t a p f e r © odh
MICROBIOLOGY
O R IG IN A L R E S E A R C H ARTICLE
published: 10 February 2014
doi: 10.3389/f m icb.2014.00043
Microsporidia-nematode associations in methane seeps
reveal basal fungal parasitism in the deep sea
A m ir S a p ir1, A dler R. D illm an 1t, Stephanie A. Connon2, Benjamin M. Grupe3, Jeroen Ingels4,
M an u el M undo-O cam po5, Lisa A. Levin3, James G. B aldw in6, Victoria J. Orphan 2 and
Paul W. Sternberg1*
1 Howard Hughes Medical Institute and Division o f Biology, California Institute o f Technology, Pasadena, CA, USA
2 Division o f Geological and Planetary Sciences, California Institute o f Technology, Pasadena, CA, USA
3 Integrative Oceanography Division, Center for Marine Biodiversity and Conservation, Scripps Institution o f Oceanography, La Jolla, CA, USA
4 Marine Life Support Systems, Plymouth Marine Laboratory, Prospect Place, UK
5 Department o f Agricultural Biotecnology, Nematology Laboratory, CIIDIR-IPN Unidad Sinaloa, Sinaloa, Mexico
6 Department o f Nematology, University o f California, Riverside, CA, USA
E d ited b y:
Hongyue Dang, Xiamen University,
China
R ev ie w e d by:
Catherine Texier, Université Blaise
Pascal, France
Ann J. Vanreusel, Ghent University,
Belgium
* Correspondence:
Paul W. Sternberg, Howard Hughes
Medical Institute and Division o f
Biology, California Institute o f
Technology, 1200 East California
Blvd, Pasadena, CA 91125, USA
e-mail: pws@caltech.edu
* P resent address:
Adler R. Dillman, Department o f
Microbiology and Immunology,
Stanford University, Stanford, USA
The deep sea is Earth's largest h a b ita t b u t little is know n a b o u t th e nature o f deep-sea
parasitism . In co n tra s t to a fe w characterized cases o f bacterial and p ro tista n parasites,
th e e xiste n ce and biological sig n ifica n ce o f deep-sea parasitic fu n g i is y e t to be
understood. Here w e re p o rt th e d isco ve ry o f a fu n g u s-re la te d parasitic m icro spo ridium ,
N e m a to c e n a to r m a risp ro fu n d i n. gen. n. sp. th a t in fe cts b e n th ic n e m a to d e s a t m ethane
seeps on th e Pacific Ocean floor. This infection is sp e cie s-sp e cific and has been
te m p o ra lly and spatially sta b le over 2 years o f sam pling, indicating an ecologically
c o n s is te n t host-parasite interaction. A high d istrib u tio n o f spores in th e reproductive
tra cts o f in fe cte d m ales and fe m a le s and th e ir absence fro m h o st n e m a to d e s' intestine s
s u g g e sts a sexual tra n sm issio n stra te g y in co n tra st to th e fecal-oral tra n sm ission o f
m o s t m icrosporidia. N. m a ris p ro fu n d i ta rg e ts th e host's body w all m u scle s causing
cell lysis, and in severe infection even m uscle fila m e n t degradation. P hylogenetic
analyses placed N. m a ris p ro fu n d i in a novel and basal clade n o t clo se ly related to any
described m icrosporidia clade, s u g g e stin g e ith e r th a t m icro sp o rid ia -n e m a to d e parasitism
occurred early in m icrosporidia evolution or th a t h o st specialization occurred late in an
a n cie nt deep-sea m icrosporidian lineage. O ur fin d in g s reveal th a t m ethane seeps su p p o rt
co m p le x e c o syste m s involving in te rkin g d o m in te ra ctio n s b e tw e e n bacteria, nem atodes,
and parasitic fu n g i and th a t m icrosporidia parasitism exists also in th e deep-sea biosphere.
Keywords: deep-sea m eth an e seeps, nem atodes hosts, deep-sea m icrosporidia parasitism , m uscle decom position,
basal fungi in th e deep sea
INTRODUCTION
A m ajo r co m p o n en t o f life in the deep sea relies o n chem osynthetic energy em itted at h y d ro th erm a l vents an d cold m eth an e
seeps, w here prokaryotes consum e sulfide o r m eth an e for p rim a ry
p ro d u c tio n su p p o rtin g oasis-like ecosystem s rich in biom ass
(Sibuet an d O lu, 1998; M artin et al., 2008). In m eth an e seeps,
m eth an e oxidation coupled w ith sulfate re d u ctio n is m ed iated by
anaerobic consortia o f archaea an d bacteria (K nittel an d Boetius,
2009). The level o f m eth an e, a p o te n t green house gas released to
the w ater colum n from seeps, is d eterm in ed in p a rt b y th e grow th
and activity o f m ethan e-fu eled au to tro p h s an d th e eukaryotes
th a t graze u p o n them (T h u rb e r et al., 2012). In c o n trast to the
w ell-established in teractio n s betw een p rokaryotes an d anim als at
vents an d seeps, little is k now n ab o u t the n atu re, abundance,
life strategies, an d ecological significance o f eukaryotic m ic ro o r­
ganism s living in chem osynthetic ecosystem s (Takishita et al.,
2007).
D ue to sam pling challenges, lim ited access, and difficulties
o f grow ing an d m ain ta in in g deep-sea organism s in the lab, o u r
know ledge o f deep-sea fungal life is lim ited an d relies p rim arily
w w w .fro n tiers in .o rg
o n the study o f a few cultureable species an d on culture-free
approaches such as m etag en o m ic sam pling. Fungi recovered
from the deep sea include free-living yeasts th a t were rep o rted
to d o m in ate fungal diversity in the deep oceans (Bass et al.,
2007) (N agaham a et al., 2011) and various filam entous species
(B urgaud et al., 2009; T h aler et al., 2012). A lth o u g h som e deepsea fungi are closely related to know n terrestrial species, recent
studies o f fungal diversity at h y d ro th erm a l vents (B urgaud et al.,
2011) an d cold seep hab itats (T haler et al., 2012) resulted in
th e discovery o f novel fungal clades suggested to be endem ic to
th e deep sea. Even th o u g h the m ajo rity o f fungi sam pled from
c h em o a u to tro p h ic en v iro n m en ts are th o u g h t to be free-living,
a few yeast species o f the genus R hodotonilahave were fo u n d
in association w ith deep-sea tu b ew o rm s an d clam s (N agaham a
et al., 2003). The possible effect o f these free-living and anim alassociated fungi in deep-sea b en th ic ecosystem s is yet to be
characterized. In con trast, the path o g en ic effect o f a m usselinfecting parasitic fungus fo u n d in a Fiji Basin h y d ro th erm al vent
(Van D over et al., 2007) d em o n strates the significant role parasitic
fungi can play in b en th ic ecosystem dynam ics. Sequence-based
February 2014 | Volum e 5 | A rticle 43 | 1
Sapir et al.
fungal surveys o f cold seeps (N agaham a et al., 2011) revealed th a t
the m o st frequently recovered clones were o f early diverging basal
fungi, including tw o species o f Rozella, an endoparasitic genus
th at, along w ith m icro sp o rid ia are considered to be th e earliest
b ran ch in g taxa o f sequenced fu n g i (James et al., 2006; CapellaG utierrez et al., 2012). The details o f Rozella parasitism an d its
biological significance in th e context o f m eth an e seep ecosystems
are yet to be understoo d .
