1.1. Life cycle

The NWS life cycle involves a complete metamorphosis (eggs, three larvae instars, pupae and fly). The pupae and adults make up the free-living phase which is critically influenced by environmental conditions. On the other hand, eggs and larvae develop under host temperature. Its cycle spans for 24 to 60 days, depending mostly on the temperature and humidity^1)(4)(6)(7. Adult gravid females lay their eggs on the dried edges of wounds and bodily orifices of animal hosts. Between 12 to 24 hours later the larvae hatch and begin feeding from the animal’s flesh and fluids⁵⁾⁽⁸. Larvae go through three stages of instar (L1, L2 and L3), for about 4 to 8 days after which L3 larvae matures and leaves the wound. As it falls it screws into the ground to pupate⁵⁾⁽⁹ and later adults emerge. Time between the falling of the larvae and the emergence of the adult depends on environmental and climatic conditions. After 24 hours of emergence males are sexually mature and are polygamous, mating 5-6 times during their life¹⁰. On the other hand, NWS fly females are monogamous¹¹, able for mating two to five days after emerging. They are autogenous (i.e., females can produce eggs without a first protein meal) at least for the first reproductive cycle, being ready for oviposition at least four days after mating¹².

1.2. Geographic distribution, habitat preferences, seasonality and abundance

The NWS fly is a tropical-subtropical species endemic to the Americas, present in almost all biomes since long time ago. A phylogeographic analysis including NWS samples from South and Central America, and the Caribbean support the hypothesis of a North to South spread that must had begun during the Pleistocene, and indicates it must had reached its current geographical range during the Holocene¹³. The NWS demographic history as revealed by mtDNA sequences supports that the human settlement of the Americas modified its habitat by introducing a novel host that could have enlarged the fly populations, a process reinforced by livestock introduction 500 years ago¹³. The screwworm prefers warm humid climate and is not homogeneously distributed¹⁴, it is mainly associated with water courses¹⁵ inhabiting the ecotone between forests and grasslands, and semi-open forests^{16)(17)(18)(19}. The Pampas biome is dominated by grasslands and riverine forests, which during the spring-summer season with warmer temperature constitute an ideal habitat for the NWS fly. In addition, the hillside forests and wetlands complement this favourable environmental setting. Another factor that could support the NWS fly development in the region is the landscape modification introduced by the current forestation intensification. Naturally, its abundance increases due to warm-blooded animal density¹⁴, something extremely high, because of livestock, in the Pampas biome. There are also high abundance differences between sites within the same habitat, with a split among feeding, mating and oviposition sites¹⁸⁾⁽²⁰, indicating its sensitivity to micro-environmental variations. Field observations and artificial habitat reproduction indicate that newly emerged individuals fly out the forest seeking for food and rest, preferring flowering trees²¹⁾⁽²². Mark-release-recapture data have also shown that NWS females have preferences for forests, but they fly in nearby grasslands looking for hosts to lay eggs²³.

Historically, the NWS fly was geographically distributed from the southern United States to central Argentina, including the Caribbean islands²⁴. The Edwards Plateau in Texas was indicated as its northernmost limit, but registered up to north Iowa (43° parallel) in the warmer months every year²⁵⁾⁽²⁶. The southernmost limit was indicated at the 45° parallel, Province of Chubut, Argentina²⁷. The central region of Argentina, like Uruguay, is a transitional zone where the NWS fly behaves seasonally, and at the north of parallel 29° is year-round present²⁸. Mean winter temperature below 9°C is considered a threshold determining the NWS fly distribution^{10)(25)(29)(30)(31} and emergence of adults is ~1% at -6°C³². Its high dispersal ability shapes its distribution in the north hemisphere extending its range northward from overwintering subtropical areas³²⁾⁽³³. The main hypothesis is that a small propagule with few individuals can re-invade ‘marginal’ areas when conditions favor its survival and reproduction return³⁴⁾⁽³⁵.

Many authors evaluated its flight capacity, Barrett²⁵ indicates that a NWS adult fly can move 56 km in its life, whereas Mayer and Aztemi³⁶ estimated a dispersal rate of 3 km/day when conditions are favorable, and finally Bush and others³⁷ estimated that most adults fly around 16 km/day, but some outliers flying 290 km were reported. More recently, in an experiment using marked flies conducted between Argentina and Uruguay in order to test the Uruguay River as a natural barrier to dispersal, individuals were recovered at 13.9 km and 15 km from the releasing point, respectively⁷. Despite its great flight ability and high reproductive potential, the NWS fly density is relatively low, around 200 flies/km^2(37)(38. More recent studies carried out in a tropical region estimated between 10 to 120 flies/km² and a dispersion that did not exceed 7 km³⁹, indicating that it is able to double the population in about 100 days⁴⁰. However, the relationship between its abundance and the climate has been difficult to establish; some studies did not find a significant correlation⁴¹⁾⁽⁴² but others did⁴³⁾⁽⁴⁴. Parman³² suggested that drier soils benefit pupae emergence in winter, while humid soils reduce pupae viability at any time of the year. But Thomas⁴⁵⁾⁽⁴⁶ reported a high resistance of pupae to drowning, and did not find negative effects of moisture on adult emergence in southern Mexico. On the opposite side, mortality of mature larvae, pupae and adults has been reported as high due to desiccation⁴⁷⁾⁽⁴⁸. In Argentina and Uruguay, as expected, the mortality of larvae and pupae is higher during winter⁷⁾⁽²⁸. In the central region of Argentina, it was shown that adult hatching percentage increases with temperature (from 47% at 14°C to 97% at 24°C), and flooding generate the death of pupae due to lack of oxygenation⁴⁹. In Uruguay, no adult emergence was registered during the severe winter of 2016, and the first emergence was registered in spring (after 33 days of pupation) supporting a seasonal behaviour, but fertile adults were recovered during the less severe winter of 2017. This emergence rate difference was also evident between the north and south of the country, revealing the temperature influence on the species occurrence, emergence and pupal period span⁷.

