are released in semi- or isolated infested areas. Competitiveness is described by several indices: monitoring of the wild adult population, the level of sterility in wild females, or the level of sterility in eggs collected from dissected infested natural or artificial fruit in the field. Unlike anopheline mosquitoes, fruit flies respond to different types of attractants, which make mark-release-recapture tests more feasible to determine competitiveness, and its related components, dispersal and survival.
The history of experimental genetic control of mosquitoes is replete with stories of males that performed well in cage mating studies, but performed poorly in the field e.g. [56, 57]. In contrast are examples in which laboratory competitiveness was consistent with adequate field performance [58]. As candidate release strains must necessarily pass laboratory tests before distribution, it serves as a useful quality control character whose use should be continued.