Sterile Insect Technique: Lessons From the Past

Abstract

When E.F. Knipling conceived of the release of sexually sterile insects to suppress wild populations, he laid down several fundamental qualities that characterized suitable target species—some of which mosquitoes generally violate—including high reproductive rates and large population numbers. Regardless of this, their global importance in public health has led numerous research teams to attempt to use the mosquito sterile insect technique against several species. Because of the degree of financial commitment required for suppression programs, most releases have consisted of preliminary investigations of male performance, population characteristics, and production methods. Those that have accomplished suppression provide important insights regarding the challenges of production, dispersal, and immigration. Insights gained from these studies remain relevant today, regardless of the genetic control technology being applied. In this article, I highlight studies that were notable for the insights that were gained, the intrinsic difficulties that mosquitoes present, and synthesize these into recommendations for successful applications of the sterile insect technique and newer technologies to mosquitoes.

Mosquitoes Versus Knipling

From the conceptualization of the sterile insect technique (SIT), E.F. Knipling recognized its tremendous potential, but he also cautioned of its limitations (Knipling 1959). Although he is often considered the father of SIT, he realized that there were characteristics of the target species, technology, and biology that distinguished suitable from poor target species. The zeal with which many are pursuing SIT technology often causes them to overlook the fact that some species and program objectives simply are not suitable targets for conventional SIT as a stand-alone population suppression or elimination method. Expansion of the basic SIT idea and more detailed modeling of SIT was first performed by Lawson (1967).

Generally, based on simple calculations and common sense, Knipling identified five requirements for suitable species (Knipling 1955). Paraphrasing and expanding a bit:

1. An economical method of rearing millions of insects must exist;

2. The insect can be dispersed to penetrate even inaccessible places where virgin females occur;

3. The sterilization method must not seriously harm mating competitiveness or longevity;

4. Females must mate only once, or fertilization uses sperm from more than one insemination randomly;

5. The insect must have a low population size, biotic potential or be controllable by artificial means.

The ability to conduct SIT against the screwworm, Cochliomyia hominivorax (Coquerel: Diptera: Calliphoridae) (Vargas-Terán et al. 2005) without a female elimination method perhaps caused Knipling to minimize the importance of ‘sexing’ among the capabilities in the above list. He advised that the first four hurdles must be considered surmountable before evaluating the last, though one might question the order of evaluation because the last is perhaps the factor least amenable to technological solutions.

He also recognized that introducing sterility into wild populations could suppress them more effectively than direct killing, e.g., using insecticides (Knipling 1959). He reiterated that if chemicals or other means were available to introduce sterility into populations without releasing sterile males that it would potentially be a less expensive and complicated method than SIT. This insight perhaps anticipated the modern application of insect growth regulators that potentially have an amplifying effect at larval sites, such as releasing males carrying pyroproxifen (Brelsfoard et al. 2019) and gene drive (Burt and Crisanti 2018), neither of which directly kill the exposed individuals but may not require releases of large numbers of mosquitoes.

There were three possible applications that Knipling proposed: 1) controlling pests that were present in low numbers; 2) eliminating small introductions before establishment; and 3) as a complement to other methods. All of these applications have been used against agricultural pests in addition to establishing barriers to invasion, such as that provided by sterile screwworm releases in Panama (Anon n.d.), and as a prophylactic measure to preclude outbreaks such as is performed with releases of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann: Diptera: Tephritidae), in Florida and California (USDA/APHIS n.d.).

It was more than a decade after the seminal concepts and demonstrations of SIT were developed that applications to public health rather than agriculture were articulated (Knipling et al. 1968). Perhaps one of the reasons for the interval is that Knipling foresaw that “…this method will be difficult and costly under the most favorable circumstances…” (Knipling 1955), so high priority target species and feasible strategies must be carefully selected.

There are several technical variations of genetic control that have been, and are being developed, many of which will be described in this collection of papers. I will refer to classical SIT as production and release of male mosquitoes that have been sterilized by irradiation, chemosterilants, or cytoplasmic incompatibility (CI). Cytoplasmic sterility is one of the oldest to have been field tested (Laven 1967) and which has become popularized more recently (e.g., Zhang et al. 2015, Kittayapong et al. 2019, Crawford et al. 2020). The most widely used Oxitec technology using a lethal gene that kills offspring not treated with tetracycline is also a derivative (Phuc et al. 2007). All of these technologies have one use: suppressing mosquito populations, possibly to the point of elimination. Failure to eliminate the population requires repeated releases to maintain suppression. Furthermore, suppressing a single vector species will often require a continued integrated vector management (IVM) program to reduce vectors of other pathogens or to control pest mosquitoes, therefore, the role of SIT in the overall management of mosquitoes must be kept in context.

