The LC50 values for 1st, 2nd and 3rd instar larvae of WF06 UNSEL were not
significantly different, 2.63, 2.91 and 3.76 µg ml-1 respectively (P>0.01) (Figure 4.3). However, for 4th instar larvae the LC50 was not determined as at the highest bioassay
concentration of 100 µg ml-1, the mortality was only 13 %.
L C50 ( µ g m l -1 ) M IC50 (µ g m l -1 ) (A) (B)
The MIC50 value for 1st instar larvae of WF06 UNSEL, 0.90 µg ml-1, was
significantly lower than the respective LC50 value and significantly lower than the
MIC50 values of 2nd (2.07 µg ml-1), 3rd (1.88 µg ml-1) and 4th (9.21 µg ml-1) instar
larvae of WF06 UNSEL (P<0.01). There was no significant difference between the respective MIC50 and LC50 values for 2nd and 3rd instar larvae (P>0.01). The MIC50
value for 4th instar larvae was significantly higher than 1st, 2nd and 3rd instar larvae (P<0.01). 0 1 2 3 4 5 6 7 1st 2nd 3rd a a ac 0 2 4 6 8 10 12 14 16 18 1st 2nd 3rd 4th Larval instar b a a c
Figure 4.3: Toxicity of Vip3A to larval stages of the WF06 UNSEL H. virescens population: (A) LC50 (± 95 % CI); (B) MIC50 (± 95 % CI). Values with same lower
case letter are not significantly different from each other(P>0.01), (see also Appendix 1, Tables A.1.5 and A.1.6.)
M IC50 (µ g m l -1 ) L C50 ( µ g m l -1 ) (A) (B)
4.4 Discussion
Previous laboratory studies with Vip3A or Vip3-related toxins have not compared variation in susceptibility, only presenting data on the toxicity range to various lepidopteran larvae (Estruch et al., 1996; Yu et al., 1997; Selvapandiyan et al., 2001; Lee et al., 2003; Fang et al., 2007). In the present study, there was generally little variability in the susceptibility to Vip3A, Cry1Ab and Cry1Ac in the field-derived and laboratory populations of H. virescens examined. Variation within each toxin for both LC50 and MIC50 values was less than 7-fold. The exceptions were the laboratory
population NCSU and field-derived population 9607VR that had higher MIC50 values
for Cry1Ab and Cry1Ac resulting in up to 13- and 17-fold susceptibility compared to the remaining populations. MIC50 values were on average 2- to 5-fold lower than the
LC50 values for Vip3A, Cry1Ab and Cry1Ac demonstrating that not all surviving
larvae successfully developed on the diet. None of the data suggested that resistance to Vip3A or Cry1Ab / Cry1Ac was present in the populations examined. The apparent low variability in susceptibility may also have been due in part as a result of these populations becoming established as laboratory cultures. With limited individuals taken from the field, the populations may have gone through genetic bottlenecks, even with a single generation (Table 4.1), possibly eliminating rare genes or genes that may have imposed fitness costs in the field that have no impact in the laboratory, and vice versa. Population bottlenecks and small populations are expected to decrease genetic variance and reduced evolutionary potential (Willi et al., 2006). The behaviour of the populations may have altered in the laboratory environment, for example, mating preferences or the use of artificial diet as opposed to natural food (e.g. cotton plants). These laboratory effects may have altered the response of individuals to the toxins.
Using moult inhibition data may present a more realistic view of the toxicity of Vip3A, Cry1Ab and Cry1Ac, as the vast majority of surviving 1st instar larvae fail to successfully develop on artificial diet (personal observation) and such data can help to highlight antifeedant effects and/or other sub lethal effects of the toxin. Gore et al. (2005) found that H. virescens showed some avoidance of Cry1Ac and Cry2Ab in bioassays in choice experiments, selectively feeding on non-toxin treated diet
compared with Cry1Ac treated diet, although avoidance was less prominent with Cry2Ab.
Moult inhibition data is useful provided identification of the instar stage is determined accurately, and not just based on larval size. Head capsule width is the most accurate way of determining instar stage, although it has been argued that this can take too much time for use in bioassays (Blanco et al., 2005). However, with practice, consistent identification of larval instars can be achieved through observation of head capsule size by eye rather than the need to measure all individuals (personal experience). Head capsule size is fairly distinct when looking at reference larvae for all stages, particularly for 1st to 4th larval instars.
The variation in susceptibility to Bt toxins determined for the populations in the present study appears to be typical for H. virescens when compared to other work. Jackson et al., (2007) found that susceptibility to Vip3A in a laboratory insecticide susceptible population of H. virescens (YDK) and three Cry1Ac resistant populations (YHD2, KCBhyb and CXC) were similar with LC50 values ranging from 179 µg ml-1
to 210 µg ml-1. This indicated that these Cry1Ac resistant populations did not show any signs of cross-resistance to Vip3A. The Vip3A LC50 values reported in the above
work are approximately 100-fold greater than those calculated in the present study (based on a mean Vip3A LC50 of 2 µg ml-1). This difference may, in part, be because
the Vip3A was prepared in distilled water in the present study rather than in an ammonium carbonate buffer as in Jackson et al. (2007). This illustrates the potential dangers of direct comparisons made on absolute values gathered from differing bioassay methodology as opposed to relative comparisons to respective reference populations (González-Cabrera et al., 2001; Liao et al., 2002; Ali et al., 2006).
