Kristina Barz
email: kristina.barz@awi.de

Hans-Jürgen Hirche

H.-D K Franke

H.-J Barz

Hirche

Ecology of mysid shrimps in the Bornholm Basin (central Baltic Sea)

Keywords: Mysis mixta, Neomysis integer, Praunus sp., Mesopodopsis slabberi, Diet, Life cycle, Abundance, Central Baltic Sea, Bornholm Basin

Mysid shrimps are an important trophic link in the food web of the Baltic Sea. In 2002 and 2003 we investigated species composition, distribution, life cycle and prey in the Bornholm Basin (central Baltic). Three species and one genus were identiWed: Mysis mixta, Neomysis integer, Mesopodopsis slabberi and Praunus sp. The dominant M. mixta and N. integer occurred in the whole study area but were more abundant in the peripheral, more shallow regions. Size frequency distribution indicated one generation for M. mixta and probably two for N. integer. Main prey of all size classes in both species was the cladoceran Bosmina coregoni maritima during summer and autumn. In spring and winter also copepods of the species Temora longicornis and Pseudocalanus acuspes were important prey items. Detritus and phytoplankton cells were of minor importance and many individuals had empty guts. Mysid shrimps in the deep basin might be advected from more shallow areas, as the abundance of all species was much lower than in coastal regions. The deep Bornholm Basin does not seem to be a favourable habitat, probably due to the inaccessibility of the oxygen deWcient bottom layer. Under current hydrographic conditions mysids are probably not important predators of zooplankton and thus not competitors of planktivorous Wsh in the Bornholm Basin.

Introduction

Mysid shrimps are widespread all over the world and occupy diverse habitats and water depths [START_REF] Mauchline | The biology of mysids and euphausiids[END_REF]. Several species are living in the Baltic Sea, where special hydrographic conditions may limit their distribution [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF]. The Baltic Sea is the largest enclosed brackish water system worldwide and is characterized by a series of deep basins separated by shallow sills. The salinity is one main factor causing regional diVerences. It decreases from west to east and from south to north and also forms vertical gradients in the deep basins. The Bornholm Basin (BB) in the central Baltic Sea has a strong stratiWcation with a permanent halocline and a summer thermocline. Below the halocline decomposition processes cause anoxic conditions. The pycnoclines form a three-storeyed ecosystem of water masses with diVerent thermal and haline combinations [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF] which is inhabited by the organisms according to their physiological requirements. For example, the zooplankton community in summer is dominated by cladocerans above the thermocline and copepods below it [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF]. Various species-speciWc and ontogenetic intraspeciWc diVerences in the vertical distribution were found [START_REF] Barz | Seasonal development of scyphozoan medusae and the predatory impact of Aurelia aurita on the zooplankton community in the Bornholm Basin (central Baltic Sea)[END_REF][START_REF] Renz | Life cycle of Pseudocalanus acuspes Giesbrecht (Copepoda, Calanoida) in the Central Baltic Sea: I. Seasonal and spatial distribution[END_REF][START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF][START_REF] Schulz | Living below the halocline: strategies of deep-living species in the highly stratiWed brackish Bornholm Basin (central Baltic Basin)[END_REF]. As many species reach the limits of their distributional and reproductive ranges at diVerent isohalines, species composition is changing all over the Baltic along this gradient.

Salinity in combination with temperature, depth and oxygen concentration at the bottom is also limiting the distribution of mysids (e.g. [START_REF] Salemaa | Life histories, distribution and abundance of Mysis mixta and Mysis relicta in the northern Baltic Sea[END_REF]). Hence, only few of the species occurring in the Baltic are distributed eastwards of the Arkona Basin [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF]. Mysid shrimps are an important element of the food web in the Baltic Sea (e.g. [START_REF] Arndt | Neomysis integer (Leach) in the chain of boddens south of Darß/Zingst (western Baltic)-ecophysiology and population dynamics[END_REF]Rudstam and 123 Hansson 1990). Many species are omnivorous and using pelagic and benthic food they form a link between these environments [START_REF] Mauchline | The biology of mysids and euphausiids[END_REF]. Thus Neomysis integer forms large swarms in the southern Baltic and feeds mainly on zooplankton [START_REF] Jansen | Untersuchungen zur Populationsentwicklung von Neomysis integer (Leach) in der Darß-Zingster Boddenkette[END_REF][START_REF] Arndt | Neomysis integer (Leach) in the chain of boddens south of Darß/Zingst (western Baltic)-ecophysiology and population dynamics[END_REF]. Mysis mixta is one of the major zooplanktivores in coastal areas of the northern Baltic Proper and may consume 20-50% of the zooplankton production annually [START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF]). This makes mysids important competitors of planktivorous adult and larval Wsh [START_REF] Rudstam | On the ecology of Mysis mixta (Crustacea, Mysidacea) in a coastal area of the northern Baltic proper[END_REF]). Mysids feed also on Wsh eggs and larvae and may therefore have an additional eVect on the Wsh stocks (Torniainen and Lehtiniemi 2008). In turn, they serve as an essential food source, for example herring [START_REF] Möllmann | Feeding ecology central Baltic Sea herring and sprat[END_REF]).

The BB is the spawning ground for the commercially important Wsh species cod and sprat [START_REF] Köster | Developing Baltic cod recruitment models. I. Resolving spatial and temporal dynamics of spawning stock and recruitment for cod, herring, and sprat[END_REF][START_REF] Köster | Baltic cod recruitment-the impact of climate variability on key processes[END_REF]. Together with medusae [START_REF] Barz | Seasonal development of scyphozoan medusae and the predatory impact of Aurelia aurita on the zooplankton community in the Bornholm Basin (central Baltic Sea)[END_REF][START_REF] Barz | Scyphozoa in the Bornholm Basin (central Baltic Sea)-the role of advection[END_REF]) and ctenophores [START_REF] Haslob | Invading Mnemiopsis leidyi as a potential threat to Baltic Wsh[END_REF]) mysids are the only abundant invertebrate predators in this region and may have a signiWcant inXuence on the populations of Wsh. Previous studies on mysids in the Baltic were mostly located in the northern area (reviewed by [START_REF] Rudstam | On the ecology of Mysis mixta (Crustacea, Mysidacea) in a coastal area of the northern Baltic proper[END_REF][START_REF] Viherluoto | Food utilisation of pelagic mysids, Mysis mixta and M. relicta, during their growing season in the northern Baltic Sea[END_REF]. For the BB only [START_REF] Salemaa | The distribution and abundance of Mysis populations in the Baltic Sea[END_REF] and [START_REF] Margonski | The distribution, abundance and biomass of Mysis mixta and Neomysis integer (Crustacea: Mysidacea) in the open waters of the southern Baltic Sea[END_REF] gave an overview on occurrence and abundance. Our study within German GLOBEC provided the opportunity to analyse species composition and their spatial and seasonal distribution on a narrow station grid in the BB. Analysis of the food spectrum of diVerent size classes of dominant species should provide insight into the trophic role of mysid shrimps in the BB and potential competition with Wsh.

