DCI 2036 No.

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27 September 2013

Developmental and Comparative Immunology xxx (2013) xxx–xxx

1

Contents lists available at ScienceDirect

Developmental and Comparative Immunology

journal homepage: www.elsevier.com/locate/dci

2 Short communication
6
4 Antimicrobial peptides play a functional role in bumblebee
7
5 anti-trypanosome defense
8 Q1 Soni Deshwal, Eamonn B. Mallon ⇑
9 Q2 Biology Department, University of Leicester, Leicester LE1 7RH, UK

10
11
a r t i c l e i n f o a b s t r a c t
1
2 3
5
14 Article history: Bumblebees, amongst the most important of pollinators, are under enormous population pressures. One 26
15 Received 25 July 2013 of these is disease. The bumblebee and its gut trypanosome Crithidia bombi are one of the fundamental 27
16 Revised 10 September 2013 models of ecological immunology. Although there is previous evidence of increased immune gene expres- 28
17 Accepted 12 September 2013
sion upon Crithidia infection, recent work has focussed on the bumblebee’s gut microbiota. Here, by 29
18 Available online xxxx
knocking down gene expression using RNAi, we show for the first time that antimicrobial peptides 30
(AMPs) have a functional role in anti-Crithidia defense. 31
19 Keywords:
Ó 2013 Elsevier Ltd. All rights reserved. 32
20 Crithidia bombi
21 Antimicrobial peptides
22 RNAi
23 Gut microbiota
24
33
34
35 1. Introduction 2000), and the ability to learn floral cues is impaired in infected 60
workers (Gegear et al., 2006). The virulence (i.e. parasite induced 61
36 Bumblebees are among the most important wild and agricul- host-death) of the parasite is condition-dependent. For example, 62
37 tural pollinators with, for example, 25 major crops grown within mortality rates of infected bumblebees increases by up to 50% after 63
38 the E.U. pollinated by bumblebees (Cameron et al., 2011; Corbet 10–15 h under starvation conditions, relative to non-infected, 64
39 et al., 1991). Throughout the northern hemisphere, bumblebee starved controls (Brown et al., 2000). 65
40 populations have being decreasing precipitously (Cameron et al., Compared to the extensive ecological understanding we have of 66
41 2011; Goulson et al., 2005). Although this is probably the result the B. terrestrisC. bombi system, its dynamics at the molecular 67
42 of multiple factors, a key component is disease (Cameron et al., level are only beginning to be revealed. Standing levels of prophe- 68
43 2011). noloxidase increase upon infection with C. bombi (Brown et al., 69
44 The bumblebee (Bombus terrestris) is an annual, eusocial insect 2003a). Several quantitative trait loci are linked to immune 70
45 and host to a variety of parasites including the trypanosomatid, defence against this parasite (Wilfert et al., 2007). Four studies 71
46 Crithidia bombi (Trypanosomatidae, Zoomastigophorea) (Lipa and have looked at differences in bumblebee gene expression upon 72
47 Triggiani, 1980). The bumblebee/Crithidia system is one of the infection (Brunner et al., 2013; Riddell et al., 2009, 2011; Schlüns 73
48 main models for the study of ecological immunology (Schmid- et al., 2010). Chief among the genes found have been antimicrobial 74
49 Hempel, 2001). C. bombi colonizes the bumblebee’s gut and begins peptides (AMPs). 75
50 releasing transmission stages into the faeces 2–3 days after infec- As well as the above evidence of AMP expression in response to 76
51 tion. Parasitemia peaks around 7–10 days into the infection Crithidia infections, there is growing evidence of expression of 77
52 (Schmid-Hempel and Schmid-Hempel, 1993). C. bombi prevalence AMPs and their efficacy in response to medically important trypan- 78
53 in B. terrestris populations ranges from 10% to 30% but can be much osomes in vector insects (McGwire and Kulkarni, 2010). AMPs at- 79
54 higher (80%) (Shykoff and Schmid-Hempel, 1991) with increased tack trypanosomes by numerous mechanism with most focussed 80
55 inter-colony transmission as the season progresses (Imhoof and on the cell membranes of trypanosomes (Harrington, 2011; 81
56 Schmid-Hempel, 1999). A variety of fitness effects are seen at the McGwire and Kulkarni, 2010; McGwire et al., 2003). 82
57 colony level due to infection: queens have reduced success in col- However the role of the immune system in Crithidia defense has 83
58 ony founding (Brown et al., 2003b), colonies have smaller worker been called into question. Recently it has been shown that gut mic- 84
59 populations and produce fewer sexual offspring (Brown et al., robiota has a central role in the bumblebee’s response to Crithidia 85
(Koch and Schmid-Hempel, 2011). Although there is data on 86