M icrosporidia is a p hylum o f fungal obligate in tracellular
eukaryotic parasites th a t infect a w ide range o f hosts from m o st
m etazoan phyla. U nd erstan d in g m icro sp o rid ia biology is o f m e d ­
ical and agricultural interests due to the m o rta lity th ey cause
am ong im m u n o co m p ro m ise d h u m an s, th eir use in b io co n tro l
against agriculturally d am aging insects (Lacey et al., 2001), and
the p roposed role o f m icro sp o rid ia infection in th e colony col­
lapse disorder o f honeybees (review ed in Texier et al., 2010). M any
species o f m icrosporid ia are k n o w n to infect the h o st’s intestine,
inclu ding the h u m a n p athogens th a t can cause severe an d p e r­
sistent d iarrh ea in im m u n o co m p ro m ise d patien ts (Lewthwaite
et al., 2005). M icrosp o rid ia cycle betw een intracellular phases
w here a cell-wall deficient m e ro n t replicates an d differentiates
to form sporonts. The spo ro n ts differentiate further, u n d erg o ­
ing sporulation (sporogony plus sporogenesis) to eventually form
spores (Keeling and Fast, 2002). The spores can either be released
in to the en v iro n m en t to be ingested b y a new h o st (ho rizo n tal
transm issio n) o r passed from p a re n t to offspring via the eggs
(vertical transm issio n ). In m o st studied cases, spore e n try into
a naïve h o st an d association w ith targ et cells leads to the acti­
vatio n o f an infection ap p aratu s— the p o lar filam ent th a t in a
n o n-activated state rem ain s coiled inside the spore. The fila­
m en t, w hen extruded from the spore to fo rm the po lar tube,
pierces the h o st cell m em b ran e, injecting nuclei and sporoplasm in to the h o st cell’s cytoplasm to initiate a new infection
cycle (Xu and Weiss, 2005). D u rin g th e ir evolution tow ard obli­
gate parasitism , the m ito c h o n d ria o f m icro sp o rid ia have evolved
in to m itosom es— m ito ch o n d rially derived organelles th a t have
retained som e m etabolic fu n ctio n b u t lost the ability to p roduce
energy via oxidative p h o sp h o ry latio n (K atinka et al., 2001; B urri
et al., 2006; Tsaousis et al., 2008). T hus, m icro sp o rid ia b io e n ­
ergetics relies either on anaerobic substrate-level m etabolism ,
on ATP supply from the host, or b o th . F u rth erm o re, w hole
genom e sequencing o f selected m icro sp o rid ia species revealed an
extrem e degree o f genom e co m p actio n an d re d u ctio n w ith som e
species h arb o rin g the sm allest genom es a m o n g k now n eukaryotes
(Keeling et al., 2005; C uom o et al., 2012). So far, only one case of
m icrosporidia infection in the deep sea is re p o rted b u t is actually
carried o u t by the m etazo an parasite M yxosporea (D u b in a and
Isakov, 1976). A lthoug h little is kn o w n a b o u t m icro sp o rid ia in the
deep sea, m icro sp o rid ia were recently show n b y u ltrastru c tu ral
and m olecular approaches to be genuine parasites o f hosts in v ar­
ious m arin e habitats in clu d in g shallow w ater fish an d nem atodes
in the low tide zone (A bdel-G haffar et al., 2011; A rdila-G arcia and
Fast, 2012).
N em atoda is perh ap s the m o st a b u n d a n t and versatile an i­
m al phylum on e arth w ith ad ap ta tio n s to a vast sp ectru m of
habitats including extrem e env iro n m en ts such as the frozen
A ntarctic D ry Valleys (A dam s et al., 2007) an d even th e E a rth ’s
Frontiers in M icrobiology | A q u a tic M ic ro b io lo g y
M icrosporidia parasitism In deep-sea m ethane seeps
cru st (B orgonie et al., 2011). D espite th eir ecological diversity,
n em ato d es all have a n o n -seg m en te d b o d y co m prised o f an
en d o d erm al alim en tary system, a m eso d erm a l m uscle an d re p ro ­
ductive system, an d an ecto d erm al hyp o d erm is covered by a
th ick extracellular cuticle. The n em ato d e cuticle restricts in ter­
action o f n em ato d es w ith th e ir e n v iro n m en t m ain ly to orifices
such as the m o u th , anus, som e sensory organs (e.g., am phids),
an d th e vulva and cloaca o f fem ales an d m ales respectively.
T he cuticle and b o d y orifices are the sites w here n em atodeassociated m icro o rg an ism s can be fo u n d , often as fo od sources
or associated sym bionts. For exam ple, the ectosym biosis betw een
m arin e n em ato d es and cuticle-associated sulfur-oxidizing bacte­
ria is w ell-established (Polz et al., 1992; B ulgheresi et al., 2011),
an d som etim es even n em ato d e-b acteria endosym bioses are estab­
lished (M usat et al., 2007; T chesunov et al., 2012). B acterivorous
n em ato d es are pro b ab ly the m o st a b u n d a n t m eio fau n a (anim als
ran g in g in size betw een 32 (xrn an d 1 m m ) living in m an y deepsea hab itats (Ingels et al., 2011; Pawlowski et al., 2011) including
h y d ro th erm a l vents an d m eth an e seeps, w here n em ato d e diets
include c h em o a u to tro p h ic bacteria (V anreusel et al., 2010a,b).
N em atode abundance in the deep oceans m akes th em p o te n ­
tial hosts for endoparasitic m icroorganism s. However, to o u r
know ledge this type o f parasitism has n o t been re p o rted yet.
Surveying for possible m icro o rg a n ism -n em ato d e in teractions
in the deep sea, we fo u n d m icro sp o rid ia parasitism to be a novel,
genuine, an d tem p o rally stable life strategy at H ydrate Ridge
m ethan e seeps in the Pacific O cean. Phylogenetic analyses concor­
dan tly show th a t the m icro sp o rid ia we describe con stitute a novel,
basal clade o f deep-sea m icro sp o rid a. O u r stu d y suggests th a t a
separate b ra n c h o f basal m icro sp o rid ian s has evolved in the deep
sea an d highlights the p o ten tial effect o f m icro sp o rid ia parasitism
on deep-sea c h em o a u to tro p h ic ecosystems.
RESULTS
M IC R O SPO R ID IA P A R A SITISM AT HYDRATE RIDGE M ETH A N E SEEPS
W hile surveying for possible sym bioses betw een m icroorganism s
an d deep-sea nem atodes, we fo u n d a b u n d a n t n em ato de assem ­
blages associated w ith carbonate rocks an d sulfide-oxidizing bac­
terial m ats at H ydrate Ridge (H R) m eth an e seeps, 85 km off the
coast o f O regon at 587-810 m w ater d ep th (F igures 1A, S I). In
this low oxygen e n v iro n m en t (m in im u m O 2 co n cen tratio n of
a b o u t 0.25 m l/L) n em ato d es com prise 80% o r m o re o f all m eio ­
fau n a (G uilini et al., 2012). We discovered th a t the second m ost
a b u n d a n t n em ato d e species collected o n carbonate rocks n ear an
active seepage (F igure 2) is associated w ith orange p ig m ented sul­
fide oxidizing b acterial m ats (F igure 1C) an d h arb o rs rod-like
m icrobes in its reproductive system (F igures 3A -C ). Based on
sm all rib o so m al su b u n it (SSU) rD N A analysis we identified the
n em ato d e as a Desmodora species (Figure S4). D etailed m o rp h o ­
logical study ind icated th a t th e n em ato d e is D esmodora marci
(Superfam ily D esm odoroidea, fam ily D esm o d o rid ae, F igures IF ,
S2-S3) a species originally described from h y d ro th erm al vents in
the W est Pacific Lau Basin n e a r Fiji (H ine H in a site) (Verschelde
et al., 1998) an d we refer to it as D. marci th ro u g h o u t. The
o riginal d escription o f this species also m en tio n s the pres­
ence o f m icro -o rg an ism s associated w ith the n em ato de cuticle.
M o rp h ologically-distinct rod-like m icrobes were also detected
February 2014 | Volum e 5 | A rticle 43 | 2
Sapir e t al.
