Genomic characterisation of an entomopathogenic strain of Serratia ureilytica in the critically endangered phasmid Dryococelus australis

Abstract

Between 2014 and 2019, unexpected mortalities were observed in a colony of Dryococelus australis, an endangered stick-insect kept at the Melbourne Zoo for a breeding and conservation program. Pure cultures of Serratia spp. were obtained from the haemolymph of moribund and recently deceased individuals. The combined bacteriological and histopathological observations suggested an infectious cause of these mortalities. Genotyping of Serratia sp. isolated from the insects and their environment revealed a predominant strain profile. A representative isolate, AM923, was entirely sequenced and compared to 616 publicly available Serratia spp. genomes, including 37 associated with insects. The genomes were distributed into 3 distinct groups, with 63% of the insect-associated isolates within a single clade (clade A) containing AM923, separated from most environmental/plant-associated strains (clade B) and human isolates (clade C). Average nucleotide identity and phylogenetic analyses identified AM923 as S. ureilytica and revealed similarities with putatively entomopathogenic strains. An experimental infection model in honey bees (Apis mellifera) confirmed the pathogenic potential of AM923. A urease operon was found in most insect isolates and a PCR assay, based on the ureB gene sequence, was used to confirm the presence of AM923 in experimentally infected bees. This species-specific PCR could be applied to detect entomopathogenic Serratia spp. in infected insects or their environment.

Fig 1. Pulsed field gel electrophoresis analysis of Serratia spp. isolated from haemolymph of captive D. australis between August and October 2014.

# Isolates (VW347 and VW348) from insects collected prior to this study and provided by Zoos Victoria. *Strains representative of the pulsotypes A and B and selected for complete genome sequencing. Molecular weight marker: lambda PFG ladder (New England Biolabs).

Fig 2. Dendrogram of PFGE profiles, pigmentation phenotypes and isolation sources.

Twenty two Serratia spp. isolated from D. australis. between August and October 2014 and Serratia spp. isolated from the insect environment in February 2015 were compared. Vertical bars represent restriction endonuclease cleavage fragments. # VW347 and VW348 represent isolates from insects collected prior to this study and provided by Zoos Victoria. Boxes indicate the two representative isolates selected for genome sequencing.

Fig 3. Cladogram of 131 complete Serratia spp. genomes.

An unrooted tree was constructed from the alignment of polymorphisms produced by REALPHY using RAxML (GTR model and Gamma parameter). Red nodes: S. marcescens, orange nodes: S. ureilytica, other Serratia species: shades of brown-green. D. australis isolates AM923 and AM1004 are indicated in blue and green, respectively.

Fig 4. Phylogenetic analysis of concatenated alignments of 14 housekeeping genes from 616 genomes of the genus Serratia.

Panel A: mid-rooted tree built from 322 non-identical sequences, including 256 S. marcescens, S. nematodiphila and S. ureilytica isolates, with RAxML and the GTR + Gamma substitution model. Bootstrap support values were inferred from 100 replicates and represented by branch thickness on the complete tree. Branches are drawn to scale and colour-coded according to the clade: red, clade A; brown-green, clade B; blue, clade C. Nodes are colour-coded according to the origin of the strain. Insets showing the groups containing the Melbourne Zoo isolates AM923 (Panel B) and AM1004 (Panel C) with bootstrap support values >50% indicated on the branches. The scale bars indicate the number of substitutions per site.

Fig 5. Genome map of strain AM923.

This map displays loci that were putatively acquired horizontally and CDSs predominantly found in the genomes of Serratia spp. isolated from insects. Features positioned in concentric circles, from outer to inner tracks as follows: 1–2, dark teal, forward CDSs; light teal, reverse CDSs; mauve, tRNA; yellow, rRNA; 3, dark red, predicted prophages (F), Integrative Conjugative Transposon (ICE); 4, red shades, putative HGT region predicted by Alien Hunter; 5, blue, CDSs predominantly found in insect isolates; 6, G+C%; 7, G+C skew. Inset, map of urease locus (top) and fructose dehydrogenase locus (bottom).

Fig 6. Kaplan-Meier survival curves in honey bees challenged with a live culture of S. ureilytica.

Strain AM923 (red), E. coli K12 (blue), or exposed to sterile PBS and the sucrose/water mix as uninoculated negative controls (dark grey). Semi-transparent zone indicates a 95% confidence interval.