Q3 ⇑ Corresponding author. Tel.: +44 01162523488; fax: +44 116 2523333. transcription supporting a role for AMPs in Crithidia defense, they 87

E-mail addresses: sd318@student.le.ac.uk (S. Deshwal), ebm3@le.ac.uk (E.B.

have never been shown to have a functional role. Along with the 88
Mallon). confusion of the role of the immune system in anti-Crithidia 89

0145-305X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dci.2013.09.004

Please cite this article in press as: Deshwal, S., Mallon, E.B. Antimicrobial peptides play a functional role in bumblebee anti-trypanosome defense. Dev.
Comp. Immunol. (2013), http://dx.doi.org/10.1016/j.dci.2013.09.004
 DCI 2036 No. of Pages 4, Model 5G
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2 S. Deshwal, E.B. Mallon / Developmental and Comparative Immunology xxx (2013) xxx–xxx

90 defense generally (Hauton and Smith, 2007; Koch and Schmid- Nautilus (11). Five bees were used for cell counts (each RNAi group) 148
91 Hempel, 2011), this presents a need for a clear display of the func- and six for qPCR (RNAi Defensin and RNAi Nautilus only). Three 149
92 tional role of AMPs in response to Crithidia. Here, we knocked uninfected and elution buffer injected bees was used as control 150
93 down Defensin and Abaecin expression and show that bumblebees samples for qPCR. 151
94 thus treated, are less able to defend themselves against Crithidia After injections, bees were kept separately and fed with 50% di- 152
95 compared to controls. luted nectar and pollen for 24 h. Bees were then fed 20 ll of a 153
1000 cells/ll Crithidia inoculum (8 bees each for infected RNAi 154
Defensin and RNAi Nautilus groups and 5 bees for RNAi Abaecin) 155
96 2. Material and methods
or 20 ll of meliose solution (3 bees each for uninfected RNAi 156
Defensin and RNAi Nautilus groups only). Bees used for cell counts 157
97 2.1. Bumblebees and Crithidia
were left for a further 5 days. Crithidia cells were counted in 10 ll 158
samples of each bee’s faeces using a haemocytometer. Bees used 159
98 A B. terrestris colony was obtained from Kopert Biosystems UK.
for qPCR were sacrificed 24 h after infection (Riddell et al., 2011). 160
99 Bumblebees were maintained in standard conditions: 27 °C, 60%
100 humidity under red light. They were fed 50% meliose solution
101 and pollen ad libitum. C. bombi was collected from wild queens of 2.4. Quantatitive PCR (qPCR) 161

102 B. terrestris. Queens were captured from the botanical gardens of

103 the University of Leicester in March 2013. Faeces was collected Total RNA was extracted from whole bees using RNAeasy mini 162

104 and examined for presence of C. bombi cells. 10 colony workers kit (Qiagen). cDNA was synthesized from RNA template by using 163

105 were infected with a mixture of faeces and meliose to produce C. Tetro cDNA Synthesis kit (Bioline). Onelg of RNA template, 164

