A gene for social immunity in the burying beetle Nicrophorus vespilloides?

Some group-living species exhibit social immunity, where the immune system of one individual can protect others in the group from infection. In burying beetles this is part of parental care. Larvae feed on vertebrate carcasses which their parents smear with exudates that inhibit microbial growth. We have sequenced the transcriptome of the burying beetle Nicrophorus vespilloides and identified six genes that encode lysozymes – a type of antimicrobial enzyme that has previously been implicated in social immunity in burying beetles. When females start breeding and producing antimicrobial anal exudates, we found that the expression of one of these genes was increased by ∼1000 times to become one of the most abundant transcripts in the transcriptome. We conclude that we have likely identified a gene for social immunity, and that it was recruited during evolution from a previous function in personal immunity.


Introduction 46 47
Insects occupy some of the most microbe-rich environments in nature and have 48 evolved diverse immunological defences to overcome the challenge that microbes 49 pose to their fitness [1,2]. In some group-living species, individuals are selected to 50 defend other individuals, as well as themselves, from potential pathogens. This is 51 social immunity in the broad sense, and it is seen in transient animal societies such as 52 animal families as well as more permanent animal societies such as the eusocial 53 insects and group-living primates [3]. Social immunity can take a range of forms, 54 from the collective behaviour that causes social fever in bees, to the production of 55 antibacterial substances by parents to defend offspring or a breeding resource [2][3][4]. 56 Yet, while the mechanisms underlying personal immunity in insects are increasingly 57 well-described [5,6], relatively little is known about the mechanisms underlying social 58 immunity. Nor is it clear whether social immune function might have originally been 59 derived from personal immune function. 60 61 In burying beetles (Nicrophorus spp), social immunity is a vital part of 62 parental care. These insects breed on small vertebrate carcasses which they shave, roll 63 into a ball and smear with anal exudates. These exudates have strong antimicrobial 64 properties [7,8] and promote larval survival [9]. The strength of antimicrobial activity 65 in anal exudates is proportional to the perceived microbial threat but increasing levels 66 of antimicrobial activity comes at a fitness cost to adults [10] and trades-off against 67 personal immunity [11]. Antimicrobial activity in the anal exudates is thus carefully 68 modulated. It is virtually non-existent in non-breeding individuals [8], is induced by 69 reproduction and the presentation of a carcass [8] and reaches its maximum strength 70 when the larvae arrive at the carcass shortly after hatching in the soil surrounding the 71 carcass [11]. 72 73 How has social immunity evolved in the burying beetle? One hypothesis is 74 that elements of the personal immune response have been recruited to control the 75 microbiota in the wider environment. Lysozymes, which are enzymes that can kill 76 bacteria by hydrolysing structural polysaccharides in their cell walls, are a likely 77 candidate because they are ubiquitous in nature and have key roles in personal 78 immunity [5,12]. In insects that feed on microbe-rich resources (e.g. Drosophila, 79 house-fly), lysozymes in the gut are thought not only to have an immune function but 80 also to digest bacteria [13,14]. Perhaps in burying beetles, lysozymes that were 81 originally confined to the gut are now exuded and applied to the carcass to limit 82 microbial growth during reproduction. Supporting this hypothesis is the finding that a 83 key active antimicrobial substance in the burying beetle's anal exudates has 84 lysozyme-like properties [8,9]  The beetles used in this experiment were bred in 2014 and descended from field-97 collected beetles trapped earlier that year from two sites in Cambridgeshire, UK. The 98 field-collected beetles were interbred to create a large, genetically diverse population. 99 This population was maintained with full parental care and no inbreeding for several 100 generations before the start of this experiment. 101