M icrosporidia parasitism in deep-sea m e thane seeps
2 »
A c tiv e see p
125° 10'0"W
Infected
D. marci
125° 8 ’0"W
125°6'0*W
N o n-infecte d
D. marci
125°4'0"W
O th e r n e m a to d e s
125° 2,0”W
125° 0 '0 'W
124° 58‘0"W
British Columbia
Pacific O cean
Oregon
125° 10'0"W
125° 8'0"W
125* W W
125° 4 ,0"W
125° 2'0"W
125°0'0"W
Prochaetosoma sp. 10
FIGURE 1 I H ydrate Ridge m eth an e seeps are inhabited by D esm odo ra
and Prochaetosom a nem atodes. (A) A m ap of Hydrate Ridge w ith th e 2010
and 2011 sam pling site s and location w h e re infected and non-infected D.
marci w o rm s w e re found. On th e global m ap, a red sq u are m arks HR w h e re
w e found infected D. marci n e m a to d es. A red arrow m arks th e location of th e
Lau Basin hydrotherm al v en t s y ste m s w h e re D. marci w a s previously
repo rted (Verschelde e t al., 1998). (B) In situ photo of a typical HR carbonate
rock. (C) O range bacterial m at (arrow) on a c arb o n ate rock within an active
w w w .fro n tiers in .o rg
124° 58’0°W
Desmodora marci
m e th a n e s e e p (arrowhead) collected by hydraulic suction. The dista n ce
b e tw e e n laser points is 10 cm . (D) A D. marci w orm (arrow) a sso c iated with
th e bacterial m at. The im age w a s taken on th e ship after sam pling. (E) SEM
m icrograph of th e head of Prochaetosoma sp. 10 w ith typical long cephalic
a d hesion tu b e s c o n n ec te d to adhesive glands, presum eably u sed for
locom otion (arrow). (F) SEM m icrograph of th e head of D. marci w ith tw o
cryptospiral am phid se n s o ry organs (1.2 tu rn s, arrow heads) and four
subm edian cephalic papilla (arrow). Scale bars are 10|xm (E,F),
February 2014 | V olum e 5 | A rtic le 43 | 3
Sapir e t al.
M icrosporidia parasitism in deep-sea m e thane seeps
Infected m ales
Infected fem ales
n=17
I
I
E
z
B
B
-
fEao
=5 o
cc ^
—
^
I03 uLUl
C
4500 4000
3500
3000
2500
2000
1500
1000
500
0
oS p e c ie s
*
^
J 3
&
FIGURE 2 I D esm odo ra nem ato des d o m in a te H ydrate Ridge carbonate
rock ecosystem s. (A) H ydrate Ridge South E4 c arb o n ate rocks th a t w e re
sam p led and p ro c e sse d on th e ship in A ugust 2010. The com bined surface
area of th e s e rocks is 0.1615 m 2 (B) Animal d en sities at Hydrate Ridge
south E4 rock calculated a s Individuals per com bined E4 rock. N em atode
num b ers re p re sen t th e average of th re e sam p les 1/120th of th e total
fraction recovered from th e rocks. "O ther anim als" are th e total num ber of
melofauna and m acrofauna (mainly Polychaeta) found In E4 rock.
Desmodora and Prochaetosoma w o rm s are 8 3.6% of th e total m elo and
m acrofauna anim als living on E4 rock. Bars re p re sen t stan d ard errors.
in another, closely related n em ato d e from the H R assemblage
th a t was identified as a Prochaetosoma species (superfam ily
D esm odoroidea, fam ily D raco n em atid ae) an d we refer to it as
Prochaetosoma sp. 10 (F igures IE , S2-S3). The m o st a b u n d a n t
n em atode was identified as an o th e r Desmodora species (affinity
w ith Desmodora alberti), closely related to D. marci. We refer to
this species as D esmodora sp. 9. Desmodora alberti was also o rigi­
nally described from h y d ro th erm a l v en t hab itats at the East Pacific
Rise (Verschelde et al., 1998). T his n em ato d e show ed n o signs of
infection by spore-form in g m icrobes (n = 40).
Because the m icrobes exhibited a nucleus-like stru ctu re
(F igures 3D,E; M ovie S I), th e possibility o f fungal in fection was
exam ined b y the use o f general fungal p rim ers an d b y C alcofluor
W hite, a dye th a t stains the fungal cell wall. A lth o u g h th e lack
o f PC R p ro d u c t suggested th a t the w ell-characterized species
o f fungi are n o t involved, a positive spore-restrictive C alcofluor
W hite staining indicated infection b y a sp o re-fo rm in g fu n ­
gus or p ro tist (F igure 3F). A m ong fu ngus-related organism s,
m icrosporidia were recently characterized as n em ato d e p a ra ­
sites (Troem el et al., 2008; A rdila-G arcia an d Fast, 2012). We
used m icrosporidia-specific p rim ers (G hosh an d Weiss, 2009)
Frontiers in M icrobiology | A q u a tic M ic ro b io lo g y
F
G
\
Y
E
Y
/
FIGURE 3 I N . m arisp ro fu ndi is associated w ith D. m a rc i reproductive
organs. (A) A diagram of infected D. marci m ale and fem ale. G onads, m ale
vas d e fe re n s, and fem ale u terus a re labeled in blue. M icrosporidia sp o res
are labeled in red. The bar graphs sh o w th e tissue-specific pattern s of
sp o re distribution in m ales and fem ales, n u m b e rs are not exclusive. (B)
Nomarski DIC of a m oderately infected fem ale. E, eg g s; V, vulva; arrow,
microsporidia sp o re s. (C) N. marisprofundi infection of m ale reproductive
organs. M icrosporidia sp o re s are distributed a t th e end of th e v a s d e fe re n s
(arrow) and at th e dorsal posterior side; S, m ale spicule; magnification a s in
B. (D) N. marisprofundi sp o re s d isse c te d from a fem ale genital tract and
stained by 4 /,6-diamidino-2-phenylindole (DAPI). (E) DAPI staining (blue)
revealed nucleus-like structure inside th e sp o re s (arrowhead) magnification
a s in (D). (F) Calcofluor w hite staining (blue) of N. marisprofundi sp o re s
(arrows) attached to a d is se c te d w orm cuticle. (G ) In situ hybridization with
N. marisprofundi SSU rRNA probe (red) and DAPI staining (blue) of an
infected fem ale fixed by paraform aldehyde to d e te c t specifically th e
v egetative s ta g e s (se e M aterials and M ethods section in Supporting
Information). V egetative s ta g e s (arrow heads) a re positioned in th e vicinity
of sp o res. E developing em bryo. Scale bars are 20p.m (B,F,G); 5|x m (D);
insert 2.5 p.m (G).
February 2014 | Volum e 5 | A rticle 43 | 4
Sap¡r e t al.
and fo u n d one rD N A SSU am plicon specific to infected D.
marci th a t is sim ilar b y BLAST to k n o w n m icro sp o rid ia SSU
rD N A sequences. We nam e this m icro sp o rid iu m N em atocenator
m arisprofundi n. gen. n. sp., Latin for “n em ato d e eater o f the
deep sea” an d w ill refer to it as N . m arisprofundi th ro u g h o u t (see
fo rm al genus an d species d escriptions in th e m icro sp o rid ia tax o n ­
om y section). We used the sam e m icrosporidia-specific p rim ers
on a few spo res-h arb o rin g Prochaetosoma sp. 10 nem atodes (five
m ales and one fem ale o u t o f five ad u lt m ales an d three ad u lt
fem ales analyzed) an d fo u n d th a t these w orm s are infected w ith
a species o f m icrosporid ia closely related to N . m arisprofundi we
n am ed N em atocenator sp. 1 (F igure 6). As Prochaetosoma sp. 10
nem atodes were h a rd to so rt due to th eir sm all size and low a b u n ­
dance in sam ples (F igure 2), we focused on th e D. marci and N.
marisprofundi interaction.