106 bombi cells for further experiment. 10 mM of dNTP mix, 5X RT buffer, Ribosafe RNAse inhibitor 2 ll 165
and tetro Reverse Transciptase 2 ll were used and the reaction vol- 166
ume was made up to 40 ll with DEPC water. A combination of Oli- 167
107 2.2. RNA interference
godT and Random Hexamers primers (1 ll each) was used to 168
improve the reverse transcription efficiency of RNA templates. 169
108 The sequences of Defensin (FJ161699.1), Abaecin (GU233780)
The prepared samples were incubated for 10 min at 25 °C followed 170
109 and the Nautilus (X56161.1) genes were retrieved from NCBI. Dro-
by 45 °C for 30 min. The reaction was terminated by incubating 171
110 sophila Nautilus shows no similarity to the genes of B. terrestris and
samples at 85 °C for 5 min. 172
111 hence is used as a RNAi control. All the primers were designed
2 ll of cDNA was added to a qPCR reaction mix containing 10 ll 173
112 using Primer3 (version 0.4.0). To amplify Defensin and Abaecin,
2X SYBR Green JumpStart Taq ReadyMix (Sigma–Aldrich, UK) 174
113 DNA was extracted from bumblebee using Qiagen DNA mini kit.
7.6 ll ddH2O and 0.4 ll of the forward and reverse primer mix 175
114 For Nautilus, w118 flies were kindly provided by Dr. Ezio Rosato,
(5 lm each). Two sets of primers were used, Defensin, and Actin 176
115 Department of Genetics, University of Leicester. Polymerase chain
(XM_003394442) (Table S1). Actin was used as an endogenous 177
116 reaction (PCR) was performed using 1 ll of DNA template,
control (reference gene). Actin has previously been used in this role 178
117 200 lM of each primer, 2.5 lM of MgCl2, and York Bioscience
in gene expression studies of bumblebee immunology (You et al., 179
118 Taq DNA Polymerase (dNTPs and buffer included) in a 25 ll reac-
2010). For each sample three technical replicates were run. qPCR 180
119 tion using PCR conditions 95 °C for 3 min and 35 cycles of 95 °C
was carried out on a PTC-200MJ research machine thermal cycler 181
120 for 30 s denaturation, 30 s annealing, 72 °C for 30 s extension.
with a Chromo 4 continuous fluorescence detector, using standard 182
121 The annealing temperature was varied according to the primers
settings and Opticon software v4.7.97.A for Windows 2000 Profes- 183
122 used (Table S1). Amplified products were checked for size on 1%
sional. The qPCR was carried out using protocol: 95 °C for 4 min 184
123 agarose gel and bands extracted using WizardÒ SV Gel and PCR
followed by 40 cycles of 30 s for 95 °C denaturation, 30 s for 185
124 Clean-Up System.
60 °C annealing and 30 s products were checked products were 186
125 T7 promoter sequence 50 -TAATACGACTCACTATAGGGAGA-30
checked products were checked products were checked products 187
126 was added to the purified amplification products of the genes
were checked for 72 °C extension. The relative expression ratio 188
127 (Defensin and Nautilus) using PCR to generate a transcription
(Pfaffl, 2001) was calculated as 189
128 template. The primers were synthesized by adding T7 promoter se- 190
DC ptarget ðcontrolsampleÞ
129 quence to the 50 end of the gene specific primers. 100 lM T7 pro- ðEtarget Þ
130 moter-containing PCR primers (sense and antisense) were used in ratio ¼
ðEreference ÞDC preference ðcontrolsampleÞ 192
131 a single PCR reaction to generate transcription template for both
132 strands of the dsRNA. Since, the first cycles of PCR used only the where E is qPCR efficiency and Cp is the crossing point of each tran- 193
133 30 half of the primers, the gene-specific part, the annealing temper- script, target refers to the target gene, reference is Actin, and control 194
134 ature for the first 5 PCR cycles was 5 °C higher than the calculated is the uninfected, elution buffer injected samples. 195
135 Tm for the gene-specific region of the primer. After 5 cycles, the
136 annealing temperature for the entire primer (Table S1) was used 3. Results and discussion 196
137 for the following cycles: 95 °C for 30 s denaturation, 30 s annealing
138 and 72 °C for 30 s extension for 35 cycles. The bands were visual- There was a significant difference between treatments in levels 197
139 ized and extracted as above. 1 lg of DNA with T7 promoter of Crithidia infections (F2,12 = 28.95, p < 0.0001). RNAi Defensin 198
140 overhangs was used to construct dsRNA, in vitro, using MEGA- treated bees had higher levels of Crithidia infection compared to 199
141 scriptÒ T7 Kit (Invitrogen) as per manufacturer’s instructions. The RNAi Nautilus controls (Tukey HSD post hoc test: p = 0.0008). RNAi 200
142 concentration of dsRNA (RNAi) was determined at 260 nm using Abaecin bumblebees also have higher levels of Crithidia infection 201
143 a Nanodrop ND-1000 Spectrophotometer. compared to RNAi Nautilus controls (Tukey HSD post hoc test: 202
p < 0.0001). See Fig. 1. 203
144 2.3. RNAi injection and C. bombi infection To confirm that it was actually decreased immune gene expres- 204
sion that caused this increased parasitemia, we measured Defensin 205
145 Thirty bees in total were used in this experiment. Twenty-seven expression in our RNAi Defensin vs. RNAi Nautilus samples. Treat- 206
146 bees were injected (16.6 ll of elution buffer containing 1 lg of ment had a significant effect on Defensin expression levels 207
147 dsRNA) with either RNAi Defensin (11) RNAi Abaecin (5) or RNAi (F1,8 = 14.27, p = 0.0054). RNAi Defensin treated bees had lower 208