In order to investigate the role of weather in the eradication of the NWS fly, Gutierrez and Ponti⁵⁰ parameterized a physiologically based demographic model using public data on developmental times, fecundity and mortality rates on temperature to characterize its year-round persistence range. They showed a strong influence of winter temperature and rainfall on NWS outbreaks in Texas, USA, from 1962 to 1982, and Libya, from 1988 to 1992, and determined that the optimal temperature to complete the life cycle is 27.2°C, also determining that the putative lower and upper thermal thresholds are 14.5 and 43.5°C, respectively.

In summary, warm and humid climates have been associated with NWS fly abundance, and dry climates, both cold and hot, with low abundance^{32)(51)(52)(53)(54)(55}. This notion that climate governs NWS fly abundance is implicit in the seasonal nature of myiasis in domestic animals⁵¹. To our knowledge, no study is evaluating how the current warming will modify the NWS fly distribution and dynamics, if southern South America will be colonized and/or will cause myiasis above a problematic threshold along the year in seasonally regions like Uruguay.

1.3. Genetics and population genetics

The NWS fly genetics have been under active investigation, from cytogenetics to molecular and population genetics unlocking many aspects of its biology, as well as generating molecular tools extremely useful for the development of new control strategies.

Cytogenetic maps allow physical and genetic maps to be integrated, becoming an invaluable tool for the genetic analysis and manipulation of any species. The NWS fly has a diploid number of 12 chromosomes (2n = 12), with five pairs of autosomes and a pair of sex chromosomes (XX for females and XY for males)⁵⁶. Polytene chromosome photomaps of the NWS fly have been described⁵⁷⁾⁽⁵⁸, and more recently updated with a resolution of 1450 bands⁵⁹. The genomics era of this pest species began with the mitochondrial genome sequencing⁶⁰ and recently the whole nuclear genome sequence was described⁶¹. A major advantage of genomics data is the opportunity to deeply address questions relating to taxonomy and systematics, molecular evolution, population divergence, gene function and adaptation. An example of real time evolution, extremely relevant for control programs, is insecticide resistance. To investigate this in the NWS fly a transcriptomic approach has been used to measure metabolic detoxification enzyme families (i.e., cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases)⁶² and identified mutations in genes related with organophosphates and pyrethroids insensitivity^{63)(64)(65)(66)(67}.

Population genetics is essential in fields like evolution, systematics, ecology, conservation and wildlife management to understand the causes of genetic differences within and among species. In pest management, population genetics will allow through the inference of divergence and migration rates to quantify the degree of geographic or ecological isolation among regions, useful information to define the target area and scale of a control program. The genetic variability and population structure of the NWS fly have been characterized using cytogenetic markers^{66)(68)(69)(70)(71)(72}, isozymes^{73)(74)(75)(76)(77)(78}, RAPDs⁷⁹, RFLP, PCR-RFLP and sequences of mtDNA^{13)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89}, microsatellites^90)(91)(92, and SNPs⁹³. These 40 years of research support that NWS fly is a single highly polymorphic species.

On the continental-wide scale, the NWS fly is structured into four regional groups of populations: CG (Cuba), DRG (Dominican Republic), NAG (North Amazon Group: Jamaica, Trinidad and Tobago, Colombia, Ecuador and Venezuela) and SAG (South Amazon Group: Brazil, Paraguay, Uruguay and Argentina)⁸⁷. Population divergence models and historical demography based on mtDNA sequences support a population expansion process that has started in the north, with a first split between North/Central America and South America populations after the Last Glacial Maximum (15,300-19,000 YBP), followed by a second split between NAG and SAG in the Amazon region during Pleistocene and Holocene (9,100-11,000 YBP)¹³. Interestingly, NAG and SAG do not share mitochondrial haplotypes indicating a high degree of isolation between the northern and southern regions of South America¹³, despite connection corridors were predicted in the Atlantic Ocean coast region and through the Northwest Brazil and Peru⁹⁴. This geographic structure has been interpreted as due to a barrier at the north of the Amazon basin, although not yet described⁸⁸. SAG has low population differentiation without geographic structure for mtDNA⁸⁷⁾⁽⁸⁹, probably due to population expansion process¹³. Within SAG, populations from Uruguay were described as a single panmictic population based on mtDNA data⁸⁴ or with low divergence based on nuclear microsatellites⁹⁰.

More recently, protocols for specific gene disruption using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) technology (CRISPR/Cas9)⁹⁵ have been successfully developed in the NWS fly⁹⁶⁾⁽⁹⁷. This functional genomics approach opens a new avenue to study gene function in vivo and for the development of biotechnological based strategies for insect pest control.

2.1 Parasitic phase

Although female flies are attracted to wounds of warm-blooded animals to oviposit, they may also do so on different body openings such as nostrils and vagina¹⁰, taking on average 15 minutes to lay their eggs¹⁰. A single female can lay around 200 eggs (ranging from 10 to 500) in an average of four oviposition, and as myiasis odour is attractive to other flies (i.e. facultative opportunistic blowflies), it results in infestations of hundreds or thousands of larvae, which causes host’s weight decrease, pain, agony and suffering for several days, and eventually death if not treated¹⁰⁾⁽²³. The eggs laid by NWS female flies hatch between 12 and 24 hours. The hatching larvae (L1) tend to aggregate on wounds borders¹⁾ and feeding on the host’s flesh screwing their way into it leaving their spiracles facing the cavity opening in order to breathe¹⁰. They molt to second stage larvae (L2) 2 days after egg eclosion, and during this stage larvae gain some weight reaching 4.9 mg¹. On the third day from infestation larvae molt to their third stage (L3) reaching 16 mg, keep growing until approximately 120 mg of weight while enlarging the injury they create¹. During the last development stage, larvae produce an exudate which promotes bacterial infections and prevents the wound from healing. After 8 days from infestation almost all larvae at the third instar have left the wound falling on the ground to start the non-parasitic phase¹⁾⁽¹⁰.