Materials and methods

Sampling location, dates and hydrography

The study was carried out in the BB, which is one of the deep basins in the central Baltic Sea (Fig. 1). Samples were taken during nine cruises from April 2002 to March 2003 on a station grid of 52 stations (»31-95 m depth). Salinity, temperature and dissolved oxygen were recorded on every station with a standard CTD probe.

Abundance and distribution

Mysid shrimps were collected from a Bongo net (0.6 m diameter, 500 m mesh size) towed in double-oblique hauls integrating the whole water column from surface to »2 m above the bottom. Sampling was independent of time and daylight. Depending on weather conditions not all stations were sampled on every cruise. The samples were preserved in a 4% borax-buVered formalin-seawater solution. In the laboratory the mysids were counted and identiWed to genus or species level. Abundance was calculated for all cruises in 2002 and 2003. Only for the cruise in early July no data on abundance were available. Spatial distribution of M. mixta and N. integer was plotted for end July, August and October 2002 and January 2003. Samples with high abundance were divided with a plankton splitter until the subsample contained a minimum of 300 individuals.

In July and August 2002 additional samples with an IKMT (Isaacs-Kidd Mid-Water Trawl, 6 m 2 opening, 1,000 m mesh size) were taken for comparison with the catch eYciency of the Bongo net. A two-way ANOVA [START_REF] Sokal | Potential predation pressure of littoral mysids on herring (Clupea harengus membras L.) eggs and yolk-sac larvae[END_REF] and a matched pairs test were performed to analyse diVerences between the two net types. The volume of Wltered water in both net types was measured with a Xowmeter.

Body size

Mysid shrimps for body size measurements were collected from preserved Bongo net samples. In March 2003 not enough M. mixta for adequate size analyses were available. N. integer was less abundant, especially during the spring and summer cruises. Therefore, size analyses were not performed in July and August 2002. Total length (TL) was measured from the apex of the rostrum to the posterior end of the telson. All individuals were measured under a stereomicroscope to the nearest millimetre below.

Prey composition

Mysid shrimps for stomach content analyses were collected from preserved Bongo net samples. In March 2003 not enough animals of M. mixta for adequate prey analyses After dissection with a Wne scalpel, the separated stomach and gut were transferred to an object slide in a glycerine-water solution (1:1). Under a dissection microscope, the stomachs were opened with Wne dissection needles and prey fragments separated. For better comparison with prey data from [START_REF] Viherluoto | Food utilisation of pelagic mysids, Mysis mixta and M. relicta, during their growing season in the northern Baltic Sea[END_REF] and [START_REF] Lehtiniemi | Feeding diVerences among common littoral mysids, Neomysis integer, Praunus Xexuosus and P. inermis[END_REF], M. mixta and N. integer were grouped into four size classes (3-6, 7-12, 13-17, 18-21 mm).

Main focus of prey analyses was on mesozooplankton species. For their easier identiWcation in the guts, a digital picture catalogue with fragments (mandibles, antennae, carapaces, characteristic body parts) of potential food items was established. By comparing the undigested fragments from the guts with pictures of potential prey, it was mostly possible to identify the gut content to species level. Thereby only the characteristic parts were used for identiWcation and counting, e.g. two mandibles for one copepod and one pair of antennae for one Bosmina coregoni maritima. However, some mandibles or carapace fragments could not be assigned to prey species and were counted and pooled as unidentiWed objects. The amount (%) of guts containing only or additional material composed of detritus, sand and/or phytoplankton cells was also recorded.

Results

Hydrography

The hydrography of the central Baltic is characterized by stratiWcation. From July to September 2002, a thermocline was found between 15 and 25 m, separating the surface layer from the colder (5-6°C) intermediate water layer. Temperature in the surface layer increased from 16°C in the beginning of July to the maximum summer temperature of 20.8°C in August. In November the surface water had cooled down to 9°C and merged with the intermediate water. Salinity was rather constant between the surface and the halocline ( 7) and increased beneath the halocline up to 15.5. Oxygen concentration decreased rapidly below the halocline to values <2 ml l ¡1 . ProWles of temperature, salinity and oxygen during our sampling period are published, e.g. in [START_REF] Feistel | Warm waters of summer 2002 in the deep Baltic Proper[END_REF], [START_REF] Barz | Seasonal development of scyphozoan medusae and the predatory impact of Aurelia aurita on the zooplankton community in the Bornholm Basin (central Baltic Sea)[END_REF], [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF] and [START_REF] Mohrholz | The impact of exceptionally warm summer inXow events on the environmental conditions in the Bornholm Basin[END_REF].

In late summer 2002, two exceptionally warm inXow events were observed in the BB. One appeared in the BB mainly in September 2002 [START_REF] Feistel | Warm waters of summer 2002 in the deep Baltic Proper[END_REF] and was distributed in the entire BB between 50 and 70 m by November [START_REF] Mohrholz | The impact of exceptionally warm summer inXow events on the environmental conditions in the Bornholm Basin[END_REF]). An autumn inXow reached the BB in November 2002 and was detected in 80-90 m. The temperature maxima of both inXows were measured in the BB during our cruises in October and November 2002 [START_REF] Feistel | Warm waters of summer 2002 in the deep Baltic Proper[END_REF].

Species composition, seasonal development, abundance and distribution

During the study period three species and one genus of mysid shrimps were identiWed in the BB: Mysis mixta, Neomysis integer, Mesopodopsis slabberi and Praunus sp.

M. mixta and N. integer occurred during the whole study period, whereas the other species appeared only sporadically (Fig. 2). From April to October 2002, M. mixta was the most abundant species. Its maximum abundance was reached in October (7.5 § 20.5 ind. 100 m ¡3 ) and decreased strongly afterwards. N. integer showed the same seasonality with lower values until October (max. 1.9 § 4.9 ind. 100 m ¡3 ) but was the most abundant species in November 2002 and January 2003 (Fig. 2). Praunus sp. was observed in April andJuly 2002 andJanuary 2003 (sts. 45, 17 and19, respectively) in low values (max. 0.5 ind. m ¡2 ). Some specimens of M. slabberi appeared in November 2002 on sta. 18 (0.3 ind. m ¡2 ) and were still found in January 2003 (sts. 13, 18, 30; max. 0.9 ind. m ¡2 ). In March 2003 it was not longer detected.

On single stations, the abundance of M. mixta was highest in August and October (Fig. 3). In July they were found nearly in the entire basin with a tendency towards Bornholm Island, whereas in August and October they were mainly located at more shallow stations (31-65 m) in the north-eastern and the southern peripheral regions of the study area (Fig. 3). Maximum abundance was found in October on st. 44 (57.1 ind. m ¡2 ). N. integer was caught in the whole basin with higher numbers in the southern area in August and October. In January 2003, N. integer was mainly distributed oV Bornholm Island (Fig. 3). Maximum abundance was found in October and January (7 ind. m ¡2 ) on sts. 43a and 19, respectively. In April and June 2002 and March 2003 all species were low in numbers and no distribution patterns were recognized.

