Reports
giraulti or N. longicornis result in F2
hybrid males exhibiting up to ~90%
lethality during larval development
while N. giraulti and N. longicornis
hybrids only exhibit ~8% hybrid lethality (6–10).
In this study, we scored the average
number of hybrid and non-hybrid eggs,
larvae, pupae and adults produced by
Robert M. Brucker1* and Seth R. Bordenstein1,2*
Nasonia females (N = 48 females each
1
2
parasitizing one S. bullata host per
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA. Department of
developmental period) and determined
Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN 37232, USA.
that 78% of hybrid lethality occurs
*Corresponding author. E-mail: [email protected] (R.M.B.); [email protected] (S.R.B.)
between the first and the fourth instar
larval stages (Fig. 1A). There was a
Although the gut microbiome influences numerous aspects of organismal fitness,
slight asymmetry in the number of
its role in animal evolution and the origin of new species is largely unknown. Here
surviving of N. vitripennis/N. giraulti
we present evidence that beneficial bacterial communities in the guts of closelyand N. giraulti/N. vitripennis hybrids
related species of the genus Nasonia form species-specific, phylosymbiotic
(F2 hybrid genotype denotes grandfaassemblages that cause lethality in interspecific hybrids. Bacterial constituents and
ther/grandmother) but the difference
abundance are irregular in hybrids relative to parental controls, and antibiotic
was not statistically significant (P =
curing of the gut bacteria significantly rescues hybrid survival. Moreover, feeding
0.36, Mann Whitney U test). In conbacteria to germ-free hybrids reinstates lethality and recapitulates the expression of
trast, hybrids of the younger sister speinnate immune genes observed in conventionally-reared hybrids. We conclude that
cies N. giraulti and N. longicornis
in this animal complex, the gut microbiome and host genome represent a coadapted
exhibited little to no F2 hybrid lethali“hologenome” that breaks down during hybridization, promoting hybrid lethality
ty, as previously reported (6) (fig.
and assisting speciation.
S1A). The hybrid lethality between N.
vitripennis and N. giraulti is often diagnosed by larval melanization—a
Although the gut microbiome influences numerous fitness traits in animals, little attention has been given to how the microbiome is structured prominent immune response to pathogens in arthropods (Fig. 1B). We
between closely related species and how the microbiome contributes to postulated that since parental Nasonia species assemble phylosymbiotic
the origin of new species. By incorporating the microbiome into the gut microbiomes (4) (Fig. 2), the melanization and lethality in larval
biological species concept (1) and the Bateson-Dobzhansky-Muller hybrids result in part from altered gut microbiomes.
First, we tested the hypothesis that bacterial community differences
model of hybrid incompatibilities (2), theoretical evidence specifies that
occur
between larval hybrids and non-hybrids during the 2nd instar larnegative epistasis between host genes and host microbiome can accelerval stage, just prior to the point of F2 hybrid male lethality. We focused
ate the evolution of hybrid lethality and sterility (3).
We recently established that when environmental factors such as diet on the N. vitripennis/N. giraulti hybrid microbiota, as this genotype elicare controlled for, species of the Nasonia wasp complex harbor phylo- its elevated lethality compared to the reciprocal cross; however, this
symbiotic gut microbiotas—a term introduced here to denote microbial asymmetry was insignificant in our experiments. As hypothesized, the
community relationships that recapitulate the phylogeny of their host (4). microbiota of N. vitripennis/N. giraulti hybrids was unlike that of either
Similar to phylogenomics, phylosymbiosis asserts that the relationships parental species in both bacterial abundance (Fig. 2B) and diversity (Fig.
of microbiomes across host species maintain an ancestral signal of the 2C), whereas the negative control N. longicornis/N. giraulti hybrids that
host's evolution. The phylosymbiotic signal could be a consequence of survive (6) (fig. S1A) had a parental-like microbiota. Both hybridizahost immune genes that rapidly evolve in a continual arms race with tions resulted in an expansion of low abundant, novel, operational taxonomic units (OTUs, P = 0.06, chi square test with Yates correction, fig.
components of the microbiome.