Despite the specific requirement of living tissues from warm-blooded animals for its parasitic phase, the NWS fly is extremely versatile considering the diversity of hosts where it can complete its development. This wide host range, from wildlife to domestic animals and humans, as well as where it can oviposit, including all kinds of wounds, even a tick bite, and bodily orifices illustrate its high consequence parasitic ability.

2.2. Free living phase

Once mature, L3 larvae reach the ground, screws into it to pupate, and after a variable period of days -depending on environment and mainly on soil conditions- they complete the metamorphosis to the adult form. It has been described that L3 larvae can screw into depths from 0.5 to 6 cm⁷⁾⁽⁹. So far it has been established that the depth to which L3 larvae can reach to pupate may vary due to soil type⁹⁾⁽¹⁰, ground vegetal cover¹⁰ and environmental temperature³². Although high moisture soils favour pupae survival, flooded grounds diminish it⁴¹. Warm seasons with higher temperatures shorten the pupation span⁷⁾⁽¹⁰, but persistent extremely high temperatures negatively impact pupae survival⁴¹. Therefore, the whole NWS life cycle is usually complete within 18 days at 29°C or 24 days at 22°C, and although the screwworm lacks a dormant overwintering stage (i.e. diapause), it can even extend the pupae phase for more than 50 days when facing harsh environmental conditions^1)(4)(6. For instance, recent experiments conducted in Uruguay recovered viable flies after 57 days of pupation⁷.

Flies normally emerge early in the morning, and usually females show up first. Initially, the flies’ cuticle looks pale and soft, and their wings wrinkled and folded. After reaching the surface, they do not move for 15-20 minutes, while spreading their wings⁹⁸. Some hours later, their exoskeleton hardens and gains a deep blue to bluish metallic colour, having on their thorax three perceptible black stripes, with the one in the middle shorter than the ones on the sides¹⁰. Males reach sexual maturity at 24 hours from emergence and are polygamous¹⁰. Females accept the male between two to five days after emergence^1)(10)(99, and copulation generally occurs during daylight period and lasts between 1.6 to 3.8 minutes. The oviposition occurs between four to six days after copulation, when NWS females feels strongly attracted to animal’s wounds and start searching for a host to discharge their eggs^{1)(10)(99)(100}.

2.3. Livestock

The screwworm was a chronic problem in southern USA^26)(31)(101. Only in Texas, during 1935, there were 3.2 million NWS-related myiasis cases in cattle, of which about 15% of infested animals died¹⁰². Currently, NWS infestations remain a serious problem for animal health in South America. Costa-Junior and others¹⁰³ reviewed the literature on occurrence of both larvae (myiasis-causing development stage) and adults in Brazil concluding that NWS fly is the most important cause of primary myiasis in livestock, pets and human beings, and it is distributed throughout the country. Other epidemiological reports in South America have shown the prevalence and incidence of the NWS fly, indicating its importance in a tropical area in Ecuador¹⁰⁴, and have revealed that is widespread in Venezuela representing a serious threat to the livestock industry, as well as to the human population⁵⁵.

In Uruguay, the NWS fly has been reported in the whole territory and it is difficult, if not impossible, to find a livestock farm without myiasis during spring-summer-autumn. This epidemiological situation makes the systematic investigation of the NWS fly prevalence necessary, as well as its population dynamics, in order to fine-tune any control efforts. A survey of the larvae from naturally infested wounds in ruminants (i.e. sheep and bovines) conducted in Uruguay, from November 1985 to May 1988, showed that nearly 88% were identified as C. hominivorax, while the remainder were assigned to Chrysomyia albiceps and Cochliomyia macellaria, both facultative myiasis-causing blowflies. The main injuries were observed in sheep hooves (~27%) and navels in new-born calves (~7%), and the authors highlighted the high incidence of myiasis on foot diseases of sheep during the investigation period. Interestingly, three myiasis with only larvae from secondary species (C. macellaria (n=2), C. albiceps (n=1)) were registered in this survey¹⁰⁵. Another survey conducted from January to May 1988 in Uruguay, based on a questionnaire to livestock producers, indicated that myiasis was reported in all the surveyed farms and the prevalence was 4.5% in cattle and 6.2% in sheep⁵⁴. Also, the Official Veterinary Services of Uruguay (DGSG) has made numerous efforts to determine the impact of NWS. In 2006, a survey was carried out using the 2005 national livestock database (DICOSE) as the sampling frame¹⁰⁶. A total of 150 from the 49.431 registered Uruguayan farms were selected by simple random sampling according to each of four strata of production type: sheep (predominantly), mixed (beef cattle and sheep), beef, and dairy farms. Data was collected through personal interviews with the livestock owners between July and November 2006. The myiasis estimated prevalence was 3.4% in cattle and 5.7% in sheep, in close agreement with the previous report, whereas mortality was 0.06% in cattle and 1.25% in sheep. It is remarkable that livestock owners declared to plan management practices, such as cattle castration and sheep docking, shearing and births, when the presence of the NWS fly is low. More recently, from August to November 2014, DGSG performed a new survey using DICOSE 2013 to select 650 from the 45.059 total farms by a stratified random sampling of three strat¹. Again, data was collected through personal interviews and showed that 76.6% of livestock owners plan the management practices trying to avoid the NWS fly season. The myiasis estimated prevalence was 3.7% in cattle and 8.4% in sheep, whereas mortality was 0.1% in cattle and 2.5% in sheep. The three surveys agree to indicate a pronounced seasonal variation for NWS presence, being at its minimum during winter and at its maximum in the summer, as expected. Despite the methodological differences in each survey, a trend in the prevalence for sheep and cattle is observed at national level.

