Table 2.

Published examples reporting an additive or less than additive result for insecticidal protein combinations

Number of examples	Insecticidal proteins	Insect	Additivity model or empirical test	Assessment method used	Model assumption concerns	Other comments	Reference
5	Vip3Aa, Vip3Ae, Vip3Af, Cry1Aa, Cry1Ac, Cry1Ca	Tobacco budworm (Heliothis virescens)	CA	Tabashnik eq. 5a	CA model not justified by dose–response slopes (used slopes which varied by ~2–4-fold)	upper end of dose–response not well-represented; precision very poor for LC90 values	Lemes et al. (2014)
1	Vip3Aa, Cry1Ca	Fall armyworm (Spodoptera frugiperda)	CA	Tabashnik eq. 5	―b	―	Lemes et al. (2014)
2	Cry1Ac, Cry1Fa	Cotton bollworm (Helicoverpa armigera)	CA	Tabashnik eq. 5	CA model not justified by dose–response slopes (dose–response could not be determined for 1 of the 2 proteins)	―	Ibargutxi et al. (2008)
1	Cry1Ac, Cry1Fa	Earias insulana	CA	Tabashnik eq. 5	―	―	Ibargutxi et al. (2008)
3	Cry1Ac, Cry2Ab	Earias insulana	CA	Tabashnik eq. 5	CA model not justified by dose–response slopes (used slopes which varied by 1.8-fold)	―	Ibargutxi et al. (2008)
1	Cry1Ac, Cry2Ab	Cotton bollworm (Helicoverpa armigera)	CA	Tabashnik eq. 5	―	Measured growth inhibition	Ibargutxi et al. (2008)
1	Cry1Ac, Cry2Ab	Earias insulana	CA	Tabashnik eq. 5	―	Measured growth inhibition; high heterogeneity across protein responses	Ibargutxi et al. (2008)
2	Cry1Ac, Cry1Fa	Earias insulana	CA	Tabashnik eq. 5	―	Measured growth inhibition	Ibargutxi et al. (2008)
1	Cry1Aa, Cry1Ab	Gypsy moth (L. dispar)	CA	Tabashnik eq. 5	―	Force-feeding bioassay; measured weight in 4th instar larvae	Lee et al. (1996)
6	Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca	Pink stem borer (Sesamia inferens)	CA	Tabashnik eq. 5	―	multiple insects (10) per test arena; purity of proteins not shown; some details of diet incorporation not shown/ referenced	Gao et al. (2010)
3	Cry1Aa, Cry1Ab, Cry1Ac	Cotton bollworm (Helicoverpa armigera)	CA	Tabashnik eq. 5	―	used droplet-feeding method	Li and Bouwer (2014)
4	Cry1Aa, Cry1Ab, Cry2Aa, Cry9Aa	Cotton bollworm (Helicoverpa armigera)	CA	Tabashnik eq. 5	―	―	Li and Bouwer (2014)
5	Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da	Bean shoot borer (Epinotia aporema)	CA	Tabashnik eq. 5	―	―	Sauka et al. (2007)
9	Cry1AbMod, Cry1Ac, Cry2Ab	Pink bollworm (Pectinophora gossypiella)	RA	Colby methodc with Fisher’s exact test	―	9 out of 11 combinations statistically insignificant.; 7 out of 11 qualitatively greater, but only 3 at plus 13% or greater	Tabashnik et al. (2013)
34	Cry1Ac, Cry2Ab	Cotton bollworm (Helicoverpa armigera) susceptible strain	RA	Colby method with t-test	―	only 2 out of 36 combinations statistically significant.; 7 out of 36 qualitatively greater	Wei et al. (2015)
1	Cry1Ab, Cry9A	Diamondback moth (Plutella xylostella)	RA	Colby method	―	Only 1 combination used; purity of substances not shown; used leaf-dip method	Mittal et al. (2007)
3	Cry1Ac, Cry2Aa	Cotton bollworm (Helicoverpa armigera)	CA, RA	Tabashnik eq. 5 or Colby method	―	―	Liao et al. (2002)
1	Cry1Ab, Cry2Aa	Mediterranean flour moth (Ephestia kuehniella)	CA	Tabashnik eq. 5	―	purity information not given; lyophilized proteins adsorbed to crushed peanuts; multiple insects (10) per test arena	Azizoglu et al. (2016)
1	Cry1Ab, Cry2Aa	Indian meal moth (Plodia interpunctella)	CA	Tabashnik eq. 5	―	―	Azizoglu et al. (2016)
4	Cry1Aa, Cry1Ab, Cry9Ca	Spruce budworm (Choristoneura fumiferana)	CA	Tabashnik eq. 5	Slopes of dose-responses not described	used droplet-feeding method vs. 6th instars and frass-failure response	Pang et al. (2002)
1	Cry1Ab, mCry3A	European corn borer (Ostrinia nubilalis)	Empirical	Low dose/high dose combinations with ANOVA	―	―	Raybould (et al. 2012a)
1	Cry1Ab, mCry3A	Colorado potato beetle (Leptinotarsa decemlineata)	Empirical	Low dose/high dose combinations with ANOVA	―	―	Raybould et al. (2012a)