M. mixta was used to compare the Bongo and IKMT catch eYciency, as it was the most abundant species in July and August 2002 when both nets were deployed. In July and August the Bongo net Wltered an average water volume of 231.8 § 87.8 and 204.0 § 94.5 m 3 , respectively, whereas the volume Wltered by IKMT was 6,174.8 § 1,907.2 m 3 and 5,308.7 § 2,444.8 m 3 , respectively. In both months 16 stations were used for comparison. A two-way ANOVA showed neither an eVect of the gear (P = 0.7494), nor of the cruise date (P = 0.8186) on the abundance of caught M. mixta. This result was conWrmed by a matched pairs test when using all stations (P = 0.1336) and only stations with mysids (P = 0.1350). The values from the IKMT 123 conWrmed the higher abundance of M. mixta in the peripheral regions of the study area as found also in the Bongo net samples. The catch eYciency of the IKMT was expected to be higher than that of the Bongo due to the higher amount of Wltered water which should reduce the inXuence of patchy distribution of mysids. However, the statistical analyses showed no diVerence between the two net types, probably caused by the high variability between stations.

Size distribution

The size of M. mixta varied between 3 and 21 mm (Fig. 4). The consistently increasing median, from 5 to 17 mm between April and November 2002, clearly documents the growth of the population. In January 2003, the median was still 17 mm but there was a group of some specimens larger than in November (Fig. 4).

The size of Neomysis integer ranged from 3 to 19 mm. In April and June only few animals of variable size were measured and the smallest size class (3-6 mm) prevailed. Due to the low abundance, no size data are available for July and August. In October 2002, the median size was 5 mm and increased up to 8 mm in January 2003 (Fig. 4). In March 2003, the median was still 8 mm and the maximum size of 19 mm was reached. During the whole time, the smallest size group of 3-6 mm was found.

The median of body size of M. slabberi was 7 mm in January 2003. Due to rare occurrence only single individuals of Praunus sp. were measured (April 2002: 10 mm, January 2003: 16 and 19 mm).

Prey composition

Up to 36 identiWed prey organisms were found in the guts of individuals of M. mixta, but also many guts were empty (Table 1). UnidentiWed objects were found in almost every month and in all size classes analysed, but prevailed only in a few cases. The cladoceran Bosmina coregoni maritima and the copepod Temora longicornis were eaten by M. mixta and N. integer during the whole study period. From end of July to October 2002, B. coregoni maritima was the main prey item of M. mixta. In October also N. integer fed mostly on this species which can be easily identiWed by its typical Wrst antennae. In spring, late autumn and winter, the preferred prey items of M. mixta were T. longicornis, Pseudocalanus acuspes or B. coregoni maritima, whereas N. integer fed on T. longicornis or P. acuspes but less on Bosmina. Further prey items of both species were the cladoceran Podon spp. and the copepod Centropages hamatus. The amount of detritus, sand and/or phytoplankton in the guts of both species was higher during early spring and winter (April 2002 andJanuary-March 2003).

DiVerences in the prey composition in relation to size were hard to see. However, little diVerences were recognized for the smallest individuals. Almost all maximum values of empty guts were found in the smallest size group of both species (Table 1). The gut content of the smallest M. mixta individuals was less diverse than in the larger ones. Only 10 of 24 smallest M. mixta contained food in their guts. Detritus, sand and/or phytoplankton were of higher importance than in most other size groups. In contrast, for N. integer the ratios of The Wrst inXow appeared in the BB in September 2002 [START_REF] Feistel | Warm waters of summer 2002 in the deep Baltic Proper[END_REF] turned right and spread along the western and southern rim of the basin [START_REF] Mohrholz | The impact of exceptionally warm summer inXow events on the environmental conditions in the Bornholm Basin[END_REF]. By November it was distributed over the entire BB between 50 and 70 m [START_REF] Mohrholz | The impact of exceptionally warm summer inXow events on the environmental conditions in the Bornholm Basin[END_REF]) when the Wrst individuals of M. slabberi were caught. An autumn inXow reached the BB by November 2002 and may also have advected this unusual species, which was present until January 2003 before it disappeared. An eVect on the distribution of the other species was not assumed as they have been found at least in low numbers in the entire basin before these inXows occurred.

Our sampling was independent from daytime and daylight, but always integrating the whole water column. Hence, vertical migration within the water column should not have aVected abundance estimations. However, as previous studies, we did not sample specimens migrating between the seaXoor and the near-bottom layer. On the other hand, the anoxic bottom layer of the deep BB probably is not inhabited by mysids anyway.

M. mixta was the most abundant species from April to October 2002. This is in accordance with [START_REF] Margonski | The distribution, abundance and biomass of Mysis mixta and Neomysis integer (Crustacea: Mysidacea) in the open waters of the southern Baltic Sea[END_REF], who worked in the same area from 1993 to 1996; however, not during winter. Its declining abundance during winter concurs with observations in the northern Baltic [START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF]. M. mixta is described as a pelagic species inhabiting deeper waters. There it lives nectobenthic, is in bottom contact during daytime and performs diurnal vertical migrations [START_REF] Rudstam | Diel vertical migration and feeding patterns of Mysis mixta (Crustacea, Mysidacea) in the Baltic Sea[END_REF][START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF][START_REF] Viherluoto | Food selection and feeding behaviour of Baltic Sea mysid shrimps[END_REF][START_REF] Kotta | Vertical migrations of mysids in the Gulf of Riga[END_REF]. N. integer, Praunus spp. and M. slabberi, in contrast, live in shallow waters of the littoral zone according to [START_REF] Jansen | Untersuchungen zur Populationsentwicklung von Neomysis integer (Leach) in der Darß-Zingster Boddenkette[END_REF], [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF] and [START_REF] Viherluoto | Food selection and feeding behaviour of Baltic Sea mysid shrimps[END_REF]. In northern Baltic coastal waters, M. mixta and N. integer reached maximum abundances of 314 ind. m -2 and 24 ind. m -2 , respectively [START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF]. N. integer in the shallow water (<1 m) of the Darss-Zingst Bodden chain reached even 554 ind. m -2 [START_REF] Jansen | Untersuchungen zur Populationsentwicklung von Neomysis integer (Leach) in der Darß-Zingster Boddenkette[END_REF]. In our study highest abundance of M. mixta was found on the more shallow stations in the north-eastern and the southern peripheral regions in August (max. 25 ind. m -2 ) and October (max. 57 ind. m -2 ), N. integer reached abundance not higher than 7 ind. m -2 in the peripheral region. Thus abundance was much lower than the maxima reported above, but slightly higher than in previous studies by [START_REF] Salemaa | The distribution and abundance of Mysis populations in the Baltic Sea[END_REF] and [START_REF] Margonski | The distribution, abundance and biomass of Mysis mixta and Neomysis integer (Crustacea: Mysidacea) in the open waters of the southern Baltic Sea[END_REF] which found 1-20 M. mixta ind. m -2 near Bornholm Island and in the northeastern and southern area. Both authors proposed the absence of these two mysid species from the deep BB. [START_REF] Salemaa | Life histories, distribution and abundance of Mysis mixta and Mysis relicta in the northern Baltic Sea[END_REF][START_REF] Salemaa | The distribution and abundance of Mysis populations in the Baltic Sea[END_REF]) related these Wndings to the oxygen deWciency in the deep layers. In contrast, we found both species in low numbers distributed all over the basin, although oxygen deWciency was still acute in 2002 [START_REF] Feistel | Warm waters of summer 2002 in the deep Baltic Proper[END_REF][START_REF] Mohrholz | The impact of exceptionally warm summer inXow events on the environmental conditions in the Bornholm Basin[END_REF]) and the deep BB was not a proper habitat for M. mixta and N. integer. According to [START_REF] Salemaa | The distribution and abundance of Mysis populations in the Baltic Sea[END_REF], specimens found in this area might be advected from populations living in more favourable areas. They also suggested the existence of populations not dependent on the benthic habitat to avoid the oxygen deWciency.