The myiasis occurrence was also investigated at a narrowed scale in the Department of Artigas (North of Uruguay, 30 °S), between August 2014 and April 2015¹⁰⁷. In this survey a total of 164 livestock farms (9%), occupying 184,826 hectares (16%), with 339,227 livestock (132,877 cattle and 206,350 sheep, being 16% and 19%, respectively) were analysed. Sheep (69%) and cattle (30%) were the most affected, whereas, horses (0.5%) and pigs (0.5%) were barely affected. Within sheep, adult females (39%), and within cattle, calves (69%) were the most affected. Hooves’ myiasis were the most frequent in sheep, and calves’ navel myiasis were the most frequent in cattle. Interestingly, in this survey myiasis cases were registered during winter in the northernmost Uruguayan region. Based on livestock farms distribution, it was possible to hypothesize a link between myiasis occurrence and the landscape. Myiasis cases were higher when animals graze in riverine fields, near natural forest. Also, it was possible to establish a connection between livestock management activities, such as cattle identification with earrings, brand fire, castration, dehorning, shearing, sheep marking, etc., and the occurrence of myiasis. The traditional sheep shearing and cattle castration are the most important activities associated with myiasis occurrence.

In order to reliably determine the occurrence and local scale distribution of NWS myiasis during winter, a larvae sampling was performed from June 21 to September 19, 2015¹⁰⁷. Nine livestock farms were selected in Artigas, each with a veterinarian responsible for larvae collection and cases reported. Larvae identification was done by morphological characters (bands of spines, spikes of spines, segments without spines, posterior spiracles, and tracheal trunks) following a key¹⁰. A total of 103 myiasis were recorded, from which 101 (98%) identified larvae were C. hominivorax, and 2 (2%) were C. albiceps. Almost 80% of myiasis were registered in three farms, one in the locality of Sepultura in a spiny savanna and shrubland with natural forest. Myiasis cases were recorded in 11 of the 13 weeks covered, with higher occurrences at weeks 2 and 10, and no cases only during weeks 7 and 13. Sheep were the most affected (88%) and all identified larvae were NWS, while remaining cases were in cattle. In this study, the authors reported that 66% of sheep myiasis were due to lambs’ tail docking, 21% in hooves possibly initiated in wounds generated by foot rot, 9% in vagina probably caused by injuries from lambing, and the remaining 4% were in navels, heads and necks. Cattle myiasis were mostly in brand wounds (33%) but only half of those were caused by NWS. Screwworm-related myiasis were uniformly distributed in brand, navel, vagina, ear, scrotum and other body wounds related to usual field management activities (i.e., fire branding, lambing and castration, etc.).

2.4. Wildlife

The NWS fly also affects wildlife warm-blooded vertebrates, but its incidence and impact are poorly documented. It has been indicated that prior eradication in the USA, around 2-3% of wild animals could be infested in endemic regions¹⁰⁸. Well documented examples are the die-offs associated with myiasis of the white-tailed deer (Odocoileus virginianus texanus) in parts of the USA with fawns deaths ranging from 25% to up 80% depending on year conditions^{109)(110)(111}, and myiasis reported in feral swine (Sus scrofa) in Florida (USA) in the 1950s, where the control of swine populations was considered a priority to reduce the NWS fly incidence in deer herds¹¹². More recently, the importance of wildlife as NWS fly host was highlighted during Florida Keys (USA) outbreak in 2016, which resulted in the death of 135 endangered Key deer (Odocoileus virginianus clavium)¹¹³. In 2021, a total of 27 NWS myiasis in feral swine were reported in the north of Uruguay¹¹⁴. Therefore, myiasis surveillance in wildlife could enhance the efficiency of area-wide NWS fly management programs¹¹⁵.

2.5. Human cases

Myiasis in humans is neglected¹¹⁶ and under-reported, mainly due to negative social implications. Every human is a potential host, but those who cannot take care of themselves are especially vulnerable, like children, elders and mentally challenged persons. People living in NWS favorable areas, like the rural population of South America, are at a higher risk. In Uruguay it is not a notifiable disease and mostly outpatient treatments, so its real incidence is not known with exactitude. DGSG in its 2006 survey reported that 0.7% of the rural population is affected by NWS annually, corresponding to 818 human cases per year. But also other authors^{117)(118)(119} have been reporting human cases in urban areas, affecting children and adults. Myiasis were predominantly in the scalp and oropharyngeal region, and all cases were associated with pre-existing injuries and concomitant conditions, such as mental disorders, poor hygiene or alcoholism, among others. A recent study described the clinical and epidemiological characteristics of 63 myiasis cases in children (7 years old in average) hospitalized in a referral center in Uruguay between 2010 and 2019. It was shown that the vast majority of myiasis (98%) were caused by the NWS fly and one third of the patients presented comorbidities, with chronic malnutrition being the most frequent. About half of the cases were initiated in injuries caused by pediculosis¹²⁰.

4.1. Insecticide usage

Mutations in target sites altering enzyme sensitivity to a chemical compound are the predominant molecular mechanisms of insecticide resistance, but also the metabolic detoxification pathways play a role in processing the insecticide. Independently or combined, these mechanisms confer resistance to all insecticide classes available¹³⁶.

Organophosphate insecticides (OP), diethyl-OPs and dimethyl-OPs have been widely used to control infestations caused by NWS larvae. Non-synonym mutations (i.e., mutation that alters the amino acid sequence of a protein) in the NWS fly carboxylesterase E3 gene (also known as ChαE7) enable the hydrolysis of organophosphate substrates^62)(137. It has been found that the substitution of a glycine for aspartate at position 137 (G137D) in the E3 enzyme of Diptera species confers broad spectrum resistance to OPs, mainly diethyl-OPs¹³⁸. As well as the replacement of a tryptophan by leucine/serine at position 251 (W251L or W251S) has been associated with low resistance level to diethyl-OPs and high resistance level to dimethyl-OPs and may be related to cross-resistance to pyrethroid insecticides¹³⁹. These mutations were detected in a wide region of Brazil and Uruguay, in a high occurrence in several locations⁶⁵⁾⁽⁸⁹, although whether resistant individuals due to the phenotypic expression of such mutations are circulating in this region has not been investigated.

