Wednesday, July 13, 2016

What's in a name? Would a Wolbachia by any other name, block RNA viruses as sweetly?

I've ranted about bacterial nomenclature in the past (see here), but this post is specifically about Wolbachia pipientis, and how we should name strain-level variants within a particular host-associated clade (such as wMel, for example).  Currently, there seem to be multiple ways to write these names and to present them in databases or the literature.  For example:

For variants within wMel, we find examples of the following types of strain names:

wMelPop-<insert specific substrain here after the dash>
The stuff that comes after the strain name (wMelPop) sometimes refers to the host insect from which the strain was isolated, or where it currently resides (such as in wMelPop-PYGP) or sometimes refers to how the strain was altered, through sequential passage (such as in wMelPop-CLA).  This variability in the naming schemas used in Wolbachia could lead to confusion down the road.

We are currently adding strain level information to some of the stocks that are now housed in the Bloomington Drosophila Stock Center.  Check out the page here for more. These strains were previously described and published and therefore, the names that appear should match those in the publication (at least for now).  However, when we identify new Wolbachia variants within Drosophila melanogaster, how should they be named?  Should we use the "dash" after the wMel<specificstrain> name? Do you work in Wolbachia? What makes sense to you? Please comment below.

some comments from the twittersphere:
Phenotypes can be dependend on host back ground and environment (e.g. temperature) - not good for naming.

when you refer to strain. Are you using Bloomington's (or source stock centre) stock number, or genetic mutation?

Thursday, June 30, 2016

The Wolbachia Holy Grail: loci behind CI

This obscure alpha-proteobacterium, named Wolbachia, was first discovered because of the weird ways in which it messed with insect reproduction - everything from parthenogenesis induction to male killing to the most common of effects, so-called "cytoplasmic incompatibility." In this case, Wolbachia somehow prevents uninfected females and infected males from mating. Ever since it was first described by Hertig and Wolbach back in 1924, folks have been trying to figure out the how of CI.  At the Wolbachia 2016 meetings, it seems like someone(s) has finally cracked that nut.  <DISCLAIMER: I am actually sitting on my ass in my home office and not at the #Wolb2016 meetings as they are in Australia and I'm allergic to traveling to Australia. However, I heard on the twitter that> Seth Bordenstein + Daniel LePage and John Beckman presented data on two loci - WD0631 and WD0632 - which  may be behind CI. It seems like these loci, when co-expressed in an uninfected male background, cause CI-like phenotype (not sure how much like CI - if this is just embryonic death or fecundity decrease) when crossed to an uninfected female.  Interestingly, though, this is rescued by crossing the same male (expressing the two transgenes) in an infected female (again, I don't know the extent or the phenotype in the embryos).

Now, Wolbachia is known to rescue some developmental abnormalities (see interactions between Wolbachia and sex lethal here) but this is the first time that transgenic expression of a Wolbachia gene(s) has been shown to induce a CI-like phenotype. Those folks that think lots about the mathematical models that may explain the patterns of CI across Wolbachia (the "lock and key" or "mod and rescue" models of Wolbachia CI induction) would be thinking about these loci as maybe the lock or the mod component. Immediately I started to think about some hypotheses you might have if these are in fact CI-related.

1) You should find homologs in all sequenced genomes of variants that cause CI
2) You should not find these homologs in genomes where the Wolbachia do not cause CI (that is debatable*)
3) The extent of expression of these genes should correlate with the extent of CI (stronger driven transgene, for example, should result in fewer progeny)
4) Homologs from incompatible Wolbachia should not rescue each other (if you express the strain A homolog, it should not be rescued by strain B, for example).
*maybe these homologs are behind other reproductive phenotypes as well and host genotype modulates this

Given that I am just sitting on my tuckus, and just wanted to shoot the proverbial shit for a while about this neat result, I will instead tackle the assumptions #1 and #2 above.

We (read: Danny W. Rice in my group) generated a database of orthologous groups using the publicly available Wolbachia genomes and reciprocal blast.  Here's a phylogeny, generated from a concatenated alignment of orthologs across these genomes:

Clearly the arthropod Wolbachia are more heavily represented than the others.  When I look for homologs to WD0631 and WD0632 in this dataset, here's what we find:

Table 1. Genes that are homologous to WD0631, a predicted CI-inducing gene, across Wolbachia genomes.

wSim (629 contigs)
wPip-JHB (21 contigs)
wMelPop (80 contigs)
wGmm (201 contigs)
wBol1-b (144 contigs)

Table 2. Genes that are homologous to WD0632, a predicted CI-inducing gene, across Wolbachia genomes.  

wPip-JHB (21 contigs)
wMelPop (80 contigs)
wBol1-b (144 contigs)
valsugana (110 contigs)

So, when you look at these tables some trends seem to emerge that might, at first glance, seem to address our assumptions 1 and 2.  For example, both of these homologs are not found across all CI inducing strains - where are wRi and wSim's WD0632 homologs? Also, not all of these strains cause CI exclusively - check out wBol1, the male-killing Wolbachia from Hypolimnas bolina.  You could also include wBm's Wbm0463 in the WD0632 homolog list, although it is a more distant homolog as well.  I haven't had the chance yet to dig into the meat of this dataset but there are some interesting results so far.  For those Wolbachia researchers reading this, comments below welcomed.


