the size class of meiofauna is generally defined as the portion of the community passing through a 1-mm sieve and being retained on a 32-m sieve . this community comprises protists and metazoan animals that remain small even as adults ( permanent meiofauna ) and animals that temporarily belong to this size class during their larval / juvenile development ( temporary meiofauna ) . as part of the sediment infauna , meiobenthos has been studied extensively worldwide from many different habitats , but less attention has been paid to the hard - substrate epibenthic or epizooic and epiphytal meiobenthos ( giere , 2009 ) . at cold seeps , a variety of geologically diverse , reducing habitats can be distinguished by the presence of microbial mats or macro / megafaunal communities ( see sibuet and olu , 1998 ; levin , 2005 ) . although some animals , such as thyrasid bivalves or siboglinids , inhabit the sediment with only the anterior part of their tubes extending above the sediment surface , siboglinid vestimentiferans , bathymodiolin and vesicomyid bivalves , and sponges can build large physical structures above the sediment surface to create habitat as foundation species for an associated macro- and meiofaunal community . in general , foundation species influence the abundance , composition , and structure of the associated community ( hacker and gaines , 1997 ) and can provide food resources , living space , favorable settlement conditions , refuge from predators , and/or refuge from environmental stress ( see bruno and bertness , 2001 ) . the gulf of mexico ( gom ) was the site of the first discoveries of cold seeps in the 1980s ( paull et al . , 1984 ; kennicutt et al . , 1985 ) , and most studies have been completed at seeps from the upper slope , located shallower than 1000  m , but more recently some have included deeper sites from the lower slope ( see cordes et al . tubeworm bushes , composed mainly of mixed vestimentiferan populations of lamellibrachia lymesi and seepiophila jonesi , were studied from the upper louisiana slope ( bergquist et al . , 2003 ; cordes et al . , 2005 ) . the tube surface area , taken as a measure of habitat size , increased the overall surface between 2.6- and 26-fold over the uncolonized seafloor ( bergquist et al . , 2003 ) . in deeper waters of the lower slope , studied vestimentiferan aggregations were composed primarily of escarpia laminata ( brooks et al . there are a number of foundation species of mussels on the lower slope , and beds may consist of single species such as bathymodiolus brooksi ( at atwater valley ) and bathymodiolus childressi ( mississippi canyon ) , or mixed populations of b. brooksi and b. childressi ( alaminos canyon ) or b. brooksi and bathymodiolus heckerae ( florida escarpment ) ( cordes et al . , 2007 ) . meiobenthic community studies at cold seeps are scarce and restricted mainly to assessments of abundance , biomass , and composition of higher taxa . they cover a wide geographical and depth range , from shallow - water sands at 10  m down to deep - sea muds at 5000  m. they include various types of hydrocarbon gas and oil seep ( montagna and spies , 1985 ; palmer et al . , 1988 ; shirayama and ohta , 1990 ; olu et al . , 1997 ; robinson et al . , 2004 van gaever et al . , 2006 ; sergeeva and gulin , 2007 ; sommer et al . , 2007 ) , gas , oil , and asphalt seeps ( montagna et al . , 1987 ) , gas hydrates ( sommer et al . , 2007 ) , and brine seeps ( powell et al . , 1983 , 1986 ) , but exclusively describe the infaunal meiobenthos from sediments covered by bacterial mats , vesicomyid clams , siboglinid frenulates , and sclerolinum in the periphery of mussel beds . furthermore , some sediments with discharge of methane but devoid of any visible microbial mat or animals have also been studied . in the gulf of mexico , seep meiofauna studies were conducted for the shallow brine seep sand communities at east flower garden ( powell et al . , 1983 , 1986 ; jensen , 1986 ) and the hydrocarbon seep bacterial mat communities at alaminos canyon ( 2200  m ) , green canyon ( about 700  m ) , and atwater valley ( about 2000  m ) ( robinson et al . , 2004 ) . epifaunal foraminiferan communities associated with tubeworm bushes on the upper slope were also examined in detail ( sen gupta et al . , 2007 ) , but no study on the associated metazoan meiobenthos has been carried out so far . this study examines the abundance and higher taxonomic composition of epizooic , permanent , metazoan meiobenthos associated with aggregations of tubeworms and mussels from three different locations : green canyon ( gc ) , atwater valley ( av ) , and alaminos canyon ( ac ) in the gulf of mexico . in addition , non - seep sediment cores were