Currently, in Uruguay, DILAVE-MGAP has approved 60 topical insecticides based on four kinds of active principles: phenylpyrazole (Fipronil), pyrethroid (Cypermethrin), organophosphate (Dichlorvos, Diazinon), and neonicotinoids (Imidacloprid) to control NWS larvae. These insecticides reached the MERCOSUR (N76/96) standard where a 100% efficacy must be proved, and the standard WAAVP¹⁴⁰, where a 100% therapeutic efficacy and 90% persistent efficacy must be proved. Systemic insecticides of the macrocyclic lactones (Ivermectin and Doramectin) did not demonstrate therapeutic or prophylactic activity against NWS larvae, and are still under investigation.

A survey done in Artigas, Uruguay, revealed that most farmers (91%) use commercial insecticides, while a small fraction (9%) use both commercial and homemade insecticides (usually a mix of some kind of oil with a repellent). This questionnaire also pointed to a decrease in product effectiveness and 15% of the participants stopped using certain products. In general, farmers based on their empirical knowledge suspect that treatment with spray and powder fails (~55-60% curative fails) more than liquid (~70% curative success) and ointment (~70% curative success) insecticides¹⁴¹.

4.2. History of SIT-based control programs

The idea of reducing wild populations of a pest or parasite species by releasing sterile insects (i.e., the sterile insect technique, or SIT) and thus controlling them was independently conceived by Alexander S. Serebrovskii in Russia, Federic L. Vanderplank in Tanganyika (now Tanzania), and Edward F. Knipling in the USA during the 1930s and 1940s¹⁴². Knipling observed the extreme sexual aggressiveness of the NWS fly males, and that females refuse to mate more than once, realizing that if sexual sterility could be induced in males, and if a vast number of them could be sterilized and released in the field, then NWS natural populations would be eventually suppressed^142)(143. The SIT is a genetic control approach based on the Mendelian inheritance of sterility produced by dominant lethal mutations generated by ionizing radiation¹⁴⁴, that imposes birth control to further reduce the target pest populations¹⁴⁵. Its application requires mass rearing of large numbers of the target insect under laboratory-controlled conditions, exposing them to ionizing radiation to induce sexual sterility, and releasing them successively into target wild populations on an area-wide basis. The NWS fly was the first insect parasite to be reared on an artificial diet enabling very large insect numbers for its study¹⁴⁶. The SIT is usually integrated as a component of Area-Wide Integrated Pest Management (AW-IPM) programs^145)(147 where the density of the target populations is initially reduced, eliminating already mated females with auxiliary methods such as insecticides¹⁴⁸. Four kinds of AW-IPM integrating SIT can be deployed: suppression, eradication, containment and prevention¹⁴⁹. A suppression program aims to maintain pest/parasite populations below an agreed and acceptable economic injury level and/or prevalence level. Eradication implies the elimination of a species from an area, usually an isolated local population. Nevertheless, the term eradication is restricted to the global extinction of a species. Containment is defined as the measures in and around an infested area to prevent spread of a pest/parasite, usually adopted to avoid the spread of alien species, or to consolidate progress made in an ongoing eradication program (e.g., current NWS fly program in Panama). The prevention strategy are the measures in and/or around a pest/parasite free area to avoid its introduction.

The NWS fly eradication from North and Central America is one of the most successful programs worldwide. Started in 1957 to rid the south-eastern USA and extended during the following ~40 years to eradicate it from the USA, Mexico, and Central America up to Panama^24)(150)(151. By 1984 the goal of eradicating it to Mexico’s Isthmus of Tehuantepec was achieved¹⁵². At the request of livestock producers in southern Mexico and Central America, in 1986 the eradication campaign was extended to the Yucatán Peninsula and bordering countries¹⁵³. Eradication was declared by steps as follows: Mexico 1991, Belize and Guatemala 1994, El Salvador and Honduras 1996, Nicaragua and Costa Rica 1999, and finally Panama 2006, where since 2004 a permanent barrier is maintained in the Darien region -along the Panama-Colombian border- to avoid reinfestation of endemic NWS from South America^{122)(151)(154)(155)(156}. During these campaigns it has been shown that winter temperatures and rainfalls have a great negative impact on the appearance of NWS outbreaks and could be of great help to succeed in the eradication^50)(157. Also, the typical low population density of C. hominivorax in the tropics was a key factor for the eradication¹⁵⁷.

Although the APHIS (The Animal and Plant Health Inspection Service, USA) official recognition was in 2006, it was in June 2021 that the OIE (World Organization for Animal Health) published Panama’s January 2020 self-declaration of NWS-causing myiasis freedom. In Panama, the Panama-United States Commission for the Eradication and Prevention of Screwworm, or COPEG (www.copeg.org), constructed a smaller insect mass rearing facility in the early 2000s at Pacora, and the larger facility at Tuxtla Gutiérrez was closed in 2012¹⁴². The Panama facility was inaugurated in July 2006 and after three years of installation of equipment, biological safety measures, and staff training in rearing and insect sterilization the biosecurity facility mass production of flies began in March 2009. Currently, COPEG is the only existing NWS fly production facility in operation. With a maximum production capacity of 100 million sterile insects per week, it is currently producing and releasing 20 million sterile flies per week to provide biosecurity for the Plant and maintain the Permanent Biological Prevention Barrier.

An event that deserves to be highlighted was the above-mentioned accidental introduction of the NWS fly during the late 80’s in Libya, from where the screwworm was eradicated using SIT in 1992¹⁵⁸. Although the NWS outbreak source remains unknown, it was initially suggested that it could have entered in a shipment of 236,000 live sheep from Uruguay in 1988. Uruguay as the NWS source of the Libya outbreak was discarded since the shipment arrived after the NWS cases had been already reported¹³². Despite this, the government of Libya banned the importation of live animals from Latin America, and until this date it was never re-established with Uruguay, despite the country being actively trading with other Arabian countries.

