Engineered Female-Specific Lethality for Control of Pest Lepidoptera

The sterile insect technique (SIT) is a pest control strategy involving the mass release of radiation-sterilized insects, which reduce the target population through nonviable matings. In Lepidoptera, SIT could be more broadly applicable if the deleterious effects of sterilization by irradiation could be avoided. Moreover, male-only release can improve the efficacy of SIT. Adequate methods of male-only production in Lepidoptera are currently lacking, in contrast to some Diptera. We describe a synthetic genetic system that allows male-only moth production for SIT and also replaces radiation sterilization with inherited female-specific lethality. We sequenced and characterized the <i>doublesex</i> (<i>dsx</i>) gene from the pink bollworm (<i>Pectinophora gossypiella</i>). Sex-alternate splicing from <i>dsx</i> was used to develop a conditional lethal genetic sexing system in two pest moths: the diamondback moth (<i>Plutella xylostella</i>) and pink bollworm. This system shows promise for enhancing existing pink bollworm SIT, as well as broadening SIT-type control to diamondback moth and other Lepidoptera

Engineered Repressible Lethality for Controlling the Pink Bollworm, a Lepidopteran Pest of Cotton

<div><p>The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (<em>Pectinophora gossypiella</em> Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects’ progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.</p> </div

Southern blot analysis of OX3402C.

<p>From left to right, lanes are: Dig-labelled DNA molecular weight marker II (M), OX3402C genomic DNA digested with <i>Bgl</i>II (A), <i>Nhe</i>I (B) and <i>Xho</i>I (C).</p

Survival to pupation of OX3400A pink bollworm reared in the laboratory on larval diet with or without chlortetracycline, and on cotton bolls in field cages.

<p>Survival is expressed relative to that of each group’s wild-type siblings. Error bars indicate 95% confidence intervals.</p

Survival to adulthood of transgenic pink bollworm, heterozygous for an OX1124 insertion (line A, C, D or E), reared on larval diet with chlortetracycline (grey bars) or without chlortetracycline (white bars).

<p>Survival is expressed relative to that of each group’s wild-type siblings. Error bars indicate 95% confidence intervals.</p

Schematic representation of the RIDL cassette (markers and <i>piggyBac</i> sequences omitted) of constructs used for transformation of pink bollworm: (a) OX1124; (b) OX3347; (c) OX3400; and (d) OX3402.

<p>Schematic representation of the RIDL cassette (markers and <i>piggyBac</i> sequences omitted) of constructs used for transformation of pink bollworm: (a) OX1124; (b) OX3347; (c) OX3400; and (d) OX3402.</p

Impact of OX3402C larvae on cotton bolls in the laboratory.

<p>(a) typical marks on exterior of cotton bolls (indicated by arrows) caused by OX3402C larvae, and (b) minor damage to lint caused by a OX3402C larva that had penetrated the boll (indicated by arrow) but remained stunted at approximately 1 mm in length. Equivalent wild-type larvae had pupated at this time, after reaching approximately 10 mm in length.</p

Survival to adulthood of transgenic pink bollworm, heterozygous for an OX3402 insertion (line A, C, M, P, T or U), reared on larval diet with chlortetracycline (grey bars) or without chlortetracycline (not visible as all data points are zero).

<p>Survival is expressed relative to that of each group’s wild-type siblings. Error bars indicate 95% confidence intervals.</p

Fluorescent phenotypes of OX3347A and OX3402C.

<p>Photographs showing a wild-type pupa (left) and an OX3347A pupa (right) under (a) white light and (b) green fluorescent protein (ZsGreen) excitation wavelength light; and a wild-type pupa (left) and an OX3402C pupa (right) under (c) white light and (d) red fluorescent protein (DsRed2) excitation wavelength light.</p