Modelo de gestión para implementación ejecutiva del proyecto de regeneración del boulevard de santa clara 2017

The sucrose sensitivity of M. anisopliae-infected bees was tested for pollen-starved bees two, four and six days after inoculation with M. anisopliae. A frame of sealed brood was collected from colony 150 (low varroa loads: 0.1 mites/day drop count and 0 phoretic mites 28/06/12, see chapter 2.6) and newly emerged adult bees were collected as described previously (chapter 2.2.1). Bees were inoculated over the course of a week such that on the experimental day the same aged bees would either have been inoculated two, four or six days earlier (timings for the experiment in table 4.4). On each inoculation day, three groups of 40 bees were chilled and split into two groups of 20. One group was inoculated with 0.5g of a 1:30 concentration of M. anisopliae in sorbitol, the second was sham inoculated with 0.5g of sorbitol. This meant on each inoculation day, three groups of 20 M. anisopliae inoculated bee and three groups of 20 sorbitol inoculated bees were produced. Only ten bees of each treatment in each group were needed, but twice this number were inoculated in case some bees died. The bees were then maintained at 30o_{C in darkness and provided}

with pollen, 60% sucrose and water. Bees were starved of all food, but not water, for 12 hours prior to experimentation and secured in glass tubes with PVC tape as for the CPE experiments (see 4.2.1). Ten bees from each treatment (two, four and six days after inoculation/sham inoculation) were tested on each run of the experiment which was repeated three times. This meant that 60 bees were tested in each of the three runs, 180 bees in total.

86 Table 4.4:

Day Experimental procedure:

1 Brood frame collected from colony 150 2 Newly emerged adult bees collected

5 First group of 120 bees inoculated/sham inoculated 7 Second group of 120 bees inoculated/sham inoculated 9 Third group of 120 bees inoculated/sham inoculated 11 Bees tested for sucrose sensitivity in three runs

Table showing timings for the sucrose sensitivity experiment.

Each bee in turn was stimulated with a drop of water on a cotton wool bud touched to the antennae. Whether the bee extended her proboscis or not was recorded. This was then repeated with increasing concentrations of sucrose (0.1%, 0.3%, 1%, 3%, 10% & 30%) with 10 minute intervals between each concentration. The number of concentrations each bee responded to was counted as used as the ‘gustatory response’, this could range from 0 (the bee responded to none of the concentrations) to 7 (the bee responded to all six concentrations and water).

These data were analysed in GenStat® ₍₁₄th _{edition) using ANOVA analysis with the}

treatment structure to test the effect of infection with the fungus M. anisopliae and time since inoculation on gustatory response. The blocking structure took account of when each group of bees was tested and which groups of bees were inoculated together. The data did not require transformation.

4.3 Results:

4.3.1 Testing for differences in learning behaviour in young adult bees over the course of infection with M. anisopliae:

Learning in M. anisopliae-infected and sorbitol-inoculated (control) young adult bees was evaluated two, four and six days after inoculation. In each successive training trial more bees learnt the association, irrespective of treatment, so there was a positive trend in learning ability over time for all treatments (Repeated measures ANOVA: H0 = equal means

for each time point. The logit transformed means ranged from -0.048 for trial 2 to 0.45 for trial 6. SED = 0.072. F2.67, 48.0 = 12.76, P<0.001, see also Figure 4.2). This is to be expected

87 Figure 4.2:

Learning ability of young adult bees infected with M. anisopliae at 2, 4, and 6 days after inoculation.

The proportion of bees that learnt the CPE response is shown for each of the six training trials except the first in which no bees responded to the odour stimulus. Open shapes and dotted trend lines are M. anisopliae inoculated bees, filled shapes and continuous trend lines are sham (sorbitol) inoculated bees. Error bars show +/- standard error.

There was a significant effect of M. anisopliae infection; infected bees were more able to learn the association than uninfected bees (Repeated measures ANOVA: H0 = infected mean

= control mean. Infected mean = 0.25, control mean = 0.11. SED = 0.063. F0.67, 8.67 = 4.86,

P = 0.046). There was no significant effect of the time since infection, or any interaction between treatments over the course of the experiment.

4.3.2 Testing for differences in learning behaviour in young adult and forager bees infected with M. anisopliae:

Learning in M. anisopliae-infected and sorbitol-inoculated (control) forager and young adult bees was evaluated four days after inoculation and again, with each successive trial more of the bees had learned the association, irrespective of treatment (Repeated measures ANOVA: H0 = equal means for each trial. The logit transformed means ranged from -0.38 for trial 2

88 Figure 4.3:

Learning ability of forager and young adult bees infected with the fungus M. anisopliae 4 days after inoculation. Open shapes and dotted trend lines are M. anisopliae inoculated bees, filled shapes and continuous trend lines are sham (sorbitol) inoculated bees. Error bars show +/- standard error.

There was a suggestion that the forager bees were better able to learn than the young adult bees, although this was only significant at the 10% level (Repeated measures ANOVA: H0 = forager mean = young adult mean. Forager mean = -0.023, control mean = -0.11.