Life cycle

The size frequency distribution of M. mixta suggests an annual life cycle as in the northern Baltic Sea, where juveniles are released in early spring, grow through the summer when food is abundant and mature in winter [START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF][START_REF] Salemaa | Life histories, distribution and abundance of Mysis mixta and Mysis relicta in the northern Baltic Sea[END_REF][START_REF] Rudstam | On the ecology of Mysis mixta (Crustacea, Mysidacea) in a coastal area of the northern Baltic proper[END_REF][START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF]). The two size groups found in April probably represent females (18-21 mm) from the foregoing year which recently had released their oVspring (3-6 mm).

For N. integer two generations per year were reported for the Baltic Sea [START_REF] Kinne | Neomysis vulgaris Thomson, eine autökologischbiologische Studie[END_REF][START_REF] Jansen | Untersuchungen zur Populationsentwicklung von Neomysis integer (Leach) in der Darß-Zingster Boddenkette[END_REF][START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF]). Adults of the winter generation reproduce in spring. Their recruits form the summer generation, which is growing fast and matures in July/August at a smaller size (»10 mm) than the over-wintering generation. The scarcity of size data in our study does not allow to completely reconstruct the life cycle of this species in the central Baltic. Adults of the winter generation dominate in April 2002. Unfortunately, size data on the summer generation are completely missing. However, the size distribution in October clearly shows the newly, probably by the summer generation, released brood, which would represent the winter generation. The occurrence of small individuals until January 2003 indicates a successive release of brood through the summer as also found by [START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF]. The source regions of N. integer in the study area are probably the coastal regions from where it has been advected. The size distribution indicates that small individuals might be transported to deeper regions regularly and in higher numbers than larger individuals, eventually due to diVerent ontogenetic vertical distribution and/or lower swimming eYciency.

Prey composition

Based on prey composition, mysid shrimps are classiWed as omnivorous and capable of utilizing a wide variety of food sources (e.g. [START_REF] Rudstam | Diel vertical migration and feeding patterns of Mysis mixta (Crustacea, Mysidacea) in the Baltic Sea[END_REF][START_REF] Viherluoto | Food utilisation of pelagic mysids, Mysis mixta and M. relicta, during their growing season in the northern Baltic Sea[END_REF][START_REF] Lehtiniemi | Feeding diVerences among common littoral mysids, Neomysis integer, Praunus Xexuosus and P. inermis[END_REF]. M. mixta [START_REF] Hansson | Selective predation by herring and mysids, and zooplankton community structure in a Baltic Sea coastal area[END_REF][START_REF] Mohammadian | Are marine planktonic invertebrates food limited? The functional response of Mysis mixta (Crustacea, Mysidacea) in the Baltic Sea[END_REF][START_REF] Viherluoto | Food selection and feeding behaviour of Baltic Sea mysid shrimps[END_REF] and N. integer [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF] showed a preference towards carnivory. This is conWrmed in our study where mainly 123 mesozooplankton species together with some detritus, sand and phytoplankton cells were found in their guts. The food composition of all size classes of both species mainly reXected the seasonal changes in the zooplankton composition. Temora longicornis was the main prey item of M. mixta in June and early July 2002, when CIV-CVI stages of this species reached their maximum abundance [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF][START_REF] Schulz | GLOBEC-Germanyintegrated datasets[END_REF]. During July, M. mixta switched to Bosmina coregoni maritima as main food source, although T. longicornis was still very numerous until August. B coregoni maritima was highly abundant from July to October with mass occurrence in August [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF][START_REF] Schulz | GLOBEC-Germanyintegrated datasets[END_REF]. Its high abundance in summer combined with low escape capability [START_REF] Viitasalo | Zooplanktivory in the Baltic Sea: a comparison of prey selectivity by Clupea harengus and Mysis mixta, with reference to prey escape reactions[END_REF]) made B. coregoni maritima an easy prey for M. mixta and N. integer during this time. In the plankton, B. coregoni maritima is present, however, in small numbers all year round [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF]) and occurred sporadically in the guts also in winter and spring. From November on, when the Bosmina population decreased rapidly, copepods became again a more important food source. Both mysid species fed now mainly on relatively low abundant T. longicornis and on Pseudocalanus acuspes. The main prey items, B. coregoni maritima and T. longicornis, inhabit the upper 30 m and the shallower marginal regions of the BB [START_REF] Hansen | Spatio-temporal distribution and production of calanoid copepods in the central Baltic Sea[END_REF][START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF] and therefore indicate a feeding close to the surface. The vertical distribution of P. acuspes was changing during season [START_REF] Renz | Life cycle of Pseudocalanus acuspes Giesbrecht (Copepoda, Calanoida) in the Central Baltic Sea: I. Seasonal and spatial distribution[END_REF]. As during winter, larger stages were found deep dwelling as well as close to the surface, we cannot distinguish where mysids fed on this copepod species. Anyhow, P. acuspes became more attractive and/or accessible from November on. Interestingly, the two common Acartia species A. biWlosa and A. longiremis, which were also present in the central Baltic Sea all year round [START_REF] Schulz | Spatial and temporal distribution patterns of zooplankton in the central Baltic Sea and methods to detect them[END_REF], were not found in the guts. [START_REF] Viherluoto | Temporal variability in functional responses and prey selectivity of the pelagic mysid, Mysis mixta, in natural prey assemblages[END_REF] and [START_REF] Viitasalo | Zooplanktivory in the Baltic Sea: a comparison of prey selectivity by Clupea harengus and Mysis mixta, with reference to prey escape reactions[END_REF] found negative selection for Acartia spp. by M. mixta in laboratory experiments, probably due to their high escape capability [START_REF] Viitasalo | Zooplanktivory by Praunus Xexuosus (Crustacea: Mysidacea): functional responses and prey selection in relation to prey escape responses[END_REF][START_REF] Viitasalo | Zooplanktivory in the Baltic Sea: a comparison of prey selectivity by Clupea harengus and Mysis mixta, with reference to prey escape reactions[END_REF]). In accordance with our results, copepods and cladocerans were described as the main prey items in former studies [START_REF] Hansson | Selective predation by herring and mysids, and zooplankton community structure in a Baltic Sea coastal area[END_REF][START_REF] Rudstam | Diel vertical migration and feeding patterns of Mysis mixta (Crustacea, Mysidacea) in the Baltic Sea[END_REF]) together with detritus and plant material [START_REF] Rudstam | Diel vertical migration and feeding patterns of Mysis mixta (Crustacea, Mysidacea) in the Baltic Sea[END_REF][START_REF] Lehtiniemi | Feeding diVerences among common littoral mysids, Neomysis integer, Praunus Xexuosus and P. inermis[END_REF]). An ontogenetic shift in food preference of M. mixta from sedimented phytoplankton to mesozooplankton described by [START_REF] Viherluoto | Food utilisation of pelagic mysids, Mysis mixta and M. relicta, during their growing season in the northern Baltic Sea[END_REF], which these authors ascribed to ineYcient handling of mesozooplankton by small mysids (<7 mm), was not absolutely conWrmed here. In April and July but not in June 2002 the amount of detritus, sand and/or phytoplankton was higher in the smallest size group of M. mixta, but also mesozooplankton such as B. coregoni maritima and T. longicornis was eaten in small numbers. It seems that small individuals are indeed able to handle large prey in certain cases, but also many guts remained empty. For the actually littoral species N. integer [START_REF] Lehtiniemi | Feeding diVerences among common littoral mysids, Neomysis integer, Praunus Xexuosus and P. inermis[END_REF] found no clear ontogenetic diet shifts, but benthic and planktonic material in the guts of all size classes. Besides some detritus and sand, mesozooplankton was the main food for all N. integer in our study. The absence of a clear ontogenetic diet shift in M. mixta and the lack of benthic prey in N. integer as well as the many empty guts in smallest individuals of both species make us speculate that oxygen deWciency on the bottom of the deep BB makes the feeding ground near the sediment inaccessible for all size groups investigated.