We hypothesize that the phylosymbiotic gut microbiome within spe- S3). The single major difference in the N. vitripennis/N. giraulti hybrid
cies breaks down in hybrids via epistatic interactions between the micro- microbiota was a shift in the dominant bacterial OTU from Providencia
biome and nuclear and/or cytoplasmic genomes, leading to hybrid sp. IICDBZ10 in pure species controls (81% and 96% of the reads in N.
lethality. Using the parasitoid wasp genus Nasonia, we set out to exper- vitripennis and N. giraulti, respectively) to Proteus mirabilis SNBS in
imentally determine the influence of the microbiome on hybrid lethality. the N. vitripennis/N. giraulti hybrid (86% of reads). These two species
The Nasonia genus consists of several species of haplodiploid parasitoid are natural residents of the Nasonia parental species. P. mirabilis SNBS
wasps that are readily hybridized. Nasonia vitripennis diverged approx- is the dominant species in N. longicornis, and Providencia sp.
imately one million years ago from the ancestor of Nasonia giraulti and IICDBZ10 is the dominant species in N. vitripennis and N. giraulti.
Nasonia longicornis, which themselves diverged less than 400 KYA (5). Second, to test if the N. vitripennis/N. giraulti F2 lethality in hybrids is
In the laboratory, all three species are reared on the fly host Sarcophaga conditioned upon the microbiome, we reared conventional (on a normal
bullata under identical conditions. Nasonia offspring are oviposited by S. bullata host), germ-free (without bacteria), and bacteria-inoculated
the mother inside the puparium of the fly, where the eggs develop (without bacteria first and subsequently inoculated with specific bactethrough to adulthood before emerging from the fly host in about 14 days. ria) hybrids and non-hybrids. For germ-free rearing of Nasonia, we used
In the absence of Wolbachia infections, reciprocal crosses between spe- Nasonia Rearing Medium (NRM), a liquid medium that we recently
cies produce fertile, diploid, F1 hybrid females (6). However, hybrid developed for culturing Nasonia without its S. bullata fly host (11). If
lethality is observed in F2 male offspring, as they are haploid recombi- hybrid lethality in larvae is intrinsically based on negative epistasis benants of their grandparents. Interspecies crosses of N. vitripennis and N. tween host incompatibility genes, then the null hypothesis is that N. vitripennis/N. giraulti and N. giraulti/N. vitripennis hybrid lethality occurs
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The Hologenomic Basis of Speciation:
Gut Bacteria Cause Hybrid Lethality in
the Genus Nasonia
expression patterns and the oxidative phosphorylation (OXPHOS) family of genes, a family thought to be causative in hybrid lethality, were
similar across all three-rearing conditions (Fig. 1D and supplementary
text). However, the 489 innate immune genes in Nasonia that we previously annotated (13) yielded, on average, a significant decrease in transcript levels in germ-free individuals relative to conventional or
inoculated hybrids (Fig. 1D, t test, P < 0.001). Specifically, 39.7% of the
immune genes were under-expressed by twofold or greater in germ-free
hybrids relative to conventional and inoculated hybrids, and 4.9% were
overexpressed (fig. S4 and table S2). Conventionally-reared and inoculated hybrids, in turn, have similar immune gene expression (Fig. 1D, t
test, P = 0.104). However it is important to note that immune genes may
be only one of several possible functional categories that break down
between the host and microbiome during hybrid lethality.
Causes for the postzygotic hybrid lethality in Nasonia have traditionally been attributed to host cytonuclear interactions and host geneby-environment interactions (7–10). However, this study shows that
severe hybrid lethality in larvae can also be due to gene-microbe interactions with beneficial members of the phylosymbiotic gut microbiome. In
this light, the phylosymbiotic microbiome can be understood as an addition to the coadapted genomes of a host organism rather than an arbitrary
amalgam. Linking the microbiome and host genome underscores the
hologenome as a unit of evolution and blurs the lines between what biologists typically demarcate as the environment and the genotype of a
species (11). Based on the mounting evidence for speciation by symbiosis (3), it is becoming clearer that a unified theory of evolution that considers the nuclear genome, cytoplasmic organelles, and microbiome as
interacting components in the origin of new species is an emerging frontier for biology.