A G EN UINE A N D TEM PO RARILY STABLE M IC R O SPO R ID IA
P A R A SITISM OF LIVE NEM ATODE HOSTS
Because o u r sam ples w ere o b tain ed from a deep-sea seep enviro n ­
m e n t w here m icro sp o rid ia have n o t previously been rep o rted , we
rigorously tested w heth er the observed n em ato d e-m icro sp o rid ia
in teractio n is b o th genuine an d ecologically relevant using the
follow ing procedures: (i) S am pling at H R for tw o consecutive
years we fo u n d th a t th e sam e species o f n em ato d e h o st is infected
w ith sam e species o f m icro sp o rid ia. T his sam pling dem o n strates
the spatially and tem p o rally stable in teractio n o f N . marisprofn n d i w ith its n em ato d e h o st at H R m eth an e seeps (Figure SI),
(ii) Spores w ere detected in sam ples fixed im m ediately after sam ­
pling, precluding secondary infection d u rin g tra n sp o rt o r in the
lab. (iii) Live nem atod es collected from H R th a t were k ep t alive at
4° C in sem i-dysoxic co n d itio ns in the lab were also fo u n d to h a r­
b o r spores, d em o n stratin g m icro sp o rid an association w ith in live
hosts (n = 4.8; F igure 6; M ovie S2). (iv) Lack o f infection (n =
40) in the closely related n em ato d e, Desmodora sp.9 (F igures 4,
S2) dem onstrates h o st species specificity o f N . marisprofundi.
N otably, a high level o f h o st specificity is n o t u n co m m o n am ong
m icro sp o rid ia (P orter et al., 2007). Taken together, these results
indicate th a t H R active m eth an e seeps are stable h ab itats o f ab u n ­
d a n t bacterivorous n em ato d e an d th eir m icro sp o rid ia parasites.
GEOGRAPHICALLY W ID E SPREAD PA R A SITISM AT HYDRATE RIDGE
SEEPS
To characterize b etter ho st-p arasite in teractio n s, we exam ined the
spatial and tem p o ral p attern s o f n em ato d e-m icro sp o rid ia asso­
ciation at the various m eth an e seeps d istrib u ted along H ydrate
Ridge. We sam pled enviro n m en ts represen tin g th e range of
ecosystem s at H R N o rth , South, and East Knoll, sp an n in g deep
seeps (700-800 w ater d e p th m ) overlain b y low oxygen w aters
and shallow er seeps (500-600 m ) w here oxygen co n cen tratio n is
h igher (F igures 1A, SI; Table S I). These enviro n m en ts include
carbonate rocks an d sedim ents w ith active m eth an e em issions
(active seeps) an d inactive sites 30-300 m from the n earest
active seepage. H o st n em ato d es were a b u n d a n t in carbonate
sam ples from active seeps (ab o u t 20 w o rm s/m l) b u t absent
in sam ples from inactive seeps or sedim ents (Table S I), sug­
gesting th a t sulfide oxidizing m ats and carbonates from active
w w w .fro n tiers in .o rg
M icrosporidia parasitism In deep-sea m ethane seeps
seep areas are the p referred niches for the n em ato d e h o st and
parasite.
D. marci n em ato d es were associated w ith b acterial m ats on
carb o n ate rocks in all three regions o f H R (F ig u re 1A), b u t in
c o n trast to H R S o u th an d East K noll, H R N o rth w orm s were
n o t infected (n = 141). To u n d ersta n d the dynam ics o f hostparasite d istrib u tio n in the various ecological niches o f H R we
placed substrates (w ood, rock, an d b o n e) n e a r inactive an d active
seeps for yearlong colonization experim ents. N em atodes were
extrem ely rare in substrates placed n ex t to inactive seeps b u t
recovered, in large n u m b ers, from active seeps. A t H R N o rth we
fo u n d unin fected D. marci to be th e d o m in a n t species associated
w ith carb o n ate rocks, w hereas w ood an d b o n es were p opulated
m ain ly by o th er n em ato d e species (Table S I). In H R S outh seeps,
however, D. marci w o rm s were the d o m in a n t n em ato d e species
recovered from rock, w ood, an d b o n e substrates. Infected w orm s
were fo u n d to be associated w ith all three substrates b u t the h ig h ­
est infection rate (as hig h as 63.33% o f th e exam ined anim als)
was o f n em ato d es colonizing rocks (Table S I). Taken together, the
find in g o f m icro sp o rid ia in n em ato d es collected from H R South
an d East K noll [ab o u t 1 5 k m ap art (F igure 1A)] dem onstrates
th a t m icro sp o rid ia parasitism is a characteristic o f H R ecosystems
w hereas colonization experim ents d em o n strate th e p o ten tial o f
these parasites to spread in to new ecological hab itats b y infected
hosts.
N. marisprofundi D ISTR IB U TIO N W IT H IN THE HOST SUGGESTS A
SEXUAL T R A N S M IS S IO N M E C H A N IS M
Because o f the extrem e con d itio n s at H R (e.g., low oxygen and
hig h pressure, see S u p p o rtin g In fo rm atio n ) we could n o t sustain­
ably culture H R n em ato d es an d th e m icro sp o rid ia th ey harbor.
T herefore, th e dynam ics o f infection w ere deciphered based on
p o sitio n al and m orp h o lo g ical analysis o f m icro sp o rid ia-infected
n em ato d es th a t were preserved by sh ip b o ard fixation. To estim ate
th e level o f infection w ith in the n em ato d e p o p u la tio n , we focused
o n tw o sam ples collected from the active seeps o f H R South th at
were rich in D. marci n em atodes. E ither due to sam pling bias or
th e n em ato d e life cycles, D. marci juveniles were rare in H R sam ­
ples p recluding com prehensive analysis o f infection in younger
n em ato d e stages. O ver 90% o f ad ult D. marci were alive at the
tim e o f fixation an d 50 -6 2 % o f m ale and fem ale w orm s from b o th
sam ples h a rb o re d m icro sp o rid ia (n = 106; Table SI).
M any m icro sp o rid ia, in clu d in g the terrestrial nem atode
infecting species N em atocida parisii an d N em atocida sp. 1, are
kn o w n to be ho rizo n tally tra n sm itte d by fecal-oral infection
cycles (Troem el et al., 2008). Surprisingly, despite the fu n c­
tio n al an d m o rp h o lo g ical sim ilarities o f C. elegans an d D. marci
intestines, we have never observed m icro sp o rid ia spores in the
intestine o f D. marci (n = 100), n o r did we find evidence of
g u t m alfo rm atio n s typical o f m icro sp o rid ia infections in C. ele­
gans (Estes et al., 2011). C oncurrently, free spores were neither
detected in a survey o f th e sam ples m o st densely p o p u lated w ith
infected D. marci (ab o u t 20 w o rm s/m l) n o r in o th e r anim als liv­
ing in close p ro x im ity to th e infected n em ato d es such as adult
C opepoda, Polychaeta, and M ollusca (n = 35).
Som e m icro sp o rid ia species are also vertically tra n sm itted by
fem ales to th e ir offspring as spores inside developing eggs (Becnel
February 2014 | Volum e 5 | A rticle 43 | 5
Sapir e t al.
M icrosporidia parasitism in deep-sea m e thane seeps
îc tiie
&
H y p o d e r m is
r e g io n
g
N o n c o n tr a c tile
’re g io n
Infected
C
Not infected
I n te s tin e
D e v e lo p in g
FIGURE 4 I N. m aris p ro fu n d i infection induces host m uscle
decom position. (A-C) D iagram s b a se d on m any TEM m icrographs
integrated to sh o w body wall m u scle d ecom position by N.
marisprofundi. C, cuticle; H, hypoderm is; S, sarco m e re s; MN, m uscle
nucleus; GT, genital tra c t, DE developing e g g , MNC m u scle
non-contractile region. M icrosporidia sp o re s a re re p re sen te d by gray
ovals. (D) A Nomarski DIC tra n sv e rse sectio n of an infected fem ale
harboring sp o re s (arrow). (E,F) TEM m icrographs of infected (E) and
et al., 1987) or by depo sitio n o f sporoplasm s in to developing
oocytes via spores localized w ith in ovaries (Terry et al., 1997).