taken in the vicinity of such aggregations at gc . the following questions were addressed : ( 1 ) do abundance and higher taxonomic composition differ between geographical regions ? ( 2 ) do abundance and higher taxonomic composition differ between mussel and tubeworm aggregations ? ( 3 ) is the seep epizooic metazoan meiobenthic community similar to seep infauna or non - seep sediments ? ( 4 ) are there similarities in abundance and higher taxonomic composition of seep and hydrothermal vent communities associated with mussels and tubeworms ? the study was conducted at the three hydrocarbon seep locations : green canyon 852 ( gc , depth 1400  m ) , alaminos canyon 818 ( ac , depth 2800  m ) , and atwater valley 340 ( av , depth 2200  m ) of the lower continental slope of the gulf of mexico ( this issue ) . during two cruises in 2006 and 2007 , a total of 13 samples were taken with the submersible dsv alvin ( 2006 ) and rov jason ( 2007 ) . five samples of each foundation group were collected at two different seep habitats : mussels , m - gc1 , m - gc2 , m - gc3 , m - av1 , m - ac1 and tubeworms , t - gc1 , t - gc2 , t - gc3 , t - av1 , t - av2 . three samples of non - seep sediments were taken as controls ( s - gc1 , s - gc2 , s - gc3 ) in close vicinity ( < 3-m distance ) to seep megafauna communities ( table 1 ) . epifauna collections were carried out with the quantitative sampling devices mussel pot ( a hydraulically actuated sampling pot lined with a net , 531  cm diameter of sample area ; for further detail of the collection device see cordes et al . , 2010 ) for mussel aggregations and bushmaster jr . ( a hydraulically actuated , custom - built sampling device lined with a net and having a 2800-cm diameter of sample area ; for further details , see urcuyo et al . , 2003 ; infauna of non - seep sediment were collected with push cores ( 6.3  cm diameter , 30  cm length ) . samples were separately put into isolated , previously cleaned plastic boxes on the basket of dsv alvin or rov jason , transported to the surface , and recovered on deck of the rv atlantis in 2006 or noaa ship ron brown in 2007 . on board , the macro- and megafauna of bushmaster and mussel pot samples were carefully rinsed with cold 32-m - filtered seawater before we removed them from the samples in order to avoid loss of smaller fauna . mussels and tubeworms of each collection were identified and counted ( table 1 ) . the samples were sieved through a 1-mm sieve to separate macro- from meiofauna . before sieving the samples through a 32-m sieve , we measured the volume of sediment of the entire sample < 1  mm . the larger size fractions were retained for complementary studies by collaborators ( see cordes et al . , 2010 ) . in order to estimate the sediment depth distribution of meiobenthos in the push corer samples , we checked the fraction deeper than 5  cm carefully on board of the ship . because one sample lacked any specimens , and two samples contained only a single nematode , we took the upper 5  cm of these samples , and fixed them in 4% buffered formalin without sieving . the push core sample s - gc1 was split into three parts along the entire length , and one part ( 52  ml ) was used for the present study . the other two samples , s - gc2 and s - gc3 , were split into half , and these parts ( 78  ml ) were used for the present analyses . to extract meiofauna from the sediment , we used a density centrifugation technique with a medium consisting of a silicapolymer ( levasil ) mixed with kaolin ( mcintyre and warwick , 1984 ; veit - koehler et al . , 2008 ) . except for sample t - av1 , all other samples were totally processed and the entire meiofauna community was counted and identified to higher taxon level . sample t - av1 was extremely large ( 7.5  l sediment including meiofauna after sieving through a 1-mm net ) ; therefore , we carefully mixed the entire sample in a bucket , let it settle , randomly took a subsample of 217  ml , and estimated the total abundance from this subsample . we noticed the presence of crustacean nauplii but did not include them in further analyses because they could not be assigned to a specific higher crustacean taxon . we also recorded the protist meiobenthos , but did not include them in this study of permanent metazoan meiobenthos . total abundance of meiobenthos was standardized to 10-cm sample area and additionally to 10-cm surface area of mussel shells and tubeworm tubes . the surfaces of mussels and tubeworms were estimated for the main foundation species b. brooksi , b. childressi , b. heckerae , e. laminata , and lamellibrachia spp . by measurements of lengths and widths for each individual in the collection ( see cordes et al . , 2010 , for methods ) . to