Currently, there is no official control program for the NWS fly in Uruguay or other country in South America. The strategies to reduce myiasis’ destructive effects rely on farm owner’s decisions, both chemical treatment and animal management (i.e., dehorning, castration, caravanning, calving, branding). Also, on zoos and parks responsible authorities, as well as veterinarians and medical doctors from private clinics and hospitals. The implementation of an AW- IPM based on SIT for the NWS fly in Uruguay has been largely discussed and is under evaluation. Some actions have been taken in order to evaluate the SIT usage in this region, and during 2007-2009 the governments of Brazil, Paraguay and Uruguay, supported by IDB (International Development Bank) and COMEXA (Mexico-American Commission for the Eradication of the Screwworm), carried out a demonstration on the Brazil-Uruguay border. This pilot program was conceived and undertaken to establish the basis for future programmes in the MERCOSUR countries^{127)(159)(160}, and encompassed two phases: Phase 1) preparation, human resource training, and society communication; Phase 2) pilot dispersion of sterile flies. The main purpose was not the parasite eradication, but technology transfer and SIT field test on the region.

The first action of this pilot program was a mating compatibility study among the factory J06-strain, originally from Jamaica, and wild populations from Uruguay. Then, the field work was executed between January 23 and May 15, 2009, spanning 17 weeks, in an area covering 100x60km (100x30km in each country) with centroid in the cities of Artigas (30°24’24.54” S, 56°28’39.34” O, Uruguay) and Quaraí (30°23’33.13” S, 56°27’13.40” O, Brazil). Pre-dispersion happened the first two weeks, dispersion during the next 13 weeks, and post-dispersion the last two weeks. Sterile flies were sent from Tuxtla Gutierrez, State of Chiapas, Mexico, by COMEXA, and quality controls were done when they arrived at Artigas Airport in order to evaluate that delivery did not affect the sterile pupae.

For NWS fly monitoring, egg masses were collected in wounded animals, in addition to 10 gummed paper traps with Swormlure-4 (i.e., lure used in traps that release a strong odor to attract screwworm flies, a chemical mix including dimethyl disulfide, benzoic acid, indole, and phenol) as bait were distributed throughout the area and sentinel sheep were placed in 10 farms (five in each country). Differences in egg mass quantity between both countries were interpreted as due to a higher NWS fly population in Uruguay, which would occur as a function of the higher livestock density, mainly sheep, that doubled the Brazilian sheep stock in the region.

The sterility per week, measured as the proportion of sterile/fertile egg masses in sentinel animals, reached in the pilot area was directly related to the native NWS fly population and its oviposition rate, ranging from 1.53% in the second week of dispersion to 25.45% in week 11. When analysing the data within each country separately, the maximum sterility was in Brazil, with 40.7% in week 11. The demonstrative test was considered a success¹²⁷ and the outcomes were promising, especially when considering that sterile flies were released during summer in the highest density peak of wild flies.

After this pilot project to demonstrate the SIT effectiveness in the Uruguay-Brazilian border, sterile flies were used to investigate natural barriers to NWS fly between Argentina and Uruguay in 2018⁷. In collaboration with SENASA (Servicio Nacional de Salud y Calidad Agroalimentaria), Argentina, and the advice from USDA-ARS and USDA-APHIS scientists, and assistance from COPEG technicians, the role of the Uruguay River as a potential barrier for NWS fly crossing was evaluated. NWS sterile pupae shipped from COPEG, Panama, were marked with distinctive colors and released on both sides of the river in the region of Fray Bentos (releasing point: 32°53’23.57” S, 57°59’33.83” W, Uruguay) and Puerto Unzué (release point: 32°55’36.41” S, 58°14’23.60” W, Argentina). No marked flies crossing the Uruguay River were captured in three traps disposed in each country, at 4.3 km, 12.9 km and 15 km away from the release point in Uruguay, and at 6.2 km, 5.2 km and 13.9 km away from the release point in Argentina. The authors hypothesized that rivers can serve as barriers and riparian habitats along rivers can serve as corridors for the movement and dispersal of flies⁷. But it is necessary to take into account that the recapture percentages of stained flies were low, as previously found in Panama (156): 0.08% (8/10,000) of 1^st, 0.05% (5/10,000) of 2^nd and 0.07% (7/10,000) of 3^rd dispersions in Uruguay, and 0.07% (7/10,000) of 1^st, 0.03% (3/10,000) of 2^nd and 0.02% (2/10,000) of 3^rd dispersion in Argentina.

4.3. Biotechnology applied to NWS control

4.3.2. CRISPR/Cas-based technology

The advance of genetic engineering brings the possibility of more complex genetic approaches for insect pest control. In this regard, the incorporation of CRISPR/Cas9-based systems⁹⁵ could significantly improve the existing toolkit of NWS molecular methods. Briefly, Cas9 is an endonuclease that produces a targeted double-strand break in a DNA sequence guided by a single-stranded RNA complementary to the DNA target sequence (Figure 2a). The generated double strand break can be repaired by two types of mechanisms: the non-homologous end joining pathway (NHEJ), in which case the process commonly results in the introduction of deletions and/or insertions (collectively called indels) at the break site. Or, if supplied with a repair template, with a sequence complementarity to either side of the damaged region, homologous recombination (HDR) can occur to repair the break incorporating the template (Figure 2b).

Paulo and others⁹⁶ established the first protocol to generate site-specific modifications in the NWS fly genome using the CRISPR/Cas9 system, targeting and disrupting the transformer gene (Chtra), producing intersexual female flies that show different levels of masculinization in their genitalia, while male adults show normal phenotype. As an important sex determination gene, required for normal female development, Chtra could be an interesting potential target for genetic control systems. In addition, the recently published whole-genome assembly of the NWS fly⁶¹ could allow the identification of other potential target genes that could be used in future genetic control programs based on CRISPR/Cas9.

The CRISPR/Cas9-based gene-editing system can be implemented in NWS management control for improving transgenic sexing strains designed for use in potential SIT programs. The use of this new technology avoids the concerns regarding the transgene being inserted in a random unpredictable place of the genome.