Thoughts after back-and-forths on the twitter-sphere:
1st - I would definitely agree that this discovery in no way claims to be the only mechanism behind CI and there could be many others, variations on this theme or highly divergent
2nd - Your definition of "homolog" most assuredly would change this list - I have posted stringent orthologs from a reciprocal blast search - removing paralogs within genomes (of which there are plenty).
3nd - Thanks to Greg Hurst for clarifying that wBol1 causes CI as well as male-killing (altered in text now).

Wednesday, May 25, 2016

Learning to fly, with microbes

It's summer and that means time for research and reading groups here at IU Bloomington.  Given the interest in Drosophila across the department, I thought it might be fun to start a fly microbiome reading group this summer. We've got a really dynamic and diverse group -- everyone from stock center folks to genomics gurus and graduate students working in flies.  If you're already working in the fly, and haven't thought about the effect of the microbial community - shame on you!; seriously, have you had your head in a hole for the last decade? Beyond ignoring Wolbachia infection and effects on host phenotypes (such as insulin signaling, oogenesis, and others), ignoring the entire microbial community is really not defendable any more. 

If you haven't been interested in the fly microbiome, because it's depauperate (what a nasty word, aye?) and generally inconstant, I hope to change your mind.

Here are some of the papers we've been reading lately:

Chandler JA, Morgan Lang J, Bhatnagar S, Eisen JA, Kopp A (2011) Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System. PLoS Genet 7(9): e1002272. doi:10.1371/journal.pgen.1002272

The introduction to the system - here they surveyed wild-caught flies and flies from a few labs for bacterial composition and found little correspondence in OTUs between wild and lab flies and a surprising about of difference in bacteria colonizing flies of the same stock from different labs.

Blum JE, Fischer CN, Miles J, Handelsman J. 2013. Frequent replenishment sustains the beneficial microbiome of Drosophila melanogaster. mBio 4(6):e00860-13. doi:10.1128/mBio.00860-13

This study focused on lab flies and looked at the ability of the bacterial community associated with the fly to grow in the fly food. They hypothesize that this may be the major way in which these microbes get to the next generation (at least in the lab).

Newell, P. D., & Douglas, A. E. (2014). Interspecies interactions determine the impact of the gut microbiota on nutrient allocation in Drosophila melanogaster. Applied and environmental microbiology, 80(2), 788-796.

Here they use a gnotobiotic system to show that microbes affect the host differently when grown together. Also who is there (which Lactobacillus or Acetobacter species) matters.

Today I'm reading up on some more of Angela Douglas' work, and checking out John Chaston's new(ish) pub:

Chaston JM, Dobson AJ, Newell PD, Douglas AE. 2016. Host genetic control of the microbiota mediates the Drosophila nutritional phenotype. Appl Environ Microbiol 82:671–679. doi:10.1128/AEM.03301-15.

Here, they leverage the Drosophila genetic reference panel - a panel of inbred, originally wild-caught, flies that have had their genome sequenced.  This allows you to correlate or associate particular alleles in the fly with phenotypes, in this case, nutritional indicies (such as triglyceride levels) and with microbiome composition.  They establish gnotobiotic flies for these DGRP stocks, allowing them to control for microbiome input, and look at the colonized flies after they eclose.  

It is immediately obvious that host genetic background has an effect. 
Figure 1. Bacterial communities and phenotypic traits of 79 Drosophila lines from DGRP. (A) Microbiota composition was assessed by pyrosequencing with OTUs called at 97% sequence identity (see also Data Set S1 in the supplemental material). (B to E) Nutritional indices (in micrograms per milligram [dry weight]), with data represented as means ± standard errors of the means (SEMs) (error bars). In each panel, Drosophila lines are ordered by the sum of Acetobacter andLactobacillus species (A) or by mean nutritional index value (B to E)

They found a big effect with one species (Acetobaceter tropicalis) and go on to test these differences using mono-associated flies - again, showing just how easy it is to test these hypotheses in the fly system. 

Next they performed their GWAS experiment; they associated particular alleles with these backgrounds and found that flies with SNPs in genes related to neural processes (such as paralytic, calnexin 14D, and dunce) were enriched in their association with community composition.  Interesting! They conclude that maybe there is a gut-brain axis link in fly.  I found this part to be cool because I immediately thought of Wolbachia colonization of the neural tissues -- is a Wolbachia colonization in these flies affecting microbiome composition and are these alleles altering Wolbachia titer which in turn could affect microbiome composition? Unfortunately, they did not look into this, at least in this pub; some of the DGRP is colonized by Wolbachia but they are all different strains, isolated from different females, and Chaston et al., chucked the Wolbachia reads from their 16S rRNA gene analyses when they made their amplicons.  Sigh. Even within the microbiome community, Wolbachia is ignored yet again!

Fear not, dear reader, several people in the Wolbachia community sure are interested to see how this prevalent endosymbiont might alter host microbial ecology.  I know that some interesting datasets are coming soon from both the Teixeira and Frydman labs - stay tuned!