Sexual transm ission has been hypothesized to occur in som e
species o f m icrosporid ia, b u t is n o t th o u g h t to be co m m o n or
im p o rta n t in tran sm issio n , even in observed cases (G oertz and
H och, 2011). In contrast to this p arad ig m , we have n o t detected
spores in the fem ale gonads o r inside eggs (n = 94 eggs dis­
sected from 12 heavily infected fem ales) b u t rath e r in th e uterus,
betw een the eggs. M oreover, at least five infected D. marci m ales
h ad spores in the vas deferens an d cloaca (F igure 3C ). The m o st
Frontiers in M icrobiology | A q u a tic M ic ro b io lo g y
non-infected (F), w orm s. C, cuticle; M Fs, m u scle filam ents; E,
developing e g g . Developing sp o re s (arrow heads) a re localized near
d e ta ch e d m uscle filam ents (arrow). (G) Sporoblast (SP) and sp o re s in
a cyst-like h o st tis s u e (arrow heads) C, cuticle; E, developing egg. (H)
M uscle filam ents (arrow) and nucleus (arrowhead) decom position in a
severely infected host. (I) M uscle filam ents (arrow heads)
c o n su m e d by
N. marisprofundi. Scale bars are 20 |x m (D); 10|xm (A); 1 p,m
2 |xm (E-G ); 0.5 |j m (I).
com pelling explanation o f these observations is th a t N . marisprofn n d i is tran sm itte d sexually, alth o u g h we c an n o t rule o u t a
vertical tran sm issio n as described fo r Sporanauta periverm is, a
m icro sp o rid iu m recently fo u n d to infect n em ato d es in the low
tide zone o f B o u n d ary Bay beach, C anada (A rdila-G arcia and
Fast, 2012).
N. marsiprofundi DECOMPOSES THE HOST'S BODY W ALL MUSCLES
M icro sp o rid ia have a com plex intracellular life cycle restru c tu r­
ing organelles an d o th er co m p o n en ts o f th e infected h o st cell
February 2014 | Volum e 5 | A rticle 43 | 6
Sap¡r e t al.
to su p p o rt the parasite m etabolism , developm ent, an d re p ro ­
d u ctio n (Troem el et al., 2008). To u n d e rsta n d the infection
dynam ics, we used in situ fluorescent h y bridization (FISH) w ith
N . m arisprofundi SSU rRN A probes an d T ransm ission E lectron
M icroscopy (TEM ). Fluorescent signal localization to the sporecon taining areas (F igu re 3G) d em o n stra ted th a t th e intracellular
stages are co-localized w ith spores in the fem ale h o st u terus
o r adjacent m uscle cells. TEM analysis o f m an y th in sections
o f three infected fem ales an d one n o n -in fected fem ale co n tro l
revealed th a t the parasite targets p rim arily b o d y wall m uscle cells
and possibly th e adjacent hypoderm is; yet parasites are absent
from oocytes, developing eggs, an d th e in testine (F igures 4A -F ).
Infection often induces th e fo rm atio n o f a cyst-like stru ctu re
com prising intracellular stages (sporonts an d sporoblasts) and
disorganized tissue, presu m ab ly c o m p o n en ts o f the infected cells
(F ig u res4 G ; S4; M ovie S I). Typically, a n em ato d e b o d y wall
m uscle cell com prises a contractile region, p rim arily u n derlying
the cuticle, and an in n e r n o n -co n tractile region. The c o n trac­
tile region includes repeating units o f th in and th ick filam ents
th a t are b u n d led in a stereotypic o rg anization to generate the
m echanical force req u ired for th e w o rm ’s lo co m o tio n . The sta n ­
d ard cellular processes are taking place in th e n o n -co n tractile
region w here organelles such as nucleus an d m ito c h o n d ria are
distributed. A lthough the n u m b e r o f anim als analyzed b y TEM
w as lim ited by the challenges o f sam ple p rep aratio n , th e few a n i­
m als exam ined exhibited a correlation betw een the n u m b e r and
d istrib u tio n o f spores an d the severity o f targ et cell decom posi­
tion. In m oderately-infected w orm s, w here spores are confined
in to a cyst-like structu re, m uscle deco m p o sitio n was restricted
to the n o n -co n tractile p a rt o f the b o d y wall m uscle (F igure 4E).
In m ore severe cases o f infection, w here spores are spread in the
entire m id -b o d y o f the host, deco m p o sitio n o f m uscle filam ents
was observed (Figures 4 H ,I). T hin cross sections th ro u g h devel­
oping and m atu re spores revealed th eir u ltrastru c tu ra l details
inclu ding tw o nuclei, a p o ste rio r vesicle, an d three to five coils
o f the polar filam ents (n = 15) (F igure 5).
Collectively, these analyses suggest th a tN . marisprofiindi infec­
tio n starts by invasion th ro u g h th e genitals (i.e., vulva an d cloaca),
targeting the b o d y wall m uscle cells w here the parasites com plete
intracellular phases, m ain ly in the n o n -co n tra c tin g region o f the
m uscle cells (F igure 4E). Spores th en translocate to the genital
tra c t for a new infection cycle (F igure 5).
Nematocenator PARASITES ARE PRESUMABLY FO UN D IN G M EM B ER S
OF A N E W BASAL M IC R O SPO R ID IA CLADE
To u n d erstan d the ev o lu tio n ary origin o f N . m arisprofundi, we
have tested phylogenetic relationships to k now n m icro sp o rid ia
species th a t occupy m arin e, fresh w ater, an d terrestrial habitats.
Based on m olecular data an d h o st association, m icro sp o rid ia
can be divided in to five clades (V ossbrinck an d D eb ru n n erV ossbrinck, 2005). D ue to th e scarcity o f N . sp. l ’s nem ato d e
host, we could recover only a p o rtio n o f its rD N A , w hich
w as used to show its close relationship w ith N . marisprofiindi
(F igure 6). We p erfo rm ed com prehensive phylogenetic analy­
ses w ith N. marisprofiindi rD N A and inclu d ed representative
species from all five clades, in cluding th e five species th a t
exhibit the highest sequence sim ilarity to N . marisprofiindi by
w w w .fro n tiers in .o rg
M icrosporidia parasitism In deep-sea m ethane seeps
FIGURE 5 I N. m arisp ro fu ndi spore m orphology. (A -D ) TEM m icrographs
of 70 nm longitudinal c ro ss sectio n s show ing N. marisprofundi spore
morphology. (A) W hole m ature spore with a typical colls of th e polar
filam ents. Coli num ber varies b e tw e e n th re e and five (n = 15 sp o res
Imaged), no sp o re dim orphism has been observed. PV, posterior vacuole
(arrow); N, nucleus; PFs, four colls of th e polar filam ents (lines); AD,
anterior disc (arrowhead), GT genital tract. (B) A c lose up of th e central
region show ing th e tw o nuclei (N) and four Isot Ila r polar filam ent colls
(arrows) (C) The lamellar polaroplast (arrow) underneath th e anchoring disc
(arrowhead) at th e anterior end of th e spore. (D) Exospore (Ex) Endospore
(En), plasm a m em brane (Pm) at th e spore periphery, and a row of
ribosom es adjacent to a nucleus (arrow heads). N om enclature of spore
c o m p o n e n ts Is ba se d on (Vavra and Larsson, 1999). Scale bars are 1 p m
(A); 200 nm (B); 100 nm (C,D).