Additionally, the CRISPR/Cas9 technology can be used to design a gene drive system. Gene drives are naturally occurring selfish genetic elements that can increase the odds that they will be inherited (Figure 2c). Many researchers have suggested that these elements might serve as the basis for ‘synthetic gene drives’ capable of spreading engineered traits through wild populations¹⁶⁶. Austin Burt¹⁶⁷ was the first to propose gene drives based on site-specific ‘homing’ endonuclease. To build a CRISPR/Cas9 based gene drive, both Cas9 and the RNA guide must be inserted as a genetic construct in place of a sequence it can cut. If it can cut this sequence in organisms with one modified and one natural locus, reliably inducing the cell to copy the construct, and avoid being too costly to the organism, it will spread through susceptible wide populations. The drive can spread this genetic construct or disrupt existing genes¹⁶⁶.

Figure 2: Main outcomes of genome-editing by CRISPR/Cas9. A) Target-specific single guide RNA (sgRNA, in green) form a complex with the endonuclease CRISPR-associated protein 9 (Cas9, in gray), and upon recognition of a DNA sequence (complementary to the sgRNA), just before a 5’-end recognition PAM sequence (NGG, where “N” is any nucleotide), the Cas9 promotes double-strand breaks (DSB) in double-stranded DNA (dsDNA) 3-4 nt 5’ of the PAM. B) DSBs repair pathways triggered by gene-specific nuclease: In 1, a mutation is generated, a gene is inactivated. Quasi-random mutation at the target site, indistinguishable from the natural mutation, no DNA fragments are left in the final product. In 2, a targeted nucleotide change edits a gene using a small DNA template sequence. In 3, a DNA fragment is inserted. The transferred fragment can be recognized, and additional DNA fragments remain in the final products. C) Genealogies comparing the spread of genetic changes or mutations as expected by the Mendelian (leftmost) and gene drive (rightmost) inheritance mechanisms 

For NWS population control, one promising strategy is to implement a population suppression gene drive that could reduce the size of the target population by disrupting genes causing infertility or lethality only when both copies are lost^168)(169. Whether a standard gene drive will spread through a target population depends on molecular factors such as homing efficiency, fitness cost, and evolutionary stability, but it is also sensitive to specific ecological variables such as mating dynamics, generation time, and other characteristics of the population¹⁷⁰.

Because any consequences of releasing gene drives into the environment would be shared by the local if not global community, research involving gene drives capable of spreading through wild-type populations should occur only after a careful and fully transparent review process. This should include independent scientific assessments of probable impacts and fully inclusive public discussions¹⁷¹. Possible ecological effects can be assessed by performing contained field trials with organisms that have been engineered to contain the desired change but do not possess a functional drive to spread it. Finally, the prevalence of the gene drive in the environment could in principle be monitored by targeted amplification or metagenomic sequencing of environmental samples.

5.1. Environmental impact of NWS fly eradication

In order to assess the possible impacts of NWS fly eradication, the Ministry of Agriculture of Uruguay (MGAP) through a consultancy is evaluating the potential, both negative and positive, environmental and social impacts of the NWS fly eradication. A preliminary analysis indicated that the NWS fly eradication in Uruguay would not have significant negative impacts and these would not exceed social and environmental benefits. Likewise, to reduce uncertainties, it was confirmed the usefulness of gathering information at the national level of the NWS fly population size, distribution and dynamics, through the fly monitoring, as well as generating national information on the distribution and population dynamics of species through monitoring for possible ecological impacts at agricultural landscapes and natural fields, before, during and after the execution of the control program. This kind of information will make it possible to evaluate different negative and positive potential socio-environmental impacts.

Potential impacts associated with ecological interactions include the likely population growth of NWS fly wildlife hosts, as well as the reduction of stress caused by myiasis in domestic and wild hosts if this ectoparasite is eradicated. The potential effect of the NWS fly as a regulator of wildlife can be evaluated as positive in the case that native warm-blooded animals are beneficiated, but negative when alien species considered pests are the beneficiaries. Special attention called the effect on feral swine¹¹⁴, and a preliminary analysis estimates a low risk since its population density depends on other more determining factors than the NWS fly. The animal stress reduction propitiated by the elimination of myiasis must be considered as positive, independently if the species is native or alien, under the current animal welfare standards.

The interaction network with other species could also be affected once the NWS fly is removed and the ecological dynamics of its predators, pathogens, commensals and/or mutualists can be negatively influenced as well as competitors can be positively influenced. No significant risk is expected for known NWS fly predators (e.g., ant, beetles, spider) given the low probability of exclusive dependence on it as a food source, since they are generalist species^174)(175. Similar to what must happen with the NWS fly commensal, mutualist and pathogenic species. For competing species, there are no records at the national level of species that could occupy the NWS niche, so the risk could be characterized as non-existent. The absence of tight specific ecological interactions, leading to a dependency relationship with the NWS fly, is supported by its seasonal population dynamics and the geographical localization of our country in the species distribution border, that may not represent an optimal area. Despite this, the absence of ecological interactions with specialist species hypothesis should be evaluated with data collected during a species monitoring planned within the eradication program under discussion by the authorities.

Another biotic environmental factor that must also be contemplated is its role as pollinator in the long-time¹⁷⁶. However, interactions with flowering plants in the role of adults as exclusive pollinators have not been identified. In summary, a preliminary risk analysis of the environmental impact of an NWS fly eradication program in Uruguay indicates that it would not result in significant alterations on ecosystems and ecological interactions. The NWS fly eradication programs have been implemented in wide areas (from USA to Panama) since the 1950s with no significant negative environmental impacts reported on the specialized literature. Although, as far as we know, no specific monitoring studies have been conducted, 70 years without reports of negative ecological impacts have contributed as an indirect proof with the implementation of control programs.

5.2. Environmental impact of the control program for the NWS fly eradication

Control strategies may involve certain activities that require to be analysed in order to determine whether they could have a negative impact on the environment. In the case of SIT-based eradication, insecticides usage during the first phase of the program and dispersion of many sterile flies were considered. Regarding the use of insecticides, that could cause potential contamination of soils and waters, on side with the mortality of non-target animals (i.e., pollinators), the potential impact is limited to the first phase of the program, because of the increasing use of topical and/or systemic insecticides for prophylaxis and myiasis treatment, as the needed way to reduce wild populations density before sterile flies’ dispersion. In a long-term, the increased usage of insecticides during this first phase will not be higher than what has been currently applied, and once Uruguay is declared free of the NWS fly its usage will decrease drastically, being necessary only to control some sporadic myiasis and suspicious cases.