EVects on Wsh stocks

The BB is an important spawning ground for cod and sprat [START_REF] Bagge | The Baltic cod[END_REF][START_REF] Köster | Developing Baltic cod recruitment models. I. Resolving spatial and temporal dynamics of spawning stock and recruitment for cod, herring, and sprat[END_REF][START_REF] Köster | Baltic cod recruitment-the impact of climate variability on key processes[END_REF]. The larvae of both species [START_REF] Groenkjaer | Ontogenetic and environmental eVects on vertical distribution of cod larvae in the Bornholm Basin, Baltic Sea[END_REF]Voss et al. 2003;[START_REF] Dickmann | Feeding ecology of Central Baltic sprat Sprattus sprattus larvae in relation to zooplankton dynamics: implications for survival[END_REF] as well as the adults of sprat and herring [START_REF] Möllmann | Feeding ecology central Baltic Sea herring and sprat[END_REF]) feed on copepods and on cladocerans and hence compete with the mysids for food in the BB. In addition, mysids feed on Wsh eggs and larvae and may therefore have an additional eVect on the Wsh stocks (Torniainen and Lehtiniemi 2008). In turn, mysids may serve as an important food resource for some size classes of herring especially in winter and autumn [START_REF] Möllmann | Feeding ecology central Baltic Sea herring and sprat[END_REF]). However, compared to coastal areas, e.g. in the northern Baltic [START_REF] Rudstam | Abundance, species composition and production of mysid shrimps in a coastal area of the northern Baltic Proper[END_REF]) all mysid species had a low abundance in the BB which is probably due to the deep and oxygen deWcient bottom layer. It seems that the Baltic mysids strongly depend on access to the sea Xoor, which due to oxygen conditions is only possible in the more shallow parts of the BB. Therefore, under current hydrographic conditions, they are not important in the trophodynamics of the deep BB. However, BB Wsh stocks like herring, which migrate for spawning to more shallow waters with higher abundance of mysids, may beneWt from additional food supply, and suVer from competition for food and/or predation on eggs and larvae. We speculate that under better oxygenated conditions, mysid shrimps could also become an important component in the food web of the BB.

Acknowledgments This study would not have been possible without the support of many colleagues, students and crew members of RV Alkor, Heincke and A. v. Humboldt during several cruises. Special thanks to Ulrike Holtz, Sabine Lenk and Sabine Graf for their help in analysing specimens. We are further grateful to Asta Audzijonytñ con-Wrming the identiWcation of small Mysis mixta specimens, Dr. habil Thomas Brey helping in statistical analyses and the anonymous reviewer for helpful comments on the manuscript. This research was funded by Bundesministerium für Bildung und Forschung (BMBF 03F0320D) in the framework of GLOBEC Germany.

Fig. 1

 1 Fig. 1 Study area in the Baltic Sea with station grid and water depth (m)

Fig. 2 Fig. 3

 23 Fig.2Mean abundance (ind. 100 m ¡3 ) and standard deviation of all species during the study period (nd no data)

Fig. 4

 4 Fig. 4 Size (total length in mm) composition (%) of the populations of Mysis mixta and Neomysis integer during the study period. TL total length, n number of individuals measured, nd no data, grey line median

Discussion

Species composition and distribution

Mysis mixta, Neomysis integer and Praunus spp. are common mysids of the Baltic Sea [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF]. Other species described for the northern Baltic like members of the recently revised Mysis relicta group (Audzijonyte and Väinölä 2005), and the Pontocaspian invader Hemimysis anomala [START_REF] Janas | Hemimysis anomala G.O. Sars 1907 (Crustacea, Mysidacea)-Wrst record in the Gulf of Gdansk (Baltic Sea)[END_REF], were not detected in our samples. The fourth species observed in our study, Mesopodopsis slabberi, is common westwards of the Arkona Basin, where it may occur in high numbers in shallow waters [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF]Wittmann 1992). Although [START_REF] Köhn | Mysidacea of the Baltic -State of the art[END_REF] suggested that mysids are rarely advected by currents, we take the possibility into consideration that two


Maria Balsamo
email: maria.balsamo@uniurb.it

Ae L Guidi

Ae M Ferraguti

Ae L Pierboni

Ae R M Kristensen

H.-D M Franke

L Balsamo

Á L Guidi

Pierboni

M Ferraguti

R M Kristensen

Diuronotus aspetos (Gastrotricha): new morphological data and description of the spermatozoon

Keywords: Diuronotus aspetos, Gastrotricha, Chaetonotida, Ultrastructure, Spermatozoon