Given this perspective, are mosquitoes suitable targets for SIT generally, and if so, which species? If suitable target mosquito species do exist, which of the possible applications of SIT make sense? Keeping in mind that a single example of successful demonstration of SIT does not necessarily constitute a sustainable control method, what lessons have been learned that inform developers which pursuits will be most productive?

Meeting the Enemy With Sufficient Force: It Is a Numbers Game

Knipling calculated that a two-fold rate of released males relative to wild males (2:1 ratio) would suppress wild populations—in theory (Knipling 1955). In fact, he recommended a more reasonable minimum of a 9-fold release rate and many programs consider 10-fold a minimum with some releasing up to 100-fold (Zheng et al. 2019) when suppression was achieved. Theoretical rates must be considered in light of practical limitations that inevitably reduce competitiveness. Reduced mating competitiveness, mortality during release and reduced longevity can all occur due to colonization, sterilizing treatment, packaging, and release, necessitating larger numbers to be released than the theoretical rates.

Immigration of fertile adults into the target population diminishes the effect of the release of sterile males, so measures to prevent immigration are usually in place. To detect an effect of SIT, immigration usually is reduced by choosing isolated target mosquito populations. Depending on the dispersal behavior of the species, these may be ecological islands such as the Nile River valley in Northern Sudan (Ageep et al. 2009, Malcolm et al. 2009), true islands (Patterson et al. 1970, Marris 2017), neighborhoods that have barriers such as roadways (Carvalho et al. 2015) or those established by conventional mosquito control (Yasuno et al. 1978). In spite of these measures, immigration into the release site is a commonly encountered problem that requires larger releases to achieve the same effect that could be achieved against highly isolated populations (Yasuno et al. 1978).

The Interplay of Biology, Technology, and Target Species Suitability

Species Biology

Because SIT suppresses only one species at a time, it has been widely accepted that the most suitable target mosquito species are those that caused widespread harm such as Aedes aegypti (L.: Diptera: Culicidae), Aedes albopictus (Skuse: Diptera: Culicidae), and several Anopheles species, so that the effort of technology development can be leveraged as extensively as possible for public health. Usually overlooked is the unfortunate fact that the very characteristics of abundance and high reproductive rates that make these species prominent vectors are the very reason why these species may be poor targets for SIT; they require large numbers to suppress them and rebound quickly in the absence of releases. Nonetheless, optimism that improved technology can overcome biological intractability, and the lure of impacting an important public health target has resulted in the current emphases and attraction of financial, technical and institutional support.

The biology of the target mosquito has a considerable effect on the ease with which SIT production technology can be developed. For example, Ae. aegypti larvae are very robust and develop at a predictable rate, the eggs can be desiccated and stored for months, and adults are less delicate than most Anopheles. It is no exaggeration to say that the ability to conduct SIT programs against Ae. aegypti, including the OX513A transgenic Oxitec version (Phuc et al. 2007), are enabled as much by the transgene phenotype as by the fact that male pupae can be separated from females by size unlike many species in which this distinct sexual dimorphism (and to a lesser extent more rapid development of males) does not occur. In the absence of this, it is likely that SIT efforts against this species would not have advanced to the degree they have. Although females of species that do not have this trait, such as Anopheles albimanus (Wiedemann: Diptera: Culicidae), have been eliminated by SIT programs, in which about 20% of the releases (Dame et al. 1974) consisted of females, it is my judgment that this proportion in an inundative release program would likely be considered unacceptable today due to pest biting, the potential for pathogen transmission and consequent ethical and community objections. In that species, the development of a genetic female-elimination strain was essential to enable increasing production for wider releases (Bailey et al. 1980). The prevailing opinion at this point in time is that for inundative releases of mosquitoes, stringent measures to eliminate females must be accomplished (Papathanos et al. 2009, Gilles et al. 2014).