During dispersal and maintenance phases the effect on air traffic due to the dispersion of millions of sterile flies could negatively affect other species, such as avifauna, because of the disturbances caused by aircrafts and the novel traffic pathways. However, its impact could be considered non-significant since the traffic bulk would be also concentrated in the first phases of the program.

Recently, genetically modified or preferentially inherited (GD, for gene drive) organisms have been proposed as a new tool that could be used to control or eradicate parasite or vector species. Gene drive systems (see subheading 5.3.2 and Figure 2c) allow the introduction of genetic elements at a higher frequency than expected, which can rapidly change genotypes in target populations with consequences on species fitness. For example, the control of the dengue vector Aedes aegypti by releasing genetically modified sterile males, described in Araújo and others¹⁶¹, in Grand Cayman (2009 and 2010), Malaysia in 2010 and Brazil in 2011, with an efficiency greater than 80% population suppression, can be approached by a GD system.

The environmental risk assessment of interventions based on novel biotechnological tools, like transgenesis and gene editing, requires having genetic and technical information about the specific strategy used to generate the organism as well as information about target and non-target species. Ecological and evolutionary data would be required such as hybridization or horizontal gene transfer between target and non-target species, the evaluation of potential target sites in the genome of non-target species, food web structure, behavioral and demographic data¹⁷⁷, as well as the mating system and gene flow between populations (e.g., dispersal ability and anthropogenic dispersal)¹⁷⁸.

Finally, yet important, the geography of Uruguay nestled in the Pampas biome, extending from southern Brazil, through the center of Argentina until the south of the Province of Buenos Aires, where no dry boundaries exist, represents a challenge to determine the releasing locations and merits an informed community consent. Release into the environment could include transboundary movement and replacement of off-target populations, with potential human/animal health impacts. Therefore, eventual regional coordination should be considered.

6.1. Current regulations

Genetic modified organisms (GMOs) are regulated in most countries and covered by international agreements such as the Cartagena Protocol under the United Nations CBD. However, excessive cautious and restrictive GMO regulations that often prevent the use of new technologies could prolong the risk of animal suffering, loss of food security, environmental imbalance using insecticides and unspecific household products. Research and development are advancing faster than regulation and CRISPR opens doors in many fields of production and environmental conservation (described below). The technology stands out for reducing time, improving the use of resources, and reducing costs to generate new varieties, involving private companies and institutions. Vegetal GMOs have been planted on millions of hectares and commercialized for a long time, but regulation is variable worldwide, with very different endpoints. Based on past experiences of innovation, it is desirable that GMO applicants and regulators interact to achieve the benefits of innovation while cautiously avoiding unacceptable risks.

In Uruguay, the existence of enacted legislation (Decree No. 353/008) that regulates the importation and various GMO uses -including research uses- within our territory- provides a safe, necessary and reliable regulatory environment for the application of biotechnological innovations. This legislation, in addition to providing a suitable environment for the application of safe biotechnologies, enables collaborations between countries in the long term. The enacted legislation also represents evidence of public deliberation on the use of genetically modified organisms within the country, and an important cultural and logistical asset that enables the use of innovative, state-of-the-art solutions^179)(180. A well-designed risk assessment helps to manage the tension between the desire for caution regarding the risk of intervention and concern about the risks of non-intervention¹⁷².

6.2. Regulatory aspects of genome editing

Genome editing by gene-specific nucleases such as CRISPR/Cas is a versatile tool that generates variations in the recipient genome at specific target sites. The CRISPR/Cas editing system is composed of two clearly differentiated elements, the nucleotide fraction usually constituted by the single guide RNA (sgRNA), and the enzymatic fraction represented by the Cas9 endonuclease (see subheading 5.3.2 and Figure 2). Once introduced into the cell the specifically designed sgRNA guides the Cas9 protein into the specific sequence in the genome to be modified by making a double cut in the target DNA. From this point on, the non-homologous end joining (NHEJ) or the direct homology repair (HDR) pathway of cellular repair systems introduce the desired mutation (Figure 2b). The NHEJ pathway promotes insertion or deletion mutations (indels) that generate, by changing the correct reading frame, a termination codon that disrupts the gene. If the edition aims to modify the gene sequence without truncating it, the cell must be provided with a repair template carrying the intended mutation and the HDR pathway is followed. The repair of DNA cleavage by gene-specific nucleases results in variants at the target site, and three types of alterations can be distinguished. In the absence of donor of DNA the NHEJ repair introduces base pair changes or small insertions/deletions resulting in frame-shift mutations that cause premature stop codons and mRNA degradation. The exact change cannot be predetermined and is almost random at the target site. When a DNA donor is present, either single stranded oligonucleotides (ssODNs) or double stranded DNA, the DNA homology repair by single-stand annealing or homology recombination occurs, respectively¹⁸¹. In the cases that large DNA elements of foreign origin are introduced it is usually considered transgenic¹⁸². It is not the case for CRISPR-based genome editing when foreign DNA is not added (e.g. knock out models), and then it is generally accepted that should not be required additional regulatory oversight than other breeding varieties. The introduction of large repair templates (e.g. knock in models) requires further regulatory oversight and specific approval. The regulatory system for genome editing differs between countries, and while some are seen as more innovative, others are more restrictive or conservative.

6.3. NWS fly editing

The development of gene-edited strains of the NWS fly by CRISPR/Cas to be used in a control program could be considered beneficial as it is species specific, and mitigates damage to the environment, economy or health of wildlife, domesticated and human life, although it is ‘invasive’ to some extent. Since GD applications aim to release organisms that become established in the environment and may spread throughout different habitats, countries have the responsibility to assess transboundary risks and liability for damage caused by such releases. It is likely that bilateral and regional measures will first establish approaches before harmonization at higher international levels¹⁷².