References and Notes
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regardless of germ-free or conventional rearing conditions. However, if
the lethality is conditional upon the microbiome, then we expect that
germ-free rearing of hybrids rescues hybrid lethality.
A comparison of results indicates a near complete rescue of hybrid
lethality in germ-free hybrids relative to pure species controls under
germ-free conditions (Fig. 1C, F1,46 = 1.207, P = 0.277 for N. vitripennis
to N. giraulti/N. vitripennis and F1,46 = 0.824, P = 0.369 for N. giraulti to
N. vitripennis/N. giraulti). Therefore, hybrids that would typically show
severe lethality under conventional rearing conditions exhibit a striking
increase in survival. In a subsequent experiment, when F1 female hybrids oviposited their F2 male hybrid offspring into germ-free S. bullata
fly hosts (GF fly host), there was also a marked increase in survival relative to hybrids that were reared on conventional (Cv) S. bullata fly hosts
(fig. S1B). In contrast, in N. longicornis/N. giraulti and N. giraulti/N.
longicornis hybrids, survival values of germ-free hybrids and non-hybrid
controls were expectedly high and similar to each other (fig. S1A, MannWhitney U test, P > 0.5).
If the NRM generally increased survival, it should also affect nonhybrid survival as it would hybrids. However, average survival rates of
controls insignificantly decreased on NRM. Further, germ-free hybrids
that were reared on NRM inoculated with bacterial strains once again
yielded higher lethality compared to parental controls (Fig. 1C). The
common Nasonia bacteria Providencia rettgeri strain IITRP2 and Proteus mirabilis strain SNBS were isolated from parental Nasonia and
added to germ-free NRM. Upon consuming a 1:1 inoculum of these
bacteria, germ-free hybrids exhibited severe lethality in comparison to
non-hybrids (Fig. 1C, F1,46 = 23.863, P < 0.001) and at levels similar to
those of conventionally-reared hybrids (Fig. 1C, Mann-Whitney U test,
P > 0.5). Furthermore, mono-inoculants of antibiotic resistant (AR) P.
rettgeri and Enterococcus faecalis str. XJALT-127-2YG1 isolated from
Nasonia, as well as green fluorescent protein (GFP) expressing E. coli,
also recapitulated significant hybrid lethality (fig. S1B, F1,46 = 36.372, P
< 0.001, fig. S2A, F1,46 = 7.281, P < 0.01, see supplemental text).
The requirement of gut bacteria for interspecific hybrid lethality in
Nasonia is remarkable given that several studies previously mapped
quantitative trait loci with hybrid lethality to the wasp chromosomes and
mitochondria (5, 7, 8, 10). Indeed, marker transmission ratio distortion
(MTRD) analyses in surviving Nasonia F2 hybrids demonstrate an allelic bias at several loci in the genome toward one parental genome, indicating that an incompatible allele from the other parental species
contributes to hybrid lethality (5, 9, 10, 12). Based on our observation
that germ-free hybrid Nasonia exhibit a significant increase in survival,
we predicted that MTRD would revert to near Mendelian inheritance
ratios in the germ-free hybrids. We selected four markers—three within
genomic regions associated with hybrid lethality and one control locus
that is expected to have 50:50 inheritance ratios—and genotyped N.
vitripennis/N. giraulti and N. giraulti/N. vitripennis hybrid Nasonia L4
instar larvae. Larvae reared conventionally yielded the expected distorted frequencies for each of the MTRD markers (5), whereas germ-free
hybrids exhibited typical Mendelian inheritance at all markers except for
MM5.03 (table S1). A large bias against v alleles remained (N. vitripennis frequency of 0.08), though the frequency was significantly higher
from the MTRD under conventional-rearing (N. vitripennis frequency of
0.20, Z-test of proportions, P < 0.001).