SED = 0.039. F0.61, 5.45 = 5.05, P = 0.051). There was also a significant interaction between

age and M. anisopliae infection; whilst infected young adult bees were better able to learn the association than uninfected controls (as in the previous experiment) (as in 4.3.3) the infected forager bees were less able to learn than uninfected controls (Repeated measures ANOVA: H0 = infected mean = control mean. Infected mean = -0.062,

control mean = -0.072. SED = 0.039. F0.61, 5.45 = 19.28, P = 0.002).

The bees from this experiment were analysed using Q-RT-PCR (see chapter 2.6). Five common bee diseases were detected; DWV, BQCV, SBV, Nosema apis and Nosema ceranae (table 4.5). Most of the bees tested positive for both Nosema species. This is likely to be because the bees were not treated against these pathogens. Several colonies failed in the winter following these experiments, potentially because of the high levels ofNosema spp. infection.

89 Table 4.5: BQCV DWV SBV N.apis N.ceranae Total tested 128 128 128 128 128 No. positive 75 126 49 105 106 No. negative 51 0 78 20 19 Inconclusive 2 2 1 3 3

Summary of PCR analysis. 128 bees from experiment 4.2.2 were analysed using RT-PCR based detection to identify the viral diseases and Nosema spp. present. Five diseases were detected, the viruses BQCV, DWV & SBV and both N.apis and N.ceranae. A few samples were inconclusive.

The multivariate analysis accounted for 95.04% of the variance within the data. There are four groups of bees that can be represented as circles equating to a 95% CIs (figure 4.4). Those four groups are: Forager bees that learned, forager bees that did not learn, young adult bees that learned and young adult bees that did not learn. The CIs around the groups that were unable to learn are larger than around those that did learn because fewer bees were unable to learn the association than those that did. The forager bees are separated from the young adult bees and the young adult bees that learned are separated from the young adult bees that did not.

Figure 4.4:

Canonical variance analysis including presence or absence of BQCV, SBV, N.apis & N.ceranae, age and whether the bees had learned the association in the sixth and final trial.

90

A bi-plot was produced to determine which factors were contributing to the separation of the data (figure 4.5). The direction of the lines produced on the biplot show the direction of data separation produced by each factor. The position and direction of the arrow point along the line is indicative of the strength and direction of the effect. The analysis suggests that the horizontal separation of the data was caused by BQCV, which was more likely to be found in the forager bees than in the young adult bees. The vertical separation, between the young adult bees that learned and those that did not, seems to be caused by a combination of SBV and N. ceranae, SBV having the greatest effect.

Figure 4.5:

Biplot from the canonical variance analysis including presence or absence of BQCV, SBV, N.apis &

N.ceranae, age and whether the bees had learned the association in the sixth and final trial.

Quantitative PCR analysis was done to quantify the absolute load of each pathogen in each bee. The DWV load for each bee, when plotted as a histogram, has a bimodal distribution such that there appear to be bees with a relatively high DWV load (centred around -9) or a relatively low DWV load (centred around 3) (see figure 4.6). This was not found for any of the other diseases detected (BQCV, SBV, N. ceranae or N. apis).

91 Figure 4.6:

Histogram showing the distribution of DWV load relative to the housekeeping gene -actin within the bees from experiment 4.2.3. The lower (or more negative) the relative load the more virus was present, see chapter 2.6.2.

REML analysis of the Q-RT-PCR and behavioural data (Table 4.6) showed that for some diseases there was a significant correlation between the disease load and age of the bee; the forager bees had higher levels of BQCV and DWV than the young adult bees. Also, for some diseases there was a significant interaction between age and disease (DWV and N. apis) but there was no effect of disease load on learning.

Table 4.6:

Disease DWV BQCV SBV N.ceranae N.apis

Learning F3,110.7 = 2.06 P = 0.109 F3,63 = 0.6 P = 0.616 F3,38 = 0.23 P = 0.877 F3,88.9 = 0.25 P = 0.861 F3,86.2 = 0.12 P = 0.948 Age F1,109.1 = 14.98 P = <0.001 F1,63 = 9.05 P = 0.004 F1,38 = 0.64 P = 0.43 F1,87.7 = 0.38 P = 0.541 F1,82.6 = 0.13 P = 0.723 Interaction F2,111 = 3.18 P = 0.045 F1,63 = 0.25 P = 0.621 F2,38 = 0.01 P = 0.991 F2,86.8 = 1.6 P = 0.207 F2,83 = 4.32 P = 0.016

Table showing REML analysis combining molecular and behavioural data for bees whose learning behaviour was tested after infection with the fungus M anisopliae. The relationships amongst each pathogen, the learning behaviour and the age of the bee were analysed. The only significant effects were on age (for DWV and BQCV) and the interaction between age and learning (for DWV and N. apis).

4.3.3 Learning behaviour of pollen-starved forager bees infected with M.