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. 2020 Dec 18;21(Suppl 2):143.
doi: 10.1186/s12863-020-00948-x.

An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT

Affiliations

An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT

Carolina Concha et al. BMC Genet. .

Abstract

Background: The New World Screwworm fly (NWS), Cochliomyia hominivorax, is an ectoparasite of warm-blooded animals and a major pest of livestock in parts of South America and the Caribbean where it remains endemic. In North and Central America it was eradicated using the Sterile Insect Technique (SIT). A control program is managed cooperatively between the governments of the United States and Panama to prevent the northward spread of NWS from infested countries in South America. This is accomplished by maintaining a permanent barrier through the release of millions of sterile male and female flies in the border between Panama and Colombia. Our research team demonstrated the utility of biotechnology-enhanced approaches for SIT by developing a male-only strain of the NWS. The strain carried a single component tetracycline repressible female lethal system where females died at late larval/pupal stages. The control program can be further improved by removing females during embryonic development as larval diet costs are significant.

Results: The strains developed carry a two-component system consisting of the Lucilia sericata bottleneck gene promoter driving expression of the tTA gene and a tTA-regulated Lshid proapoptotic effector gene. Insertion of the sex-specifically spliced intron from the C. hominivorax transformer gene within the Lshid gene ensures that only females die when insects are reared in the absence of tetracycline. In several double homozygous two-component strains and in one "All-in-one" strain that had both components in a single construct, female lethality occurred at the embryonic and/or first instar larval stages when raised on diet without tetracycline. Laboratory evaluation for phenotypes that are relevant for mass rearing in a production facility revealed that most strains had fitness characteristics similar to the wild type J06 strain that is currently reared for release in the permanent barrier. Testing of an "All in one" strain under mass rearing conditions showed that the strain maintained the fitness characteristics observed in small-scale rearing.

Conclusions: The early female lethal strains described here could be selected by the NWS Control Program for testing at large scale in the production facility to enhance the efficiency of the NWS eradication program.

Keywords: Female lethal strain; Pest control; Transgenic sexing strain.

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Conflict of interest statement

The authors declare they have no competing interests.

Figures

Fig. 1
Fig. 1
A transgenic female early lethal system for control of the New World screwworm. a Schematic diagram of the genetic constructs used to create female early lethal strains in NWS. A two-component system consisting of a Driver construct that expresses tTAo protein under the control of the Lucilia sericata bottleneck (Lsbnk) promoter and an Effector construct, composed of 21 copies of the tTA binding site, tetO, upstream of the L. sericata apoptotic hid gene. The hid gene contained two mutations (LshidAla2) that should prevent inhibition by MAPK and thus increase activity [22]. The All-in-one construct contains both Driver and Effector cassettes in the same transformation vector. b Two component transgenic lines express both red and green fluorescent markers at the late larval, pupal and adult stages. c Heterozygous and d homozygous female lethality when insects were reared in the absence of tetracycline in the diet. Eight pairs of flies were used for each test, and error bars show the standard error of the mean (n = 3)
Fig. 2
Fig. 2
Transgenic NWS lines show early female lethality. Female specific lethality of four double homozygous two-component lines and an All-in-one line (TD1) was studied at each developmental stage from egg to adult. 1000 eggs were placed in larval diet, containing the following tetracycline (Tet) regimens: 200 μg/mL Tet in the larval diet and 10 μg/mL in adult drinking water, no Tet in the larval diet and 10 μg/mL in the drinking water for one or two days, and no Tet in the larval or adult diet. The number of first, second and third instar larvae, pupae and adult male and females were counted. a The wild type J06 strain on diet lacking tetracycline, b DR2–17/EF1–16, c DR2-11A/EF1–16, d DR2–17/EF1-6B, e DR2–21/EF1–16 and f All-in-one or TD1. All experiments were repeated at least three times and the average and standard deviation (error bars) are shown
Fig. 3
Fig. 3
Fitness tests of transgenic female early lethal strains. Four homozygous two-component strains and an All-in-one strain (TD1) were evaluated for fitness parameters relevant to mass rearing and compared to the wild type J06 strain that is currently reared at the COPEG facility. These fitness parameters are a Average number of eggs laid per cage, b The percentage of eggs that hatch into first instar larvae, c The percentage eggs that develop into adults, and d the average weight of the pupae. These tests were performed with 200 μg/mL tetracycline in larval diet and 10 μg/mL in the adult drinking water. All experiments were repeated at least three times, with the average and standard deviation (error bars) shown
Fig. 4
Fig. 4
Fitness tests of an All-in-one strain under mass rearing conditions. Twice the number of eggs of the TD1 strain were seeded on diet without tetracycline than were seeded in diet containing tetracycline or relative to J06 on diet without tetracycline. a The average weight of one pupa. b The percentage of first instar larvae hatched from eggs. c The number of adults that develop from 20 L of larval diet are shown. d The number of males that develop from 20 L of larval diet are shown under the same conditions as in c

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