Using a diet incorporation assay, Ali et al. (2006) found that H. virescens varied 12- fold in susceptibility to Cry1Ac (MPV II formulation) (LC50 0.36 µg ml-1 to 4.54 µg
ml-1) in five laboratory, seven laboratory-cross and 10 field populations collected from 2002 to 2004. The difference was only 4-fold across all laboratory and field populations when LC50 values were pooled, demonstrating little change in
relative differences compared with populations collected in 1992 and 1993 (LC50 0.02
variability in toxicity of Cry1Ab to laboratory and field populations of H. virescens was reported by Luttrell et al. (1999).
Susceptibility of H. virescens populations to other Bt toxins has also demonstrated a similar pattern of low natural variability. Blanco et al. (2008) found a 3-fold variation in baseline LC50 values for Cry1F using a diet overlay method, while Ali and Luttrell
(2007) found only a 2-fold variation in mean LC50 values for Cry2Ab2 across tested
laboratory and field populations collected from 2002 to 2005 using a diet incorporation method.
In the related heliothine species H. zea the pattern of susceptibility has been reported to be more varied. Ali and Luttrell (2007) found H. zea LC50 values for Cry2Ab2
varied up to 37-fold in eight laboratory, 10 laboratory-cross and 64 field populations collected from 2002 to 2005, and only 3-fold across all laboratory and field populations based on mean LC50 values. Helicoverpa zea susceptibility to Cry1Ac
(MPV II formulation) varied over 500-fold in five laboratory, nine laboratory-cross and 57 field populations collected from 2002 to 2004 and relative susceptibility appeared to have decreased in comparison with earlier collections in 1992 and 1993 (Luttrell et al., 1999; Ali et al., 2006). This reduced susceptibility has led to the suggestion that H. zea has evolved field resistance to Cry1Ac (Bt cotton), although there have been no reports of widespread control failures or changes in efficacy of Bt cotton (Tabashnik et al., 2008a). There has also been some discussion among researchers over whether this conclusion meets the classification of field-evolved resistance (Moar et al., 2008; Tabashnik et al., 2008b).
Analysis of published monitoring data for five other major lepidopteran pests targeted by Bt crops concluded that field-evolved resistance had not occurred to Cry1Ac (Bt cotton) in H. armigera, H. virescens and P. gossypiella and not to Cry1Ab (Bt corn) in O. nubilalis, and S. nonagrioides (Tabashnik et al., 2008a).
In the present study, the LC50 and MIC50 values for Cry1Ab and Cry1Ac were similar.
Vip3A toxicity was up to 134- and 300-fold lower compared with Cry1A toxins based on LC50 and MIC50 values respectively. The lower toxicity for Vip3A could be due to
the flux through pores may differ from that of Cry1A toxins (Lee et al., 2003; Jackson
et al., 2007). Jackson et al. (2007) reported that H. virescens Vip3A toxicity was on average 110-fold lower than that of Cry1Ac in a laboratory susceptible population (YDK) using the diet incorporation method. The lower toxicity of Vip3A may also be a result of their lack of specificity to gut cells, due to the prevalent nature of their production during vegetative growth (section 2.1.3). In contrast, the H. armigera ANGR laboratory population using a surface application bioassay method found that Vip3A and Cry1Ac toxicities were not significantly different and Cry1Ab was less toxic (Liao et al., 2002).
Cry1Ac and Vip3A expression levels in Bt cotton have been shown to decrease in various plant parts throughout the growing season, potentially reducing efficacy to target pests (Greenplate, 1999; Adamczyk et al., 2001b; Olsen et al., 2005; Llewellyn
et al., 2007), and Cry1Ac expression was found to be greater in terminal foliage than
in fruiting structures (Greenplate, 1999). Observed differences in H. virescens larval survival among Bt cotton expressing Vip3A could be due to protein expression variation in different plant structures and although they would not likely survive to pupation, larval feeding may cause economic damage (Bommireddy and Leonard, 2008). Information on the effect of larval stage of H. virescens on susceptibility to Vip3A and Cry1Ab may thus help to predict the possible impact of declining expression of Bt toxins in the plant with age and/or variation in expression of Bt toxins in the plant on the survival of H. virescens larvae.
In the present study, the LC50 values for Vip3A were found to be similar against 1st,
2nd and 3rd instar larvae of the NCSU and the field-derived population WF06 of H.
virescens. Fourth instar larvae of the NCSU population had approximately 3-fold
reduced susceptibility while for WF06, the tolerance of the 4th instar larvae to Vip3A was at least 27-fold greater than the earlier instars. MIC50 estimates showed that there
was greater inhibition of 1st instar development compared with later instars in both populations. Thus, larval age effects on susceptibility to Vip3A indicated that higher concentrations are needed against 4th instar larvae, and that there is greater inhibition of 1st instar development compared with later instars.
First, 2nd, 3rd and 4th instar larvae of the NCSU population all had similar susceptibilities to Cry1Ab based on LC50 and MIC50 values. Only 1st and 4th instar
larvae showed significant effects due to moult inhibition. Thus, larval age had little effect on susceptibility to Cry1Ab indicating that the dose of toxin doesn’t have to be greater to kill or inhibit development of later instar larvae.
As larval age increases so does diet consumption, and although the toxicity of Vip3A and Cry1Ab were similar between some larval instars, differences in tolerance were present as later instar larvae consumed more diet than earlier instars (personal observation). This increased tolerance with 4th instar larvae may be a result of increased effectiveness of cell-mending or a more effective immune response in later instar larvae (Loeb et al., 2001; Heckel et al., 2007). Difference in tolerance demonstrates that later instar larvae would potentially cause more damage in a field crop situation (Liao et al., 2002).