BLAST analysis (F igure 6). Intriguingly, b o th m ax im u m like­
lih o o d an d Baysian analyses conco rd an tly place N . marispro­
fiin d i as the basal an d plausibly fo u n d in g m em b er o f a clade
h erein in terp re te d to be at the ra n k o f new genus w ithin
m icro sp o rid ia, n o t closely related to any o th er k n o w n genera. The
N em atocenator lineage is pred icted to b ra n c h at the tran sitio n
from th e m o st basal gro u p o f bryozo an -in fectin g m icrosporidia
(C an n in g et al., 2002; M o rris et al., 2005) an d species th a t infect
o th e r phyla such as nem atodes, arth ro p o d s, an d chordates. The
p o sitio n o f N em atocenator relative to o th er n em ato d e-infecting
m icro sp o rid ia (F ig u re 6; Troem el et al., 2008; A rdila-G arcia and
Fast, 2012) dem o n strates in d e p e n d e n t instances o f nem ato de
p arasitism b y m icrosporidia, w ith the deep-sea event likely occur­
rin g m u c h earlier in m icro sp o rid ia evolution or w ith h o st spe­
cialization having occurred late in this ancien t m icrosporidia
lineage.
DISCUSSION
We have discovered a tem porally-stable p arasitic association of
m icro sp o rid ia an d n em ato d es in deep-sea m eth an e seeps. O u r
results d em o n strate th a t m eth an e seep ecosystem s su p p o rt n o t
only p rokaryotes an d anim als b u t also fungal parasitism . To o u r
February 2014 | Volum e 5 | A rticle 43 | 7
Sapir et al.
M icrosporidia parasitism In deep-sea m ethane seeps
Association Key:
(&
■w
Bee
Beetle
tl
WBryozoan
y^V ^
Cricket/
G rasshoper
»
Nosema bombi
t
100 Nosema œ ranae
Nosema apis
N o se m a anis
Vittaforma
corneae IJjJ
Sporanauta perivermus\
Encephaiitozoon lacertae
■Pleistophora ovariae
Vavraia oncoperae
Glugea anomala c Q |
■Microgemma caulleryi <z&Nadelspora
c ^ >
Crustacean
Fish
Ö
Human
Lizard
ÉÉÉ
Shallow
water
nematode
ip r
C la d e IV
C la d e I
canceri
Mosquito
1 oo .— N em a tocida p a ris ii
É&
1 0 0 1N em atocida sp.1 ^ y
C la d e II
Ovavesicuia popilliae
100 f—Paranosema grylli
100*-— Antonospora scoticae
jOfAmblyospora khaliulini
■Amblyospora c in e r e i- ^ f
C la d e I
1g-^-Amblyospora indicola- jf e
ML
■Amblyospora creniferajsfe
BY
Hazardia milleri
______________________ 100/------- I. sp. 1
1 0 0 L—
'
NJ,em a tocen ato r
m a ris p ro fu n d i n. gen. n. sp.
y 00r Trichonosema aigonquinensis
WO'-Trichonosema pectinatellae ^
100
100.--------- Schroedera plumatellae I
C la d e V
' 9 9 ---------Janacekia debaisieuxi iju
■Bacilllidium vesicuioformis
V_ Pseudonosema cristatellae
Conidiobolus corronatus
Fungi
Basidiobolus ranarum
(outgroups)
D eep-sea
Nematode nem atode _
r
V_^\ioo,
0.2
FIGURE 6 I Phylogenetic analysis o f N. m arisprofundi. A com bined
maxim um likelihood and Bayesian analysis of th e small rlbosom al DNA
from sele c te d microsporidia and c lo se relatives. Two zygom ycete fungal
taxa w e re u sed a s o u tg ro u p s (see M ethods). The five major
mlcrosporldlan clad es are noted w ith Roman num erals (after Vossbrlnck
know ledge, N . m arisprofundi is the first fungal path o g en show n
w ith its h o st to be p a rt o f deep-sea m eth an e seep ecosystems. O u r
findings highlight the involvem ent o f a new player, m icrosporidia,
in deep-sea biom ass-rich c h em o au to tro p h ic fo o d webs.
We tried b u t were unable to establish a stable h ost-parasite
culture in the lab b ey o n d 6 weeks, w here w orm s were kept at
4°C in sem i-dysoxic conditio n s created b y displacing atm o sp h eric
air w ith nitrogen (see S u p p o rtin g In fo rm a tio n for details). P ost­
m o rte m analysis revealed th a t fo u r o u t o f the seven w o rm s in this
culture were infected w ith m icro sp o rid ia. We could n o t detect
any behavioral or lo co m o to ry differences am o n g the nem atodes
in this culture, b u t o u r analysis was necessarily lim ited. O u r abil­
ity to keep N . marisprofiindi infected hosts alive in th e lab o rato ry
for at least 6 weeks suggests th a t N . m arisprofiindi-induced dis­
ease m ay be m ore chronic in n atu re th a n th e acute patho lo g y
induced for exam ple b y N . parisii, w hich kills 50% o f its C. eIe­
gans h o st w ith in 127 h o f infection (Troem el et al., 2008). It is
possible, therefore, th a t N. marisprofiindi's life cycle inside h ost
Frontiers in M icrobiology | A q u a tic M ic ro b io lo g y
and Debrunner-Vossbrlnck, 2005). The ecological associations of
mlcrosporldlan taxa are noted with colored Icons. M axim um likelihood
bootstrap support values are reported above branch n o d es and
concordant Bayesian posterior probabilities are reported below each node.
Support values < 7 0 w ere not reported.
cells is significantly slower th a n th a t o f o th er m icro sp oridia and
th a t th e large n u m b e r o f spores observed in m an y infected ani­
m als is th e outco m e o f m an y m o n th s, o r even years o f infection. In
su p p o rt o f this hypothesis FISH and TEM analyses rarely resulted
in detecting developing stages inside infected cells, suggesting
th a t spores are th e p ro m in e n t stage o f the N . marisprofiindi life
cycle. The TEM analysis, however, revealed th a t the parasitic
infection induces body-w all m uscle d eg rad atio n b y d ecom pos­
ing cellular c o m p o n en ts in clu d in g m uscle filam ents. T herefore,
it is likely th a t N . marisprofiindi reduces h o st fitness significantly,
at least in severe infection cases (F ig u re 4 H ). Because the n e m a ­
tode h o st is the second m o st ab u n d a n t anim al species at som e
H R hab itats (F igure 2), m icro sp o rid ia in fection m ay have gross
in d irect effects on the H R fo o d web, for exam ple b y affecting the
abundance o f th e host, its pred ato rs, an d its b acterial prey.
M any free-living o r p h o retic terrestrial n em ato d es in clu d ­
ing C. elegans are h erm a p h ro d itic an d isolated from the w ild
prim arily as n o n -re p ro d u c in g d au er larvae (Felix an d Braendle,
February 2014 | Volum e 5 | A rticle 43 | 8
Sap¡r e t al.
2010). In contrast, H R n em ato d es are m o st likely gonochoristic (m ale-fem ale) an d are fo u n d in th e m eth an e seeps p rim arily
as rep ro d u cin g adults. The obligate sexual rep ro d u c tio n and
abu n dance o f ad u lt stages o f the n em ato d e h o st m ay shape the
transm ission strategy o f the parasite, con strain in g it tow ard ver­
tical or sexual transm issio n in co n trast to the co m m o n fecal-oral
transm ission from en v iro n m en tal spores. A h o rizo n tal tra n sm is­
sion strategy relying on an oral-fecal cycle w ould pro b ab ly have
resulted in a low ratio o f infecting spores p er to tal n u m b e r of
spores, reducing the fitness o f the parasite. R ep ro d u ctio n th a t
requires direct contact betw een organism s from the sam e species
could be a m u ch m ore efficient tran sm issio n strategy in deep-sea
env ironm en ts such as H R , w here fo o d is plen tifu l an d the n e m a ­
todes are very ab u n d an t. V ertical tran sm issio n is the strategy of
m an y m icro sp o rid ia species in cluding the recently characterized
m icro sp o rid ia th a t infect n em ato d es at the low tide zone (ArdilaG arcia and Fast, 2012). However, spore d istrib u tio n in m ale and
fem ale genitals, the conspicuous absence o f m icro sp orid ia and
spores from gonads, eggs an d eggshells, in ad d itio n to the lack
o f FISH signal in gonads and developing eggs all su p p o rt a sexual
transm ission strategy. We could n o t test this hypothesis directly
by m atin g -in d u ced infection, due to th e n o n -cu ltu ra b le n a tu re of
the sam ples.