The genus Diuronotus (Gastrotricha Chaetonotida) includes two species, D. aspetos and D. rupperti; its morphological affinity with the genus Musellifer has been pointed out. Here, new morphological data from light and electron microscopy and the description of the spermatozoon of D. aspetos are reported, with the aim of clarifying the phylogenetic position of the genus. The mouth cavity has a wreath of stout, protrusible processes. The two secondary furcal tubes are inserted ventrally and are covered with elongate scales. All the caudal tubes contain a duogland adhesive system. Three kinds of ciliated sensory receptors are described for the first time in Diuronotus. The filiform spermatozoon consists of an acrosome, a nuclearmitochondrial complex, and a flagellum. The acrosome including two long and different cones, the single, giant mitochondrion surrounding the nuclear base, and the axoneme arising from a deep nuclear 'fossa' appear as autapomorphic characters. The keeled, solid cuticular body scales and the spermatozoon with a supernumerary membrane are features shared with Musellifer delamarei. The structure of the accessory fibres is a strong spermatological similarity between the families Muselliferidae and Xenotrichulidae. Thus morphological and spermatological characters support the inclusion of D. aspetos and M. delamarei into the family Muselliferidae recently described. The comparative spermatology also suggests that Xenotrichulidae may be the sister group of Muselliferidae.

Introduction

The phylum Gastrotricha includes over 700 meiobenthicsized species, traditionally grouped into the two orders Macrodasyida and Chaetonotida, different in a number of morphological, ecological and reproductive features. All 450 species of Chaetonotida Paucitubulatina share a uniform tenpin-shaped body plan, with two caudal adhesive tubes, and a pharynx with an inverted Y-shaped lumen lacking pores (Remane 1961). Within the Chaetonotida, the families Muselliferidae and Xenotrichulidae are marine, the Chaetonotidae include both marine and freshwater species, while Dasydytidae, Dichaeturidae, Neogosseidae and Proichthydiidae are exclusively freshwater. The monophyly of the strictly freshwater families has never been questioned, and has gained support from an inclusive cladistic analysis of the Gastrotricha based on 81 morphological and reproductive traits (Hochberg and Litvaitis 2000). On the contrary, the phylogenetic relationships within Chaetonotidae and between Chaetonotidae and Xenotrichulidae are still unclear, due in part to the uncertain status of the genus Musellifer, originally assigned to the Chaetonotidae. A sister-group relationship between Xenotrichulidae and Musellifer was hypothesized based on the similarity of the spermatozoa ultrastructure (Guidi et al. 2003;Marotta et al. 2005). However, the muscular architecture of Musellifer greatly differs from species of both Chaetonotidae and Xenotrichulidae (Leasi and Todaro 2008). The close affinity of the two genera Diuronotus and Musellifer, both marine and hermaphroditic, is supported by numerous traits of the external morphology, which has prompted Leasi and Todaro (2008) to create the new family Muselliferidae. The genus Diuronotus includes only two species, D. aspetos and D. rupperti, which have been mainly described on the basis of optical microscopy (Todaro et al. 2005). Here we provide ultrastructural details of several morphological traits of D. aspetos, and a full description of the spermatozoon. The aim of this study is to describe additional details on the anatomy of a species of Diuronotus, and to find possible additional autapomorphies useful to support the family Muselliferidae and its phylogenetic relations.

Methods

Sampling sites

The sediment samples were taken by a mini van Veen grab (225 cm 2 ) at 2.0-2.5 m water depth in the small bay of Itersla (69°26.989 0 N; 52°19.688 0 W), near the settlement of Skansen, Disko Island, West Greenland. The samples were taken on 25th August 2006 during the Arctic Biological Field Course 2006. In the subtidal sandy ripples, the sediment consists of fine, well-sorted sand with detritus. Further information can be found in Kristensen and Niilonen (1982), Kristensen and Nørrevang (1982) and Todaro et al. (2005).

Samples preparation for optical and electron microscopical analysis

The sediment samples were sorted out at Arctic Station, Qeqertarsuaq, immediately after collection. They were next treated with isotonic magnesium chloride at 4°C; the meiofauna was retained in a 32 lm mesh net and thereafter was observed with an Olympus stereomicroscope at 40-809 magnification. Five adult specimens were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), and stored in 0.1 M sucrose/sodium cacodylate buffer until the post-fixation in 1% osmium tetroxide buffered with 0.1 sodium cacodylate. They were next prepared for EM analysis. For TEM, after washing in the same buffer, three of them were dehydrated in a graded alcohol series, stained en bloc in uranyl acetate in 70% acetone, and embedded in Araldite. Semi-thick and ultrathin sections were cut with a LKB Ultrotome 2,088 V, contrasted with toluidine blue and lead citrate respectively. The semi-thick sections were observed in transmission light under VA-NOX AHBT3 Olympus optical microscope, whereas the ultrathin sections were observed under a Philips CM10 transmission electron microscope. For SEM study, two specimens were dehydrated in a graded alcohol series, then critical point-dried using CO 2 , mounted on aluminium stubs, sputter coated with gold palladium and finally observed with a Philips 515 scanning electron microscope. Three specimens (two adult and one subadult) fixed in 2% glutaraldehyde were observed under Nomarski differential interference contrast (DIC) microscope, and photographed with a Nikon Coolpix 990 digital camera. Measurements were taken on one of the two adult specimens by an ocular micrometer.

Results

The interstitial gastrotrich fauna of Skansen appeared very rich, including seven species of gastrotrichs in addition to Diuronotus aspetos, of which the present is the second absolute finding since 1979 (see Todaro et al. 2005). Two more species of chaetonotidans (Chaetonotus atrox, Halichaetonotus sp.), and five species of macrodasyidans (Mesodasys sp., Paradasys sp., Tetranchyroderma sp., Thaumastoderma sp., Turbanella sp.) were found. All these seven species, as well as two undeterminated others were reported by Danish students during an Arctic field course at Qaamassoq, Disko (Ehrhardt and Svendsen 1994). However, Diuronotus aspetos was not found, even if Qaamassoq is only twenty kilometres far from its type locality.

In the present paper new features of the general morphology and of the structure of the spermatozoon are described.

External morphology

Total body length 670 lm (369 lm in the subadult); pharynx length 190 lm (subadult 175 lm); pharingeointestinal junction at U28. The ciliary band surrounding the head edge is 22 lm wide. Widths of head/neck/trunk/ caudal base are as follows: 60/65/75/70 lm at U07/U28/ U60/U90, respectively (Fig. 1a,b). The caudal furca indents at U83. Each furcal branch measures 93 lm in length (50 lm in the subadult), and is made of a fleshy portion, 72 lm long and a primary adhesive tube, 21 lm. The secondary adhesive tubes are much longer than the primary ones (62 vs 21 lm), and arise from a conical base, 42 lm, which is inserted on the intrafurcal edge of the furcal branches (Fig. 1a).

