Examining the role of neuronal TcSNMPs for the detection of fatty acid derivatives

To test an involvement of TcSNMP1a and TcSNMP1d in the detection of compounds that are not produced by T. castaneum but might be relevant for it, also other odors were analyzed. All known odors that require a functional SNMP for

normal response kinetics are fatty-acid derivatives (Benton et al., 2007; Gomez-Diaz et al., 2013; Pregitzer et al., 2014; Ronderos et al., 2014). Hence, fatty food odors and in addition pheromone like substances were tested for the requirement of TcSNMP1a and TcSNMP1d on their detection.

First, typical fatty food odors were analyzed. Phillips et al. (1993) showed that T.

castaneum is attracted by wheat germ oil (WGO). It was hypothesized that older and damaged grain with a higher fatty acid content reflects the habitat preference of T. castaneum. For that purpose, antennal responses of RNAi treated beetles towards WGO and another compound of wheat, ß-ionone, were tested using EAG (Nixon, 1994).

The averaged EAG responses of seven beetles each and following statistical calculations showed that knockdown of TcSNMP1a and TcSNMP1d led to significantly decreased antennal responses in female beetles to wheat germ oil and ß-ionone for all tested odor concentrations (figure 4.15, right).

Also male beetles showed reduced EAG responses to WGO and ß-ionone after injection with dsRNA targeting transcripts of TcSNMP1a and TcSNMP1d (figure 4.15 left), but not as strongly reduced as in female beetles (figure 4.15, right). For both sexes and both odorants disruption of TcSNMP1d function had a stronger effect to all tested situations than knockdown of TcSNMP1a. Statistical analysis showed that this strong effect is highly significantly different when compared with the control beetles (figure 4.15).

The EAG measurements showed that SNMP1a dsRNA and SNMP1d dsRNA injected beetles of both sexes did not show a dose dependent response to different wheat germ oil concentration as obtained from the DsRed dsRNA injected control beetles (figure 4.15 upper row). These constant EAG values of the knockdown beetles are at a lower level than the response of the control group to the much diluted WGO (10^-4 dilution, figure 4.15).

Taken together, knockdown of TcSNMP1a and TcSNMP1d transcripts led to decreased antennal responses towards the food odors WGO and ß-ionone. Here, female TcSNMP1a knockdown beetles showed a stronger effect. Generally, TcSNMP1d knockdown led to a stronger effect than TcSNMP1a knockdown in all beetles.

Figure 4.15 EAG responses of RNAi treated T. castaneum to fatty food odors

Shown are EAG responses of indicated beetles (n=7) and odor dilutions, plotted as mean and s.e.m (standard error of the mean). EAG responses were measured by Karthi Balakrishnan (Forest Zoology and Forest Conservation, University Göttingen), after application of wheat germ oil (WGO) and ß-ionone diluted in silicon oil. The asterisks indicate statistically significant differences between control beetles that were injected with dsRNA against DsRed (dsred, grey bars) and knockdown beetles injected with dsRNA against TcSNMP1a (snmp1a, green bar) or TcSNMP1d (snmp1d, yellow bar), respectively. Statistical significance was calculated using ANOVA with Bonferroni post hoc test: *p< 0.05;**p <0.01;***p<0.001; n.s. no significant difference. Line ends indicate experimental groups showing significant differences as indicated.

To test whether TcSNMP1a and TcSNMP1d are required for the detection of fatty, pheromone like plant odors, geraniol and cis-3-hexenol were analyzed. Geraniol is a component of many plant oils. It has been shown that this odor is attractive to the japanese beetle Popillia japonica (Fleming, 1969) and that it is an ingredient of the Nasonov pheromone of the honey bee Apis mellifera. This highly attractive odorant blend is used by bees to find the entrance to their colony or flowers with nectar (Schmidt, 1994; Williams et al., 1981). The fatty-acid derivative cis-3-hexenol is a typical green-leaf-volatile (Abdullah and Butt, 2015).

To test for an involvement of TcSNMP1a and TcSNMP1d in the detection of geraniol and cis-3-hexenol, an analogous EAG approach as described above (chapter 4.3.1) was performed. Comparison of the obtained EAG responses of TcSNMP1a dsRNA injected male beetles (figure 4.16, left, green bars) with the EAG

responses of the control beetles (figure 4.16, left, grey bars) revealed that the measured sum potentials were in a similar range. Statistical analysis showed that there was no significant difference between TcSNMP1a dsRNA and DsRed dsRNA injected males and hereby confirming that finding. In contrast to that, TcSNMP1a dsRNA injected female beetles showed an effect (figure 4.16, right). In particular, for all tested concentrations of geraniol and cis-3-hexenol, the EAG responses were significantly decreased compared to the female control beetles.

Loss of TcSNMP1d function resulted in lower EAG responses for all tested concentrations of geraniol and cis-3-hexenol compared to control beetles (figure 4.16). This reduction was found for male and female beetles and is highly significant. In general, the effect of dsRNA injection against TcSNMP1d results in a more severe decrease of EAG responses than RNAi against TcSNMP1a.

In summary, knockdown of TcSNMP1a and TcSNMP1d led to decreased antennal responses towards the plant odors geraniol and cis-3-hexenol. Again, as for the other tested odors (chapter 4.3.1 and 4.3.2), the female beetles showed a stronger effect to theTcSNMP1a knockdown than the male beetles. Furthermore the TcSNMP1d knockdown led to a stronger effect than TcSNMP1a knockdown in both sexes.

Figure 4.16 EAG responses of RNAi treated T. castaneum to plant odors

Shown are EAG responses of indicated beetles (n=7) and odor dilutions, plotted as mean and s.e.m (standard error of the mean). EAG responses were measured by Karthi Balakrishnan (Forest

Zoology and Forest Conservation, University Göttingen), after application of geraniol and cis-3-hexenol diluted in silicon oil. The asterisks indicate statistically significant differences between control beetles that were injected with dsRNA against DsRed (dsred, grey bars) and knockdown beetles injected with dsRNA against TcSNMP1a (snmp1a, green bar) or TcSNMP1d (snmp1d, yellow bar), respectively. Statistical significance was calculated using ANOVA with Bonferroni post hoc test: *p< 0.05;**p <0.01;***p<0.001; n.s. no significant difference. Line ends indicate experimental groups showing significant differences as indicated.