M icrosporidia bioenergetics is p ro p o sed to rely on the h o st’s
ATP and, at least in som e cases, o n substrate-level p h o sp h o ry ­
lation (K atinka et al., 2001). M icro sp o rid ia are fo u n d in m any
env ironm en ts includin g terrestrial niches w here oxygen concen­
tra tio n is relatively high. However, a negative correlation betw een
N . marisprofiindi abundance in H ydrate Ridge an d oxygen levels
m ay represent an ad ap ta tio n o f N . marisprofiindi to an aero ­
bic m etabolism advantageous in oxygen-depleted environm ents
such as oxygen m in im u m zones on co n tin en tal m argins, an d in
m eth an e seeps an d h y d ro th erm a l vents. Seep sedim ents su p p o rt
anaerobic m eth an e oxidation featu rin g highly dysoxic porew aters w hereas in hy d ro th erm al vents gross tem p eratu re v ariations
result in very low levels o f dissolved oxygen in specific h abitats
(M cM ullin et al., 2000). Strikingly, D. marci has been fo u n d p re ­
viously in hy d ro th erm a l vents in the Lau Basin o f th e East Pacific
Rise (Verschelde et al., 1998), suggesting a w ide range d istrib u ­
tio n o f the ho st n em ato d e in the Pacific O cean. M oreover, the
tw o sequences m o st closely related b y BLAST search to th e h o st
D. marci SSU rD N A are o f un id en tified n em ato d e species recov­
ered from deep-sea hy d ro th erm a l vents at th e M id A tlantic Ridge
(L opez-G arcia et al., 2003) (Figure S4). This raises the possibil­
ity th a t along w ith th eir hosts, m icro sp o rid ia parasites are w ide
spread in ch em o au to tro p h ic niches b u t dispersal o f th e parasite
by spreading o f the h o st over great distances needs fu rth e r study.
A detailed characterization o f eukaryotic ho st-p arasite in te r­
actions in the deep sea is cu rren tly lim ited to a few cases such
as the m ussel-infecting epizootic fungus fo u n d in a Fiji Basin
h y d ro th erm al ven t (Van D over et al., 2007) an d trem ato d e p a r­
asitism o f m ussels at h y d ro th erm a l vents an d cold seeps (M oreira
and L opez-G arcia, 2003). O u r findings p resen t a new perspective
on the abundance, n atu re, and ecological significance o f deep-sea
parasitism by placing nem atodes, one o f th e m o st a b u n d a n t an i­
m al phyla in m any deep-sea settings, as a h o st for m icro sp o rid ia
parasites. We propose th a t parasitism is m o re w idespread in
w w w .fro n tiers in .o rg
M icrosporidia parasitism In deep-sea m ethane seeps
deep-sea enviro n m en ts th an previously considered. The ap p ar­
en t p au city o f deep-sea parasitism is due n o t only to lim itations
in sam pling (Jones, 2011), b u t because som e parasites such as
m icro sp o rid ia can be easily overlooked due to extracellular phases
th a t appear bacteria-like. M oreover, deep-sea biodiversity stu d ­
ies rely p rim arily on the use o f degenerate PCR p rim ers lim ited
b y design to detect taxa w ith conserved SSU rD N A sequences.
The lack o f am plification o f N . marisprofiindi either w ith degen­
erate “universal” eukaryotic p rim ers or w ith fungal-specific ITS
p rim ers (data n o t show n) suggests th a t broad-scale biodiver­
sity surveys o f extrem e enviro n m en ts are able to recover only
som e o f the diversity th a t occurs in these habitats. In addition
to p rim e r bias, lack o f detection m ay stem from insufficient lysis
o f the fungal spores d u rin g D N A extraction. The recent discov­
ery o f m icro sp o rid ia-in fected n em ato d es at the low er tide zone
(A rdila-G arcia an d Fast, 2012) suggests th a t m icro sp orida p a r­
asitism is a characteristic o f v arious m arin e en v iro n m ents and
is p resu m ab ly m u c h m o re w idespread in th e m arin e realm than
previously considered. M icro sp o rid ia infections o f nem atodes at
H R m eth an e seeps indicate th e p o ten tial for un id en tified cases of
fungal parasitism in deep-sea chem osynthetic environm ents.
MATERIALS AN D METHODS
SAM PLE COLLECTION A N D NEM ATODE RECOVERY
Samples for n em ato d e analyses were recovered p rim arily from
sulfide oxidizing m icrobial m ats o r authigenic carbonates col­
lected from H ydrate Ridge N o rth , H ydrate Ridge South, an d East
K noll (see F ig u re 1 an d Table SI for site co o rd in atio n) on two
research cruises on R /V A tlantis in 2010 an d 2011 using the
H O V Alvin o r the ROV Jason II. U p o n sh ipboard recovery all
sam ples were stored at 4 °C (close to in situ tem p eratu re) u n til
fu rth e r processing (w ithin 2 -7 h o f recovery). N em atodes were
hand p ick ed from w ashed an d sieved (300 p.m) sam ples using for­
ceps u n d e r a dissecting m icroscope (Leica) an d stored in cold 4%
form aldehyde o r 2.5 gluteraldehyde and filtered seaw ater buffer,
o r alternatively in a 50:50 ethanol: filtered seawater. A dditionally,
b u lk m icrobial m a t sam ples, ro ck anim als th a t were recovered by
ro ck w ashing an d in cu b atio n in sea w ater for 24 h, an d crude sed­
im en ts were collected an d stored at 4°C in 50 m l falcon tubes
for live sam ple so rtin g u p o n re tu rn to C alifornia Institute of
Technology or Scripps In stitu tio n o f O ceanography. We exam ined
th e p o ten tial o f parasite spreading in th e deep sea b y coloniza­
tio n o f infected hosts b y placing rock, w ood, an d cow bones next
to active seeps and non-active area o f H ydrate Ridge N o rth and
S o u th for a yearlong colonization ex p erim en t startin g in A ugust
2010. In Septem ber 2011 these substrates w ere collected from the
sites, w ashed in sea w ater to recover associated n em atodes th at
were fixed in eth an o l o r form alin. N ext, D. marci w orm s were
sorted an d exam ined for th e presence o f m icro sp o ridia infec­
tio n as described above. For a detailed descrip tio n o f w orm and
species n u m b e r p e r site, an d n u m b e r o f w o rm s th a t were an a­
lyzed in each assay see Table SI and S u p p o rtin g Inform ation.
To establish a dysoxic culture, live w orm s were individually
picked an d tran sferred to glass culture bottles h alf filled w ith
sea w ater an d sed im en t from H ydrate Ridge South. The bottles
were sealed w ith ru b b e r caps th e n flushed w ith n itro g en to dis­
perse residual atm o sp h eric air and k ept at 4°C in the d ark and
February 2014 | Volum e 5 | A rticle 43 | 9
Sapir et al.
visualized every w eek for a b o u t 3 m o n th s. N em atodes viability
was determ in ed based o n th eir lo co m o tio n an d after 6 weeks one
bottle containing seven live w orm s was analyzed for infection
percentage.