Mouth and buccal cavity

The mouth is a circular opening supported by a thick cuticular ring, 4 lm thick, inner and external diameters 7.5 and 10 lm respectively. Twenty-four longitudinal, cuticular ridges run along the wall of the wide mouth cavity. Twelve stout, double, cuticular processes are inserted to internal base of the mouth ring and protrude 2 lm outside (Fig. 1b-e). Each process appears to be made of two very thin sheets, overlapping each to the other, the upper one narrower and shorter (Fig. 1d,e).

Cuticular armature

The body is covered with 41 alternate columns (25 dorsal and 8 ventrolateral per side) of about 55 scales each. The scales are hemielliptic, keeled and measure in length from 2.25 lm on the head to 10.25 lm on the trunk (Fig. 2a,b). The ventral interciliary field posteriorly to the PhIJ is covered with scales 6 lm in length, arranged into 18-23 alternate columns. They are similar in morphology to the dorsal ones (Figs. 2c,3b).

The fleshy base of each primary furcal tube is entirely covered with 3-4 rings of 7-8 scales similar to the dorsal ones. The tube is enveloped by 3-4 rings of 7-8 very thin scales tightly adhering to it (Fig. 2d,g,h), and longer (11 lm) than the other furcal scales (Fig. 2h). Each secondary tube is covered with 14 rings of 4-5 scales each, looking like the dorsal ones, and decreasing in length from 8 up to 4 lm in the top 2-3 rings (Fig. 2e,f). Both the primary and the secondary tubes show a bare apex (Fig. 2f,h) and contain a duo-gland adhesive system (Fig. 2i,j).

The cuticle is composed of two layers: the thin exocuticle is formed by only one 'bilayer', while the endocuticle is thicker (0.2 lm) and fibrous (Fig. 3a). The single exocuticular 'bilayer' extends to cover the whole surface of the scale (Fig. 3c). Scales are solid, and made of two strongly electron-dense, homogeneous layers, closely tight each to the other. In transverse section the boundary line between the two layers appears strongly electron-dense and striated (Fig. 3a). The prominent keel of each dorsal scale greatly increases posteriorly its height taking the shape of a talon (Fig. 3b). In each scale column, scales adhering to the cuticle regularly alternate to pedunculated scales (Fig. 3c-e). For a

Sensory organs

The head bears dorsally and laterally two kinds of ciliary sensory organs. Scattered, single cilia, about 20 lm long, with the base protected by a small, cuticular cup, surround the mouth ring: they are probably mechanical receptors (Fig. 3g). Some other sensory receptors, with a putative chemioreceptive function, look like small bulges; each of them includes at least five short cilia arising from a single epidermal cell and slightly protruding outside (Fig. 3h). A third type of sensory, possibly mechanoreceptive, organ is represented by single, scattered, and very short cilia, which lie beneath the dorsal pedunculated scales, in touch with their lower side (Fig. 3i). Each cilium rises from a modified monociliated epidermal cell and is surrounded by a ring of microvilli. The complex cilium-microvilli is at the centre of a rounded, small (6 lm in diameter), bump-like, electron-dense protrusion. f Reconstruction of the whole secondary tube: scales shape and arrangement and the free apex (asterisk) are visible (SEM). g, h Close-up of the dorsal posterior end showing the primary and the secondary tube of one furcal branch (g light microscopy; h SEM). Note the very thin scales (arrow) tightly adhering to the primary tube and its free apex (asterisk). i, j TEM images showing the duo-gland adhesive system (arrows) of the furcal tubes. pt Primary tube, st secondary tube. Scale bars a-e, g = 16 lm; f, h = 8 lm; i, j = 3 lm

Ventral ciliature

The ventral ciliature arises from cuboidal, multiciliated, epidermal cells (10 cilia per cell). Each cilium stands out from a basal body with two striated rootlet fibres. An accessory centriole is absent (Fig. 3j,k).

Spermatozoon

The spermatozoon of Diuronotus aspetos is a filiform cell composed by an acrosomal region, a nucleus, and a very long flagellum. A single, giant mitochondrion surrounds the basal half of the nucleus and a supernumerary external membrane surrounds the whole cell (Fig. 4a).

The acrosome, about 18 lm long, is formed by two very elongated cones, which run parallel to each other and gradually reduce their diameter from the base, about 350 nm, to the apex, about 75 nm. The two cones have a different length, and a different texture of their contents. The larger cone is very electron-dense and uniform in aspect, whereas the smaller one shows several microfilaments (Fig. 4b-d). The two cones originate separately from different points of the apical surface of the nucleus: the larger one begins at the apex of the nucleus, whereas the other one starts at about 0.5 lm posterior to the apex of the nucleus (Fig. 4e). The whole acrosome extends anteriorly to the nuclear apex: its length is at least three-times as that of the nucleus, but the small cone is long about a half of the large one (Fig. 4a).

The nucleus is a cylinder 15 lm long, approximately 0.5 lm in diameter, with a completely condensed chromatin. It can be divided in two regions of roughly equal length: the basal one, 8 lm long, is almost completely surrounded by a single, giant mitochondrion; the apical Fig. 4 The spermatozoon of Diuronotus aspetos. a Schematic drawing of the filiform spermatic cell. b, c The two elongated cones forming the acrosome are visible (arrows): the larger one is very electron-dense and uniform in aspect, whereas the smaller one shows several microfilaments. d Apical portion of the small cone. e Detail of the insertion of the two acrosomial cones on the nucleus. The larger cone starts just at the nuclear apex, whereas the smaller one slightly behind. f, g The two nuclear regions are shown: the basal region is almost completely surrounded by a single, giant mitochondrion, the apical one is slightly larger and tapers only at the top (asterisk). h Oblique sections of the apical portion of the nucleus. i Cross section of the basal nuclear region. Note that the mitochondrion does not completely surround the nucleus, leaving a narrow space (arrow). j The flagellar axoneme sinks into a deep depression of the nuclear base, and it is surrounded by an outstanding system of nine accessory fibres. k Oblique section of the piece connecting head and tail. l A slightly oblique section of the flagellum. Note the moderately electrondense contents (arrow) into the areas toward the doublets. m Longitudinal section of the flagellum: the electron-dense contents of the accessory fibres and their thin oblique striation (arrow) are clearly visible. n Cross section of the posterior region of the flagellum where the electron-dense contents of the accessory fibres disappears. d Depression, f flagellum, lc large acrosomal cone, m mitochondrion, sc small acrosomal cone. Scale bars b, d, e-h, j, k, l = 0.5 lm; c, i, m, n = 0.25 lm region, 7 lm, appears slightly larger, and tapers only at the top (Fig. 4g-i). The axoneme sinks into a deep (ca 4 lm) depression of the nuclear base for about 2 lm (Fig. 4j,k). A conventional basal body was never found. The bases of the nuclear-mithocondrial complex and of the flagellum are spaced out by a deep groove of the double membrane. The flagellum, about 0.5 lm in diameter, is at least five times longer than the nucleus; it has a conventional axoneme surrounded by an outstanding system of nine external accessory fibres. Each accessory fibre is connected to the subfibres A and B of the corresponding doublet as well as to the subfibre B of the following doublet. The outline of the accessory fibres is denser than their internal contents, which appears electron-transparent. In grazing longitudinal sections, the dense material of the accessory fibres shows a thin (19 nm) oblique striation. The moderately electrondense contents of the areas toward the doublets (Fig. 4l,m) tends to disappear in the posterior region of the flagellum (Fig. 4n).