Some mosquitoes occur as sympatric but morphologically indistinguishable complexes of species that do not intermate freely, thus possibly requiring SIT against all of them. Given the existence of sympatric species complexes and sometimes poorly understood different genetic forms within the same species, it is natural that mating compatibility is a prominent concern when planning SIT programs. Notable examples of a previously colonized strain competing effectively in cages but mating assortatively in the wild or displaying distinct reproductive behavior have been observed (Reisen et al. 1982, Reisen et al. 1985). As a further control for the effects of irradiation, laboratory males carrying an eye color mutation were released at the same location and again were found to mate competitively with laboratory but not field females (Reisen et al. 1985). To circumvent this issue, a common precaution is to introgress the genetic background of wild collected mosquitoes into the strain to be released by repeated crossing (e.g., Kaiser et al. 1979, Munhenga et al. 2016), but this is not always done (Harris et al. 2012, Carvalho et al. 2015) and has not always been successful (Reisen et al. 1985). For SIT, in which the suppression effect is highly dependent on the immediate effect of mating success, this is of paramount importance whereas, arguably, for effectors that assort from the remainder of the genome over multiple generations such as gene drive or a male-biasing transgene (Galizi et al. 2014), this is less critical. As a generalization, populations with complex genetic structure require more careful consideration of whether this step will be necessary. An advantage of irradiation or chemosterilization to accomplish sterility is that if no sexing strain is required, such as for Ae. aegypti, a colony derived from locally collected mosquitoes can be used without further crossing thus, reducing, but not fully eliminating, this concern (Reisen et al. 1982). Colonization itself causes changes in the genotype of mosquito colonies (Norris et al. 2001), particularly related to mating in laboratory cages (Baker et al. 1979).

Many of the technologies that will be discussed in this issue aim to increase effectiveness by decreasing the number of mosquitoes that need to be produced by using more powerful effectors or by increasing the mating competitiveness of those that are released. Many factors go into attaining an effective and efficient release program including male competitiveness, the availability of a female elimination method, the intrinsic robustness of the species and the heritability of the effector.

Does Irradiation Make Males Noncompetitive?

It is often claimed that irradiated male mosquitoes do not mate competitively (e.g., Zheng et al. 2019, Crawford et al. 2020), an assertion which is used as a rationale for the use of the Incompatible Insect Technique (IIT, in which sterility is produced by Wolbachia, via cytoplasmic incompatibility) or transgenic approaches (Alphey 2002). This is an exaggeration which, ironically, is demonstrated by two recent programs, both of which asserted that rationale and experimentally disproved it. Loss of competitiveness due to irradiation is, of course, a strong outcome when very high radiation doses are used; excessively high radiation doses likely resulted in the failure of an Ae. aegypti SIT program in Pensacola, FL USA (Morlan et al. 1962) in which doses > 97 Gy—well above a near fully sterilizing dose—were used (Hallinan and Rai 1973, Yamada et al. 2020)! Optimized suppression approaches create high levels of sterility in the population rather than individuals (Lawson 1967), so a dose that is less than fully sterilizing is often recommended if it maintains competitiveness for a greater overall effect.

Two successful release programs that combined IIT and SIT (irradiation) are particularly instructive. Both used what they referred to as ‘low dose’ irradiation (Kittayapong et al. 2018, Zheng et al. 2019). The Zheng et al. project released IIT-SIT and IIT alone treated Ae. albopictus into two target areas. The purpose of irradiation was to sterilize a low proportion of contaminating females that carried Wolbachia which could create a cytoplasmic-incompatibility resistant population. This project ‘nearly eliminated’ the target populations at both sites, in one of which mosquitoes received a dose of 45 Gy. Previously, Du et al. (2019) and Balestrino et al. (2010) demonstrated 97–99% sterility using a lower dose of 40 Gy, so the dose was hardly ‘low’. Comparing the sites where male mosquitoes were sterilized by IIT alone with one in which IIT plus X-ray irradiation was used, it was difficult to detect any difference in the effect although the use of irradiation did allow releases of larger numbers, possibly compensating for reduced male competitiveness.

Similarly, Kittayapong et al. (2018) achieved ‘a significant reduction (P < 0.05) of the mean egg hatch rate (84%) and the mean number of females per household (97.30%)’ after releases of IIT-SIT treated Ae. aegypti. The irradiation dose they used (75 Gy) was higher than the 70 Gy that has been reported to achieve full (Hallinan and Rai 1973) or nearly full sterility (Yamada et al. 2020) in Ae. aegypti. Using radio-sterilized males without IIT has the advantage of avoiding possible patent complications that might accompany IIT programs (Bourtzis et al. 2006, Dobson 2011).