102
We examined the transcriptional response to breeding in N. vespilloides by comparing 103 the transcriptional profiles of a breeding female beetle and a non-breeding female of 104 the same age. We focused on females alone, because our previous work suggests that 105 they contribute more to social immunity than males [1]. Prior to each treatment, 106 beetles were given a small meal of minced beef as part of the usual protocol for beetle 107 husbandry in the lab. The "breeding" treatment initially consisted of 3 female-male 108 pairs of beetles. Each pair was placed in a breeding box with soil and a thawed mouse 109 carcass (10-16 g). These boxes were then put in a dark cupboard to simulate 110 underground conditions and the beetles were allowed to mate and begin preparing the 111 carcass. Forty-eight hours after pairing, at peak antimicrobial activity in the anal 112 exudates [2], we removed the female from each breeding box and placed them 113 individually in small plastic boxes (box dimensions, length x width x depth: 12 cm x 8 114 cm x 2 cm) where they remained for approximately 1 hour before being killed and 115 dissected. The "non-breeding" treatment consisted of 3 females that were treated in 116 exactly the same way as the "breeding" treatment except that the non-breeding 117 females were placed individually in a breeding box that did not contain a mouse 118 carcass. This was repeated on two occasions to generate 6 breeding and 6 non-119 breeding beetles. Individual beetles were euthanized with CO 2 and immediately 120 dissected to remove the gut. We focused on gut tissue because this is where the anal 121 exudates are produced. To estimate transcript abundance, we aligned reads separately from each library onto 154 the combined-read transcriptome assembly using the short read aligner bowtie 155 [6]. Abundance estimates were then produced using RSEM [7]. These steps are 156 combined into a single perl script bundled with Trinity, 157 align_and_estimate_abundance.pl. In further analyses we used estimates 7 for differential expression analysis. Differentially expressed transcripts were 161 identified using the Trinity scripts run_DE_analysis.pl and 162 analyze_diff_expr.pl with default settings. In a very small number of cases 163 it was clear that alternative haplotypes of a gene had been split into two genes during 164 the assembly and this gave a false signal of differential expression. The avoid this we 165 identified all the genes whose predicted peptides were >98% identical to another gene 166 using CD-HIT [8] and excluded them from the analysis. 167 168 Peptide and domain prediction 169 170 Trinity assembles nucleotide reads into nucleotide transcripts, and as such candidate 171 peptide sequences must be predicted post-hoc (Supplementary Figure S1). Peptide 172 predictions were generated from the combined read assembly using Transdecoder 173 [5] and the standard protocol for peptide prediction. Any transcript that did not 174 encode a predicted peptide was removed from our assembly. Differential expression of lysozymes was verified by quantitative PCR. We 186 synthesised cDNA using Promega Go-script® reverse transcriptase following the 187 standard conditions using 1ul RNA template and incubating at 25C for 5 minutes, 50C 188 for 50 minutes and 70C for 15 minutes. PCR primers were designed that amplify the 189 six lysozymes (all three lys1 isoforms were amplified by a single primer pair) and the 190 reference gene actin5C (Table S1). The quantitative PCR was performed using SYBR 191 green using the SensiFAST SYBR Hi-ROX Kit with a 10 ul reaction volume (2 ul 8   194   195 196

Results 197
The burying beetle transcriptome 198 To allow us to investigate the transcriptional response in the guts of burying beetle 199 when they breed, we first sequenced their transcriptome from the gut of a single 200 breeding and non-breeding beetle, combined the sequence reads and then assembled 201 them de novo. This process resulted in 11290 genes that encoded 26378 different 202 transcripts. This suggests that we sequenced the majority of genes in the genome, as 203 the exceptionally well-annotated Drosophila genome contains 13920 protein coding 204 genes encoding 30443 transcripts (Flybase release 6). As the guts we used for the 205 RNA extraction might contain RNA from the mouse the beetles were feeding on, or 206 nematode parasites, we used Blast to search for the most similar sequence in the Mus 207 musculus, C. elegans, Drosophila melanogaster and Tribolium castaneum genomes. 208 The top hit of 91% of the genes was another insect (Drosophila or the beetle 209 Tribolium), suggesting the levels of contamination were low ( Figure 1A). 210 211

Many genes are strongly upregulated in the guts of breeding beetles 212
By mapping reads from the breeding and non-breeding beetles to the transcriptome, 213 we found that there was a strong transcriptional response in the breeding beetles 214 ( Figure 1B). Among the most significantly differentially expressed genes (p<10 -20 ), 215 90% were upregulated in the breeding beetles ( Figure 1B; N=42, 95% binomial CI: 216 77-97%). The magnitude of these changes in transcription was often large -on 217 average the expression of these 42 most significantly differentially expressed genes 218 changed by nearly 1000 times (mean log 2 (fold change)=9.96). Furthermore, some of 219 the most strongly differentially expressed genes also had the highest total levels of 220 expression in our transcriptome ( Figure 1B). 221

