Cadherin mutation linked to resistance to Cry1Ac affects male paternity and sperm competition in Helicoverpa armigera

Abstract

Several lepidopteran pests of cotton have cadherin-based resistance to the Bacillus thuringiensis (Bt) toxin Cry1Ac. Cadherins are transmembrane proteins that mediate cell-cell adhesion and tissue morphogenesis, suggesting that fitness costs associated with cadherin mutations may be present in many aspects of life history. To evaluate whether cadherin-based resistance is associated with fitness costs reducing male paternity in Helicoverpa armigera, we examined the effects of a major cadherin resistance allele on sperm competition within and between male ejaculates. When homozygous resistant and susceptible males competed for fertilization of a homozygous resistant or susceptible female, fertilization success was high in males with a different cadherin genotype than females and low in males with the same cadherin genotype as females. Single matings between heterozygous males and susceptible females produced offspring within typical Mendelian ratios. Heterozygous males mated to resistant females, however, resulted in a disproportionate number of heterozygous offspring. While these results show that cadherin-based resistance to Cry1Ac has significant impacts on paternity in H. armigera, there was no evidence that costs associated with resistance consistently reduced male paternity. Rather, effects of cadherin-based resistance on paternity depended on interactions between male and female genotypes and differed when males or sperm competed for fertilization of females, which complicates assessment of impacts of cadherin resistance alleles on resistance evolution.

Lack of fitness costs and inheritance of resistance to Bacillus thuringiensis Cry1Ac toxin in a near-isogenic strain of Plutella xylostella (Lepidoptera: Plutellidae)

“…In a study of Pectinophora gossypiella , Cry1A resistance was associated with alteration of cadherin, a Bt toxin receptor, and with reduced sperm transfer . Furthermore, a homologue of cadherin has been reported to affect adult reproduction in H. armigera . This suggests that cadherin is not involved in the resistance to Cry1Ac toxin in our P. xylostella strain.…”

Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

“…Toxin-binding sites of cadherins have been mapped to the membrane-proximal region, and 12 this particular region of cadherin is critical not only for toxin binding but also for mediating Wu, 2015). However, due to lack of genetic manipulation tools, 25 functional knockout of these cadherin receptors associated with resistance in lepidopteran 26 insects has not yet been achieved.…”

“…These studies suggest that the CRISPR/Cas9 11 system could also be a promising tool for genetic manipulation of agricultural pest insects. 12 The cotton bollworm Helicoverpa armigera with indel mutations, PCR products were then TA-cloned and sequenced to identify the exact 1 mutations presumably resulting from efficient nonhomologous end joining (NHEJ). In total 2 we detected seven types of indel mutations from the mutated individuals (Fig.…”

“…The 9 fragment (187 bp) flanking the CRISPR target site was amplified using a pair -AAAACCAACATACCCTTAGC-3'). Direct sequencing was conducted using the reverse12 primer as the sequencing primer. Double sequencing peaks present as a cluster of bases 13 indicated a mutation event(Fig.…”

“…BBMVs were prepared from insect midguts by the differential magnesium (20 µg) from each strain were separated by SDS-PAGE (8%) and 7 transferred to polyvinylidene difluoride (PVDF) filter (Millipore Corp., Bedford, MA). Filters 8 were blocked with 2.5% BSA in PBST (pH 7.4, 25 mM Tris, 3 mM KCl, 135 mM NaCl, 0.1% 9 Tween 20) for 1 h and then detected with rabbit polyclonal antibodies (1:2000), which was 10 raised against a peptide "PRPERPDFPSQNFD" near the N-terminal of HaCad (ABGENT,11 Suzhou, China), followed by incubation with horseradish peroxidase (HRP)-conjugated goat12 anti-rabbit IgG (Sigma) (1:5000) in PBST at room temperature for 1 h. Protein was visualized 13 using SuperSignal® West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc.,14 Rockford, IL) according to the manufacturer's instructions, and photographed with Bio-Highly efficient mutagenesis induced by CRISPR/Cas9 system19 We selected HaCad as a candidate gene to examine the efficiency of CRISPR/Cas9 20 mediated genome-editing in H. armigera, and an sgRNA was designed to mediate a21 double-strand break in exon 9 of HaCad(Fig. 1A).…”