The results of these successful projects demonstrated that irradiation does not prevent the production of competitive Aedes spp. males when used at nearly fully sterilizing doses. However, any loss of competitiveness is always undesirable simply because it requires releasing more males to achieve the same effect, but this must be kept in context; a decrease in competitiveness due to irradiation alone is only one effect of production and release among many that can affect this trait.

Suppression or Elimination: A Critical Decision

The difference between ongoing suppression and elimination is not simply a matter of degree, and the importance of the choice is critical and has far-reaching consequences for downstream activities, including methods to exclude reintroduction, surveillance, and the financial commitment demanded. Funding and the threat of reintroduction after elimination will determine whether either choice is sustainable. One of the few examples of prophylactic ongoing treatments after suppression was performed using Oxitec technology in Brazil (Garziera et al. 2017) but few mosquito release programs have attempted this.

Most early classical mosquito SIT release projects (up to 2003) were identified previously (Benedict and Robinson 2003) and were updated in the list given in Supp Table S1 (online only). The programs that are often recalled as the most notable examples of success in the early releases were local elimination programs. These include the elimination of Culex pipiens (L.: Diptera: Culicidae) from a village in Myanmar (Laven 1967), Culex quinquefasciatus (L.: Diptera: Culicidae) from a Florida island (Patterson et al. 1970) and elimination of An. albimanus from Lake Apastepeque (Weidhaas et al. 1974). The indisputable binary outcome of such elimination programs accents particular success in a way that suppression programs, which are often conducted for one or two seasons and which may struggle to demonstrate suppression, simply cannot. However, elimination is an ambitious objective that is difficult to achieve, except when isolation of the release zone is high and mosquito populations are small, and therefore few programs state this as an explicit objective.

In the total absence of the target species, application of SIT can be used to preclude establishment of a vector, create prophylactic barriers, and eliminate initial introductions of an exotic species before it becomes established. These applications are the ‘low-hanging fruit’ identified by Knipling. Such low level infestations are particularly suited to the unique observation that SIT is most effective when target populations are low (Kean et al. 2008).

As described above, Knipling anticipated that ongoing suppression of a prolific species was not a desirable purpose for SIT—except perhaps in combination with other control measures. The expense of incorporating SIT against mosquitoes in this context must be carefully evaluated considering the cost-effectiveness of alternative control methods that are available and more easily implemented. For practitioners of mosquito control, a particular method of genetic control must not only be favorable over other genetic methods, it must compete against source reduction and chemical control (Mumford 2005). The use of Wolbachia-infected mosquitoes to replace target mosquitoes with non-vector forms (Hoffmann et al. 2014) in essence acknowledges this and surrenders to the prolific reproduction of mosquitoes by leveraging that characteristic to spread pathogen refractoriness in wild populations. A draw-back is that this approach does nothing to alleviate nuisance biting, and communities may eventually demand counter-intuitive adult control to improve environmental quality.

Mosquitoes are generally unsuitable targets for SIT so the application of the technology should be restricted to those with the highest chances of sustainable success. Elimination, creating barriers, and stopping early introductions are the most sustainable options. Long-term suppression using SIT makes sense only if a sustainable program can be conceived, most feasibly as part of an IVM activity.

Small-Scale Methods Cannot Simply be Multiplied

Significant cost is involved developing technology for larval production, ‘sexing’ (removing females from the releases), packaging and release. When these are species-specific, those efforts must often be independently developed, or at least adapted and proven. An example of highly specific technology is genetic sexing systems based on chromosome rearrangements (e.g., Curtis et al. 1976, Seawright et al. 1981) which must be developed for each target species. Therefore, technological advances and methods that are not species-specific will likely find wider utility such as spiking blood with a toxin to remove females (Yamada et al. 2013). Similarly, sex separation methods based on pupa size sexual dimorphism can be modified for species that have useful size differences. Contrary to the common perception that sexual size dimorphism is limited to Aedes species, this also occurs among some Anopheles species such as An. quadrimaculatus (Say: Diptera: Culicidae) and Culex spp. (Sharma et al. 1972).

Producing hundreds of thousands of mosquitoes a week places demands on rearing, sterilization and release that cannot be met with methods and equipment that are suitable for laboratory rearing. There have been many innovative devices and systems for larva production (Balestrino et al. 2012), separating pupae (Fay and Morlan 1959, Ramakrishnan et al. 1963, Balestrino et al. 2011), and sexing adults (Crawford et al. 2020). The ability to produce large numbers of mosquitoes—while not trivial—has not prevented producing sufficient numbers of mosquitoes for SIT. Management plays a prominent role in SIT programs and must often implement refinement of the methods during releases such that increases of production to target levels due to improved methods, organization, and rearing schedules during the release season are often significant (Harris et al. 2011).