test for significant differences in abundances among habitat types in the green canyon samples , data were square - root transformed and bootstrapping was used , as this is a well - proven method when working with a relatively low number of samples and high variances ( 10,000 resamplings each , t - test , 2-sided test , routine ftboot from the package computer intensive statistics ( nemeschkal , 1999 ) . results were classically bonferroni - corrected ( p=/n ; =0.05 ) . to evaluate similarity and dissimilarity among all samples , a bray abundance data from 10-cm sample area were square - root transformed , but were not standardized , to enable us to better recognize differences caused by total abundances , and similarity percentage ( simper ) analysis , analysis of similarity ( anosim ) , and multi - dimensional scaling ( mds ) ordination were performed using primer v5 ( clarke and gorley , 2001 ) . the total abundance of the permanent metazoan meiobenthos associated with mussel and tubeworm aggregations of most samples from three different locations at the northern gulf of mexico ranged from 1 to 8  1 individuals per 10-cm sample area . however , one tubeworm aggregate sample ( t - av1 ) from atwater valley ( av ) revealed a total abundance between one and two orders of magnitude higher ( 447 individuals 10  cm ) than the nine other seep samples . non - seep sediment control samples showed abundance values from 870 to 1523 individuals 10-cm sample area ( table 2 ) . green canyon ( gc ) was the only site where the number of samples was sufficient to compare statistically the abundances among mussel- and tubeworm - associated communities , and among the seep communities and adjacent non - seep sediments . we found no significant difference between mussel and tubeworm meiobenthos abundance ( p=0.190 ) , but significantly lower abundances at both seep communities than in non - seep sediments ( both : p=0.003 ) . the mussel beds at av , ac , and one sample from gc ( m - gc3 ) were built exclusively by b. brooksi . in addition , b. childressi co - occurred in two gc samples , contributing with 50% and 63.2% , respectively , to the total mussel abundance . also , the tubeworm aggregations of all collections were mixed populations of e. laminata and one or two species of lamellibrachia ( table 1 ) . as foundation species forming biogenic habitat , tubeworms and mussels considerably increase the surface area and thus the potential living space for meiobenthos . by estimating the actual surface of the foundation species , we found an increase of surface in both types of aggregations between 1.78- and 6.03-fold . the ratio of sample area to the surface area of tubes / shells was similar between the two biogenic habitat types , but was more variable in tubeworm bushes ( 1.786.03 ) than in mussel beds ( 3.075.46 ) ( table 1 ) . by assuming that the surface of foundation species was the actual living space of associated meiobenthos , we standardized the total abundance of this community to the surface area and calculated even lower densities , between only 1 and 3 individual per 10  cm . again , one tubeworm sample ( t - av1 ) contained much greater densities of meiobenthos ( 20 individuals 10  cm ) ( table 2 ) . t - tests on abundance per surface area of gc samples revealed similar results as calculations per sample area , with similarly low abundances found in the seep habitat types ( mussel and tubeworm : p=0.150 ; seep and non - seep both : p=0.003 ) . the seep metazoan meiobenthic communities were composed of the higher taxa nematoda , copepoda , ostracoda , and halacarida . in addition , nauplii larvae were found in seven out of ten samples with variable abundances but were excluded from analyses because it was impossible to assign them to a specific crustacean taxon . the protist phylum foraminifera was also represented in all seep samples . in all five tubeworm samples from the three different locations , the most prominent taxa were the nematodes , with relative abundances between 57% and 90% , followed by the copepods ( 1043% ) . ostracods and halacarids were relatively rare , often found with relative abundances below 1% and below 0.5% , respectively ( fig . the relative distribution of higher taxa was more variable in mussel bed samples . in three samples ( m - gc3 , m - av1 , m - ac1 ) , nematodes dominated ( 6682% ) , followed by copepods ( 1730% ) , and in two samples ( m - gc1 , m - gc2 ) copepods were most abundant ( 82% and 99% ) . ostracods were present in 4 out of 5 samples , and halacarids in 3 out of 5 samples . in two of these more diverse communities , ostracods and halacarids the non - seep control sediments collected in close vicinity to mussel and tubeworm