A lysozyme is highly expressed in breeding females 223
We identified lysozymes by searching for the conserved LYZ1 domain, which 224 contains the active site of C-type lysozymes. Using this approach we identified six 225 lysozymes (Figure 2A). These ranged in size from 103-214 amino acids, which is 226 within the typical size range of insect lysozymes. 227

228
To identify the gene that may be responsible for the antimicrobial activity of the anal 229 exudate of breeding females we compared the expression of the six lysozyme genes in 230 breeding and non-breeding females in the whole transcriptome data. Five of the genes 231 had similar expression levels in breeding and non-breeding beetles, while Lys6 was 232 massively upregulated -the expression level in breeding females was 1409 times 233 greater than in non-breeding females ( Figure 1B; log 2 (fold change)=10.46, p<10 -26 ). 234 In the breeding beetles Lys6 was the 14 th most abundant transcript in the entire 235 transcriptome, while in the non-breeding beetles it was only the 5967 th most abundant. 236

237
We replicated this result using quantitative PCR to measure the expression of the 238 lysozymes across six breeding and six non-breeding females ( Figure 2B). In the non-239 breeding females the different lysozymes all had similar levels of expression. As was 240 the case in the transcriptome analysis, Lys6 was strongly upregulated in breeding 241 females, with an average expression level that was 860 times than non-breeding 242 beetles (t =12.6, df = 9.99, p <10 -07 ). The expression of the five remaining lysozymes 243 was unaltered in the breeding females. Our analyses indicate that breeding induces a very strong transcriptional response in 248 female burying beetles, causing substantial upregulation of just one lysozyme gene 249 (Lys6) in their gut tissues relative to non-breeding females. This correlates with our 250 earlier phenotypic observations that the antimicrobial properties of the anal exudates 251 in females are dramatically elevated after the presentation of a carcass and the onset 252 of reproduction [8,11]. Since none of the other 5 lysozyme genes that are expressed 253 in the gut were upregulated during reproduction, it suggests that upregulation of Lys6 254 causes at least some of the change in the exudates' antimicrobial properties during 255 breeding. In addition, it provides further evidence to support the hypothesis that 256 burying beetles have recruited a component of their personal immune system, namely 257 lysozyme, to play a major role in social immunity. 258 259 Does this mean we have therefore found a gene for social immunity in the burying 260 beetle N. vespilloides? We cautiously suggest that the answer to this question is likely 261 to be yes. Previous work has identified lysozyme-like activity in the antimicrobial 262 anal exudates [8,9]. Given the very high levels of Lys6 expression were only detected immunity. The finding that a lysozyme has a role in social immunity would be 265 unsurprising. These enzymes provide a broad-spectrum defence against microbes in 266 their environment, and they are secreted onto external surfaces that are vulnerable to 267 infection, such as the gut, eyes, mucous membranes and respiratory tract [12]. It may 268 therefore be straightforward to recruit lysozymes to social immune functions. 269 Nevertheless, further experiments are now needed to confirm that the protein encoded 270 by this gene is present in the anal exudates and that it is indeed functioning in social 271 immunity. 272 273 It might be argued that bacteria form a key part of the diet of breeding burying beetles 274 or their larvae, but not of non-breeding burying beetles. Thus, a possible alternative 275 interpretation of our data is that Lys6 primarily serves a digestive function, rather than 276 an immune function, as has been suggested for the lysozymes expressed in housefly 277 or Drosophila guts. However, we think this alternative interpretation is unlikely as 278 behavioural evidence suggests that beetles prefer to feed on meat rather than on the 279 microbes living on the meat [7]. Furthermore, beetles in both treatments were given a 280 similar meat-based diet, whether they bred or not, which suggests that upregulation of 281 Lys6 in the breeding beetles was not induced simply by consuming mouse flesh. Thus, 282 although at this stage we cannot rule out the possibility that Lys6 plays some minor 283 role in digestion, this is unlikely to be its sole or even primary function. 284

285
In summary, we suggest that we have found a gene (Lys 6) for social immunity in the 286 burying beetle and that it was recruited from personal immune function in the 287 evolutionary past. The challenge for future work is to determine how this gene's 288 function is integrated with other components of the social immune system to influence 289 the microbial community on the burying beetle's carcass breeding resource. 290