1	Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1F, Cry1I, Cry2Ab, Cry9B, Cry9E	Beet armyworm (Spodoptera exigua)	CA	Tabashnik eq. 5	―	Used mixtures of washed crystals from 2 different Bt isolates; expression levels of isolate genes not determined	Konecka et al. (2012)
1	Cry1Ab, Cry1F, Vip3A	European corn borer (Ostrinia nubilalis)	RA	Colby method	―	―	Graser et al. (2017)
1	Cry1Ab, Cry1F, Vip3A	Fall armyworm (Spodoptera frugiperda)	RA	Colby method	―	―	Graser et al. (2017)
1	mCry3A, eCry3.1Ab	Colorado potato beetle (Leptinotarsa decemlineata)	RA	Colby method	―	―	Graser et al. (2017)
1	Cry1Ab, Cry1F, Vip3A, mCry3A, eCry3.1Ab	Fall armyworm (Spodoptera frugiperda)	Empirical	Low dose/high dose combinations with ANOVA	―	―	Graser et al. (2017)
1	Cry1Ab, Cry1F, Vip3A, mCry3A, eCry3.1Ab	Colorado potato beetle (Leptinotarsa decemlineata)	Empirical	Low dose/high dose combinations with ANOVA	―	―	Graser et al. (2017)
1	Cry1Ab, Cry2Ab	Corn earworm (Helicoverpa zea)	RA	Colby-like plus Chi-square test	―	used event material plus ELISA; multiple insects (4) per test arena	Greenplate et al. (2003)
1	Cry1Ab, Cry2Ab	Fall armyworm (Spodoptera frugiperda)	RA	Colby-like plus Chi-square test	―	used event material plus ELISA; multiple insects (4) per test arena	Greenplate et al. (2003)
1	Cry1Ac, Cry1F	Beet armyworm (Spodoptera exigua)	Empirical	ANOVA comparisons	No apparent interactions, but experimental design cannot support the conclusions	used event material but no ELISA	Adamczyk and Gore (2004)
1	Cry1Ac, Cry1F	Fall armyworm (Spodoptera frugiperda)	Empirical	ANOVA comparisons	No apparent interactions, but experimental design cannot support the conclusions	used event material but no ELISA	Adamczyk and Gore (2004)
1	Cry1Ac, Cry1B	Coffee leaf miner (Perileucoptera coffeella)	CA	Tabashnik eq. 5	―	used infused leaf material	Guerreiro Filho et al. (1998)
1	Cry1Ac, Cry2Ab2	Cotton bollworm (Helicoverpa armigera)	CA	Tabashnik eq. 5	Pooled data from populations which varied by 37-fold; slopes varied over six-fold; large ranges for 95% CI	used formulated MVP and lyophilized maize leaf as source material	Brévault et al. (2009)
2	Cry1Ac, Cry2Ab2, Vip3Aa19	Cotton bollworm (Helicoverpa zea)	CA	Tabashnik eq. 5	―	Measured growth inhibition; used event material plus ELISA; used a shared(?) slope parameter	Levine et al. (2016)
1	Cry2A, Cry9C	Cotton leafworm (Spodoptera litura)	Empirical	ANOVA comparisons	―	used event material plus ELISA; low expression of Cry2A; multiple insects (10) per test arena	Li et al. (2014)
1	Cry1Ab, Vip3Aa19	Tobacco budworm (Heliothis virescens)	Empirical	ANOVA plus LSMEANS	―	used event material but no ELISA; multiple insects (3) per test arena	Adamczyk and Mahaffey (2008)
1	Cry1Ab, Vip3Aa19	Corn earworm (Helicoverpa zea)	Empirical	ANOVA plus LSMEANS	―	used event material but no ELISA; multiple insects (3) per test arena	Adamczyk and Mahaffey (2008)
2	Cry1Ba, Cry1Ca, Cry1Da	Fall armyworm (Spodoptera frugiperda)	Empirical	GLM and Kruskal- Wallis test	―	―	Costa et al. (2014)
3	Cry1Aa, Cry1Ac, Cry1Ca, Vip3Aa	Oriental fruit moth (Grapholita molesta)	RA	Colby-like plus Chi-square test and Fischer’s test	―	―	Scaramal Ricietto et al. (2016)
6	Cry1Aa, Cry1Ca, Vip3Aa	Oriental fruit moth (Grapholita molesta)	CA	Tabashnik eq. 5	CA model not justified by dose-response slopes (used slopes which varied by 1.9–2.3-fold); Broad LC50 estimate for 2 components (9–11-fold); 2 components with base of 32-fold differing potency	―	Scaramal Ricietto et al. (2016)

^aEquation 5 as described in Tabashnik (1992). ${\text{LC}}_{50(m)}=[1/[(r_a{\text{/LC}}_{50(a)}) + (r_b{\text{/LC}}_{50(b)})]]$ with LC_50(m) being the expected LC₅₀ of the mixture, where LC_50(a) and LC_50(b) are the expected median lethal concentrations for the individual components, a and b, respectively. The relative proportions of a and b components are described as r_a and r_b, respectively.

^b― = none noted.

^cAs described in Colby (1967). If Component A alone gives x% effect and Component B alone gives y% effect, then under the assumption of independent action, the predicted percent response to A + B is: x + y – (xy/100).