Please cite this article in press as: Deshwal, S., Mallon, E.B. Antimicrobial peptides play a functional role in bumblebee anti-trypanosome defense. Dev.
Comp. Immunol. (2013), http://dx.doi.org/10.1016/j.dci.2013.09.004
 DCI 2036 No. of Pages 4, Model 5G
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S. Deshwal, E.B. Mallon / Developmental and Comparative Immunology xxx (2013) xxx–xxx 3

Fig. 1. Boxplotsof levels of Crithidia infection for various RNAi treated bees.

Fig. 2. Boxplot of Defensin expression for infected and uninfected RNAi Defensin and RNAi Nautilus (control) treated bees.

209 levels of Defensin expression relative to RNAi Nautilus treated con- come of a Crithidia infection is not the result of the immune system 222
210 trols. This is true in both infected and uninfected groups. Infected or the gut microbiota but rather the interaction between the para- 223
211 RNAi Defensin treated bees had similar levels of Defensin expres- site, the gut microbiota and the host immune system (Castro et al., 224
212 sion as uninfected controls (RNAi Nautilus). See Fig. 2. As would 2012; Garcia et al., 2009; Weiss et al., 2013). This interaction will 225
213 be expected (Riddell et al., 2011), infection caused a significant in- be the focus of much future research. 226
214 crease in Defensin expression (F1,8 = 11.896, p = 0.00871) for both
215 RNAi Defensin and RNAi Nautilus groups.
216 We have shown that by knocking down AMP expression we in- 4. Conclusions 227
217 crease the infection levels of Crithidia. This is evidence for the
218 central role of the bumblebee immune system in the fight against We found that reducing expression of two AMPs (Defensin and 228
219 Crithidia. This seems to contradict recent findings that found a Abaecin) lead to an increase parasitemia of C. bombi in bumblebees. 229
220 central role for gut microbiota in the bumblebee’s defense against This is strong evidence that AMPs are powerful anti-trypanosome 230
221 Crithidia (Koch and Schmid-Hempel, 2011). It is clear that the out- agents. 231

Please cite this article in press as: Deshwal, S., Mallon, E.B. Antimicrobial peptides play a functional role in bumblebee anti-trypanosome defense. Dev.
Comp. Immunol. (2013), http://dx.doi.org/10.1016/j.dci.2013.09.004
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309

Please cite this article in press as: Deshwal, S., Mallon, E.B. Antimicrobial peptides play a functional role in bumblebee anti-trypanosome defense. Dev.
Comp. Immunol. (2013), http://dx.doi.org/10.1016/j.dci.2013.09.004