NEM ATODE rD N A A M PLIFIC A TIO N A N D SEQUENCING
Small ribosom al su b u n it 18S (SSU) rD N A o f H R nem atodes was
isolated, am plified, an d sequenced as described b y H o lterm an
et al. (2006) an d in S u p p o rtin g In fo rm a tio n M aterials and
M ethods. N em atode accession n u m b e rs in NCBI: D. marci
JX463180; Desmodora sp. 9 JX463181; Prochaetosoma sp. 10
JX463182.
M icrosporidia parasitism In deep-sea m ethane seeps
T R A N S M IS S IO N A N D S C A N N IN G ELECTRON M IC ROSCOPY
N em atodes for scanning electron m icroscopy were handpicked,
fixed, an d critical p o in t d ried before im aging. For transm ission
electron m icroscopy, crude sam ples w ere fixed in 2.5% gluteraldehyde in sea w ater an d stored in fixative solution at 4°C. Before
em bedding, sam ples were w ashed in PBS three tim es, fem ale
D. marci w orm s were picked and m o u n te d o n a th in film of
2% agar on slides an d were exam ined for spores b y N om arski
D IC m icroscopy. D etailed tran sm issio n an d scanning electron
m icroscopy m eth o d s are in S u p p o rtin g In fo rm atio n M aterials
an d M ethods.
AUTHOR CONTRIBUTIONS
N. marisprofundi rD N A A M PLIFIC A TIO N A N D SEQUENCING
Individual n o n -in fected an d infected w orm s w ere so rted for PCR
analysis. A buffer-only PC R was always p erfo rm ed in parallel
as a negative co n tro l as well as D N A o f a n o n -D e sm o d o rid a
w o rm species belonging to E noplea class isolated from the same
site th a t were n o t infected b y m icrosporidia. Samples were
only used for cloning an d sequencing if th e negative controls
gave no signal by gel electrophoresis. Positive PCR p ro d u cts
were cloned in to the T O PO TA vector (Invitrogen) and inserts
were sequenced using p rim ers th a t flank th e in sert o f the vec­
tor. D etailed in fo rm a tio n on p rim ers an d m eth o d s used is in
S up p o rtin g In fo rm atio n M aterials an d M ethods. A final contig
o f 1165 b p o fN . marisprofundi SSU rD N A sequence was reconsti­
tu ted and su b m itted to G enebank- accession n u m b e r JX463178.
N em atocenator sp. 1 SSU rD N A w as recovered using the same
m eth o d o lo g y b u t we were able to o b tain only 575 bp o f h ig h q u al­
ity sequence th a t was su b m itted to G enebank- accession n u m b er
JX463179.
FISH REACTIONS OF D.marci W O R M S
Probes were designed against regions o f th e rib o so m al sequence
specific to N . marisprofiindi an d were synthesized w ith a Q uasar
570 (Cy3) 5' m odification an d H PLC pu rified b y Biosearch
Technologies, Inc. FISH was th en p erfo rm ed on infected and n o n infected w orm s essentially as described (Troem el et al., 2008) w ith
som e m odification describe in S u p p o rtin g In fo rm a tio n M aterials
and M ethods.
CALCOFLUOR W H IT E S TA IN IN G OF SPORES
C alcofluor W hite (Faluka) staining o f infected anim als and
C alcofluor W hite-based spore survey o f e n v iro n m en tal sam ­
ples collected from H ydrate Ridge is describe in S u p p o rtin g
In fo rm atio n M aterials an d M ethods.
MORPHOLOGICAL NEM ATODE STUDY
N em atode individuals from 50:50 ethanol: sea w ater sam ples were
slowly evaporated to anhydrous glycerol before m o u n tin g on glass
slides follow ing (Som erfield an d W arw ick, 1996). N em atodes
were identified u n d er a c o m p o u n d m icroscope w ith differential
interference co n trast illu m in atio n to species level using p ictorial
keys (Platt an d W arw ick, 1988), an d th e NeMys database (D eprez
et al., 2005) w ith identification keys to the fam ilies D esm odoridae
and D raconem atidae.
Frontiers in M icrobiology | A q u a tic M ic ro b io lo g y
V ictoria J. O rp h a n , Lisa A. Levin, Paul W. S ternberg, A dler R.
D illm an, an d A m ir Sapir conceived an d design the experim ents
an d analyzed the data. Lisa A. Levin, B enjam in M . G rupe, and
V ictoria J. O rp h a n collected, processed, an d analyzed the sam ples
shipboard. A m ir Sapir, A dler R. D illm an, Stephanie A. C onnon,
an d B enjam in M. G rupe processed, so rted o u t an d analyzed
n em ato d es an d o th er anim als from the sam ples in th e lab. M anuel
M u n d o -O cam p o an d James G. B aldw in co n d u cted SEM analy­
sis, Jeroen Ingels, James G. Baldw in, A dler R. D illm an and A m ir
Sapir con d u cted w o rm tax o n o m y an d analysis o f w o rm m o r­
p h o lo g y changes u p o n infection. A m ir Sapir did the FISH reac­
tions, C alcofluor W hite staining, and the TEM im aging. A dler R.
D illm an co n d u cted n em ato d e an d m icrosp o rid ia phylogeny stu d ­
ies an d w o rm diagram draw ing. A m ir Sapir, A dler R. D illm an,
V ictoria J. O rp h a n , Lisa A. Levin, an d Paul W. Sternberg w rote
the paper.
ACKNOW LEDGMENTS
We th a n k captain, crew, A lvin an d Jason II pilots, and cruise
p articip an ts o f RV A tlantis legs 15-68 an d 18-10 for assistance
w ith sam ple collection. We th a n k A lasdair M cD ow all fo r excel­
len t TEM assistance, N athalie De H auw ere an d the Flanders
M arine In stitu te for draw ing th e H R m ap, Stephen M eisenheiter
for w o rm picking, Em ily Troem el for sharing reagents, John
C u rin g to n for Latin g ra m m a r advice, Katja G uilini for shar­
ing sam ples, slides, pub lish ed data a b o u t H R n em atodes, James
Becnel for co m m en ts on th e m an u scrip t, an d G reg Rouse for E4
ro ck photo. T his w o rk was su p p o rted b y the H ow ard H ughes
M edical Institu te, w ith w hich Paul W. Sternberg is an investi­
gator, sam ple collection was su p p o rted b y NSF O CE 0826254
to Lisa A. Levin an d NSF OCE-0825791 to V ictoria J. O rp h an ,
an d an N IH U SPHS T raining G ran t (T32G M 07616) to Adler
R. D illm an. Jeroen Ingels is su p p o rted b y a M arie C urie IntraE uropean Fellowship w ith in th e 7 th E uropean C o m m u n ity
F ram ew ork P ro g ram m e (G ran t A greem ent FP7-PEO PLE-2011IEF N o 300879).
SUPPLEMENTARY MATERIAL
The S u p p lem en tary M aterial for this article can be fo u n d online
at:
h ttp ://w w w .ffo n tiersin .o rg/jo u rn al/1 0 .3 3 8 9 /fm icb.2014.
00043/abstract
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Conflict o f Interest Statem ent The authors declare that the research was con­
ducted in the absence of any com m ercial or financial relationships th at could be
construed as a potential conflict of interest.
Received: 2 9 N o ve m b er 2013; p a p er p e n d in g p ublished: 14 D ecem ber 2013; accepted:
21 la n u a r y 2014; p u b lish e d online: 10 February 2014.
C itation: Sapir A , D illm a n A R , C onnon SA, G rupe B M , Ingels /, M u n d o -O c a m p o M ,
L evin LA, B a ld w in IG , O rp h a n V J a n d S te rn b e rg P W (2014) M icro sp o rid ia -n em a to d e
associations in m eth a n e seeps reveal basal fu n g a l p a ra sitism in the deep sea. Front.
M icrobiol. 5:43. doi: 1 0.3389/fm icb.2014.00043
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February 2014 | Volum e 5 | A rticle 43 | 12