Moreover, mosquitoes usually occur in places where at least part of their range is inaccessible due to, e.g., lack of roadway access or being on private property. Therefore, there are active efforts to develop release technology beyond ground-based methods. In order to accomplish this, methods are advancing rapidly due to access to inexpensive GIS/GPS and drones. Although more expensive, aerial release methods have historically been used for agricultural SIT and the significant obstacle of compact packaging for transport has been significantly reduced. For example, release of adult Ae. aegypti from a drone has been accomplished (Bouyer et al. 2020) as have advances in packaging of adults for transport (Zhang et al. 2020), an important capability because production facilities may be located at a significant distance from release sites (Morlan et al. 1962).

Finally, a major activity of SIT programs is monitoring. When adequate monitoring is already being conducted as part of an integrated vector management activity, it may not add significant cost to add an SIT activity. When SIT is the sole control activity and monitoring is conducted solely for this program, it can be expected to consume a significant portion of the budget.

Sustainability Has Not Been Accomplished

If we accept the definition of sustainability as the ability to maintain an effect or activity over a long period of time, no classical or transgenic SIT program against mosquitoes can yet claim to have been sustainable. Even projects that reached local elimination and suppression that have been described made no claim that it was sustained past a couple of seasons (Garziera et al. 2017). It is possible that relatively small introductions after the first elimination phase might have maintained elimination status, but the cases in which this was done are the exceptions (Garziera et al. 2017). In the absence of political will to provide the necessary ongoing financial support, all programs have been of limited duration, the costs are seldom reported (except Staletovich et al. 1972) and are often obscured by donations of labor from research institutes and other programs.

It is perhaps cynical to suggest that elimination programs do not present an attractive business plan, but in my estimation, opportunities to create and maintain barriers and to eliminate isolated and small introduced populations are numerous and unlikely to be exhausted. For example, the introduction of Anopheles stephensi (Liston: Diptera: Culicidae) into east Africa (Faulde et al. 2014) may be a suitable target for an elimination program that includes SIT. These situations present those in which classical SIT is most likely to be effective and their effects sustained.

The history of mosquito SIT has been burdened with prolific species, poor choices of target mosquito populations and unsustainable levels of effort required to maintain an effect. It is possible that the hurdles that biology presents can be overcome with technical solutions. Other authors in this issue will describe technologies that build upon the seminal concepts of SIT, but overcome the fundamental barriers to sustainable programs using variations more suitable for mosquito suppression and to make populations less hazardous for disease transmission.

However, technical approaches that require sustained large-scale mosquito production and release for suppression programs will continue to be costly and must achieve methods to inexpensively produce and distribute mosquitoes to be competitive with both genetic and conventional mosquito control alternatives. Ultimately, SIT must compete cost-effectively against all control methods that are available, many of which control more than one pest species. It is difficult to argue at this point that this can be achieved without greatly improved technologies.

Unfortunately, although classical SIT may be more acceptable to the public than some transgenic approaches, it is perhaps fatally hobbled by mosquito fecundity for long-term suppression programs. These programs must continue to receive exceptional support that is adequate to demonstrate suppression—at least for a while. For some programs, it is an opportunity to demonstrate technical capability within a public good context (Crawford et al. 2020), but the ability to do this in research institutes or academic and public health organizations is limited and can be supported only if there is an expectation of a sustainable cost-effective program.

My desire for this chapter is to convey the perspective that Knipling articulated (emphasis mine):

“It is hoped that the information in this paper will stimulate further research where indicated and at the same time discourage investigations where the sterility method obviously will not be feasible. The writer is of the opinion that this method will be difficult and costly under the most favorable circumstances but it might prove practical primarily as an eradication tool for certain highly destructive pests or provide a method of preventing the buildup or spread of established infestations. Basic studies on the problem might also, in future years, lead to new and more economical ways to induce sterility and thereby extend the practicability of this approach to insect control.” (Knipling 1955)

Acknowledgments

Thanks to Bill Reisen for providing valuable comments and copies of several useful articles related to his SIT activities, many of which tested critical hypotheses. Thanks to many colleagues who provide informal accounts of experience and programmatic issues that are not always evident in manuscripts. Thanks also to Ellen Dotson and Priscila Bascuñán who provided useful comments on this manuscript.

References Cited