One explanation for a microbial basis for hybrid lethality is that negative epistasis, mismatched gene-gene interactions, occurs between
chromosomal genes and the microbiome. These interactions can accelerate the number of potential hybrid incompatibilities under the BatesonDobzhansky-Muller model of genetic incompatibilities (3). To better
understand the mechanisms behind host-microbe interactions that underscore hybrid lethality, we compared the transcriptomes of germ-free L2
instar hybrid larvae with those of conventionally-reared and bacteriainoculated L2 hybrid larvae (just prior to lethality). The genome-wide
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Acknowledgments: Sequences are available at NCBI accession number
1643290. We thank S. Bordenstein and A. Williams for technical
assistance during the development of the in vitro cultivation method;
L. Funkhouser, D. Sutherland, and C. Wogsland for their assistance
in genotyping Nasonia hybrids; R. Pauly for bioinformatic
assistance; and B. Jovanovic, L. Funkhouser, K. Jernigan, and N.
Renner for providing feedback on an earlier version of the
manuscript. We apologize in advance to colleagues whose papers we
could not cite due to space restrictions. This research was made
possible by NSF award DEB 1046149 to S.R.B.
Supplementary Materials
www.sciencemag.org/cgi/content/full/science.1240659/DC1
Materials and Methods
Supplementary Text
Figs. S1 and S2
Tables S1 to S5
References (14–26)
17 May 2013; accepted 8 July 2013
Published online 18 July 2013; 10.1126/science.1240659
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Fig. 1. The symbiotic and genetic basis of hybrid lethality. (A) Average number of F2 males (+/− SEM)
within the S. bullata host during development of egg, 1st instar larvae (L1), 4th instar larvae (L4), yellow
red-eye pupae, and eclosed adults conventionally-reared on S. bullata hosts. The F2 hybrid genotype is
indicated as paternal/maternal where v = N. vitripennis and g = N. giraulti. Mann-Whitney U test, **P <
0.001. N = 48 replicates per developmental stage. (B) Top: N. vitripennis 3rd instar larva that is healthy and
rd
alive; Bottom: hybrid v/g 3 instar larva that is melanized and dead. (C) % survival (+/− SEM) from egg to
pupae of conventionally-reared (red), germ-free (blue), and inoculated (purple; germ-free individuals
inoculated with a Providencia and Proteus bacteria in the NRM) N. vitripennis (v) and N. giraulti (g) parental
species and hybrids. Mann-Whitney U test, **P < 0.001, F1,46 = 23.863 and 12.962, ***P < 0.001, an
average of triplicate experiments with N = 48 hosts for egg and pupae counts per conventional cross and N
= 24 wells containing 12-42 larvae per germ-free and inoculated cross. (D) Average hybrid v/g gene
expression relative to conventionally-reared hybrids for the total genome, oxidative phosphorylation genes,
and immunity genes (t-test, **P < 0.001). RPKM denotes reads per kilobase per million mapped reads.
Conventional N = 14, germ-free N = 20, and inoculated N = 20 individuals sequenced and averaged.
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Fig. 2. Phylosymbiosis and speciation in Nasonia. (A) Simplified phylogeny of
Nasonia (bold lines) and S. bullata flies. N. vitripennis (v), N. giraulti (g), and N.
longicornis (l). (B) A weighted, to abundance of each OTU, UniFrac cluster analysis
depicting the microbial relationships of the three Nasonia species (bold lines) and
two hybrids for the second instar larval microbiota, as well as the unparasitized
Sarcophaga host pupa. The tip of each branch is a pie chart depicting the
abundance of each OTU within the host insects. Genera of the top ten most
abundant bacterial OTUs are listed in the key. (C) An unweighted UniFrac cluster
analyses depicting the microbial relationships; pie charts at the tip of each branch
represent bacterial diversity sampled.