aggregations at gc additionally harbored the taxon kinorhyncha . the community was composed primarily of nematodes ( 8081% ) , followed by copepods ( 1619% ) , ostracods , halacarids , and kinorhynchs ( all<1% ) . simper and anosim analyses did not demonstrate significant differences between mussel bed and tubeworm aggregation meiobenthic communities at the taxonomic level examined . there were also no significant differences among sites , despite the differences in depth ( gc 1400  m , av 2200  m , ac 2800  m ) ( table 3 ) . however , strong differences were detected between non - seep sediment communities and tubeworm- and mussel - associated communities ( > 74% bray curtis dissimilarity ) , and these differences were significant in the anosim ( r=0.64 ; p=0.040 for tubeworm / sediment ; r=0.81 ; p=0.020 for mussel / sediment ) . multi - dimensional scaling ( mds ) ordination revealed that metazoan meiobenthos from seep habitats and from adjacent non - seep sediments formed distinct groups , with the exception of sample t - av1 , which exhibited relatively high similarity to non - seep communities ( fig . the epizooic metazoan meiobenthic communities associated with tubeworm bushes and mussel beds at cold seeps in the gulf of mexico ( gom ) can be characterized as a community composed of a limited number of higher taxa , including nematoda , copepoda , ostracoda , and halacarida , occurring in remarkably low abundances . these seep communities are similar to epizooic meiobenthic vent communities associated with bathymodiolin mussels or vestimentiferan tubeworms . however , communities associated with biogenic habitats differ from the infaunal communities studied from sands of shallow - water seeps and clays of deep - water seeps , which show much higher abundances compared to the epizooic meiobenthos from our studied sites . tubeworm aggregations and mussel beds are colonized not only by meiobenthos but also by a diverse and abundant macrobenthic community at the gom cold seeps . in these same samples , mussel - associated macrofauna were present in densities between 235.5 and 1196.3 individuals per m ( 0.2 and 1.2 individuals per 10  cm ) and tubeworm - associated macrofauna were between 35.9 and 127.9 individuals per m ( 0.04 and 1.3 individuals per 10  cm ) ( cordes et al . , 2010 ) . in other samples from the upper slope , macrobenthic abundances calculated per sample area ranged from 209 to 9590 individuals per m ( 0.29 individuals per 10-cm ) ( bergquist et al . , 2003 ) , and abundances standardized to the tube surface vary from 4 to 233 individuals per m on the upper slope ( cordes et al . , 2005 ) , and 134607 individuals per m on the lower slope ( cordes et al . , 2007 ) abundances per mussel shell surface from the florida escarpment , a different site in atwater valley , and alaminos canyon were between 160 and 4458 individuals per m ( cordes et al . , 2007 ) . it appears that the macro- and megafauna are relatively well represented in such aggregations , fueled by in situ primary production , whereas small meiobenthic animals are relatively scarce . some shallow - water studies indicate that the interaction between macrofauna and meiofauna is negative for the smaller size class , because adult large animals are potentially predators and/or dislocate meiofauna by movement . in addition , the juvenile macrofauna , temporarily in the meiofauna - size class while growing up , can act as predators or competitors ( bell , 1980 ; lafsson , 2003 ) . also , a recent study at seeps on the norwegian margin revealed a negative correlation between meio- and macrofaunal abundance , and predation pressure was speculated to be the underlying cause for this pattern ( van gaever et al . , 2009 ) . however , whether the seep meiofauna community is regulated by such top - down or bottom - up processes remains to be tested . overall , the abundances and higher taxonomic composition of meiobenthos associated with tubeworm and mussel habitats from cold seeps in this study are quite similar to those at hydrothermal vents ( table 4 ) . the epizooic communities of both environments are low in abundance ( usually below 100 individuals per 10  cm ) and are mostly dominated by nematodes . in addition , communities with equal nematode to copepod distribution ( east pacific rise , 950n region , tubeworm aggregations , gollner et al , 2007 ) , copepod - dominated communities ( this study ; juan de fuca ridge , paralvinella aggregations , tsurumi et al . , 2003 ; east pacific rise 11n region , mussel aggregations , zekely et al . , 2006 ; east pacific rise , 950n region , tubeworm aggregations , gollner et al . , 2007 ) , or foraminiferan - dominated communities ( east pacific rise , 950n region , tubeworm aggregations , gollner et al . , 2007 ) similar to varying higher taxa proportions in mussel aggregations at gc of this study , the tubeworm aggregation at the east pacific rise vent site riftia field also exhibited a high variability ( gollner et al . , 2007 ) . this finding points to a patchy distribution , a common phenomenon , which other studies has been suggested to be related to the inhomogenous occurrence of food , predation , and/or displacement by larger animals ( see giere , 2009 ) . while the present study describes the epizooic meiobenthos from cold seeps , all other meiobenthic seep studies concern the infauna inhabiting seep sediments ( table 4 ) . they range from very shallow sites down to 5000-m depth , come from different geographic regions and a variety of seep types , mostly hydrocarbon gas or gas / oil seeps but also gas / oil / asphalt seeps , gas hydrates , or brine seeps . most samples were taken from sites covered by bacterial mats or colonized by siboglinid tubeworms , or were obtained from underneath clam beds , but sometimes from sites devoid of any microbial or megafaunal community . in addition to different approaches in extraction techniques and size classes included in the meiofauna fraction , large variations also occur in studies in which only part of the meiobenthic community was analyzed . some communities encompass the entire permanent ( metazoan and protist ) and temporary meiobenthos , and some include only parts . overall , so far no trends in abundance according to depth , geographic regions , seep types , or habitat types are apparent . epizooic metazoan meiobenthos from seeps ( 181 individuals per 10  cm ) and vents ( 1976 individuals per 10  cm ) , as well as vent infauna from sediments ( 11075 individuals per 10  cm ) , seem overall to be lower in abundance than infaunal meiobenthos from seeps ( 111292 individuals per 10  cm ) ( table 4 ) . low abundances of seep infauna were detected only in anoxic sediments of the black sea and in some samples from a brine seep at east flower garden banks and at the norwegian margin ( powell et al . , 1983 ; sergeeva and gulin , 2007 ; van gaever et al . , 2009 ) . all other infaunal abundances are at least above 100 individuals per 10  cm and most exceed 1000 individuals per 10  cm ( table 4 ) . the vast majority of epizooic and infaunal vent and seep meiobenthic samples are dominated by nematodes , usually followed by copepods . other dominant taxa include gnathostomulids and plathelminths in highly sulfidic brine seep samples ( powell et al . , 1983 ) and rotifers in gas hydrate samples ( sommer et al . , 2007 ) although in several meiobenthic studies of seeps the nearby non - seep deep - sea samples were found to be lower in abundance than the seep sediment samples ( olu et al . , 1997 ; robinson et al . , 2004 ; van gaever et al . , 2006 ) , our study could not confirm this trend . in general , the abundance of meiobenthos in the deep sea has been found to decrease with depth owing to a decrease in pom flux in addition to sedimentary factors such as calcium carbonate content and sorting ( see soltwedel , 2000 ) . ranges between 100 and 1000 individuals per 10  cm at shallower depths and between 10 and 100 individuals per 10  cm at deep sites are considered quite typical ( see giere , 2009 ) . a very large data set from the gom deep - sea meiobenthos , carried out at between 200- and 3000  m depth , indicated a range between 600 and 9500 individuals per 10  cm ( baguley et al . , 2006 ) . calculated from the correlation between abundance and depth , approximately 2500 individuals per 10  cm are expected in about 1500-m depth ( baguley et al . , 2006 ) . this estimate is much higher than the actual abundances ( 8701523 individuals per 10  cm ) in our comparable non - seep sediments at a similar depth of 1450  m. the more puzzling result of this study , however , was the remarkably low abundances at seep sites . the fact that meiobenthos associated with similar foundation species at vents is also low in abundance points to a commonality between seeps and vents , and is in sharp contrast to the high abundance of associated seep and vent macrobenthos . since in situ primary production obviously fuels the large - sized community , it seems unlikely that meiobenthos is bottom - up controlled . instead , the interactions with macrobenthos , such as high predation pressure and/or competition , are more likely to be underlying causes , but these hypotheses need to be tested . also , detailed studies on the species richness and diversity patterns of these epizooic deep - sea communities are currently in progress and will help to elucidate the origin and evolution of seep meiobenthos .