Discussion

The objective of this description is to provide increased morphological resolution on the enigmatic chaetonotidan Diuronotus aspetos. Our study reveals important anatomical details not present in the original description, including data on the ultrastructure of the body wall and the spermatozoon.

The body size and shape, and the number, the dimensions and the shape of the scales of these specimens are in line with the data taken on the single adult specimen originally described (Todaro et al. 2005). The mouth cavity shows a very complex structure: in fact, the stout, protrusible processes arise from the cuticular ridges; the latter are more numerous than previously reported (24 vs. 19-20) (Todaro et al. 2005). The muzzle characteristic of the family Muselliferidae appears much more developed in species of Diuronotus than in Musellifer (Hummon 1969). The fine structure of the body cuticle of both D. aspetos and M. delamarei agrees with that of the other Chaetonotida Paucitubulatina in lacking the apical zone of the endocuticle, and showing the exocuticle made of a single 'bilayer' in contrast with all the macrodasyidans in which it is made of at least two bilayers (Rieger andRieger 1977, Ruppert 1991). Both the keeled scales of Diuronotus and the spined scales of Musellifer have a solid, homogeneous structure unlike those of all the other Chaetonotida. The presence of pedunculated scales in addition to the ones fully adhering to the cuticle distinguishes Diuronotus from Musellifer. Among Chaetonotida pedunculated scales are present only in Aspidiophorus and Xenotrichula, two genera greatly differing in morphology from the Muselliferidae, and belonging to separate evolutionary lines (Hochberg and Litvaitis 2000).

The secondary tubes on the furca are the most striking feature of the genus Diuronotus. They have been proved to arise from the ventral side of the furcal base. The furcal scales are not restricted to the fleshy base of the primary tubes but extend to cover their whole length as well as that of the secondary tubes. The presence of a well-developed duo-gland system in all the tubes proves with certainty that they are adhesive tubes, and not simple cuticular structures like those present at the furcal end of other chaetonotidans (e.g., species of Dichaetura).

Of the three kinds of sensory organs detected in D. aspetos, the two cephalic ones fall within the structure of typical mechanoreceptors represented by individual or grouped cilia and described for a number of Gastrotricha (see Marotta et al. 2005 for references). The third type, which is placed in an unusual location, has a very complex structure reminiscent to that of the sensory cilia of Lepidodermella squamata (Hochberg 2001, Fig. 4): it is likely a mechanoreceptor too.

The multiciliated condition of ventral epidermal cells of Diuronotus is in line with that of all the other Chaetonotida Paucitubulatina. Diuronotus aspetos is similar to Xenotrichulidae in having a single centriole per cilium unlike most gastrotrichs which have two centrioles (Rieger 1976).

Sperm Discussion

Species of three families of Chaetonotida Paucitubulatina have been spermatologically studied: Heteroxenotrichula squamosa, Xenotrichula punctata and X. intermedia (Xenotrichulidae;Ferraguti et al. 1995), Musellifer delamarei (Muselliferidae;Guidi et al. 2003), Lepidodermella squamata and Chaetonotus maximus (Chaetonotidae; Hummon 1984; Balsamo 1992). The spermatozoa of the latter two species consist of nothing but rods of condensed chromatin enclosed in a cellular membrane, so they are very different from the filiform and complex spermatozoa of the other Chaetonotida. L. squamata and C. maximus are parthenogenic species, thus their aberrant sperm are generally interpreted as relictual structures and will not be discussed here. The spermatozoa of Diuronotus aspetos show an assemblage of plesiomorphic and apomorphic character states, and may be classified among the modified spermatozoa (sensu Franze ´n 1955). The acrosome including two long, different cones, the completely condensed chromatin, the single, giant mitochondrion around the nuclear base, and the very deep nuclear fossa hosting the axoneme base are all peculiar characters of Diuronotus spermatozoa. However, it may be reminded that Heteroxenotrichula squamosa shows two conical paracrosomal bodies in a position similar to that of the double acrosome of Diuronotus. The bodies observed in H. squamnosa, however, were not interpreted as acrosomes since (a) they are not surrounded by a plasma membrane and (b) there is a conventional acrosome. A correct interpretation of the paraacrosomal bodies in xenotrichulids and the parallel, long, double acrosomes of Diuronotus awaits the study of spermiogenesis. The presence of supernumerary membranes is a feature shared by D. aspetos and M. delamarei, while the structure of the accessory fibres and a similar geometry of the connections to the doublets are synapomorphies of D. aspetos, M. delamarei and the Xenotrichulidae. The thin, oblique striation of the dense material of the accessory fibres is similar in D. aspetos and the Xenotrichulidae (Ferraguti et al. 1995;Guidi et al. 2003).

Conclusions

A number of additional morphological features of D. aspetos and the ultrastructure of its spermatozoon are described with the aim of integrating the original description, and clarifying the phylogenetic position of the genus. Summarizing, the solid structure of the cuticular scales, and the presence of supernumerary membranes in the spermatozoon appear to be synapomorphies shared by D. aspetos and M. delamarei, supporting the assumption that the two species group in the same clade. The sharing of the peculiar structure of the flagellum between the Muselliferidae and the Xenotrichulidae suggests their sister group relationship.

Adding these new data to the morphological and spermatological sets of autapomorphies will be very useful for a phylogenetic analysis at low taxonomic level and a better resolution of systematics and taxonomy of Gastrotricha Chaetonotida.

Fig. 1

 1 Fig. 1 External morphology, mouth and buccal cavity of Diuronotus aspetos. a Habitus of an adult specimen; note the secondary tubes (light microscopy). Mouth, buccal cavity and pharynx. (b, c light microscopy; d SEM; e TEM). Note the evident cuticular mouth ring. Twelve couples of stout, double, cuticular processes protrude from the buccal cavity. bc Buccal cavity, cp cuticular processes, ph pharynx, r mouth ring, st secondary tubes. Scale bars a = 42 lm; b, c = 14 lm; d, e = 4 lm b

Fig. 2

 2 Fig. 2 Cuticular armature and furca. a, b Dorsal view showing the scales on the head and the trunk. c Detail of the ventral scales (arrows) in the interciliary field in the trunk region. d Dorsal posterior end, with the primary tubes. e Dorsal posterior end, showing the secondary tubes (light microscopy).f Reconstruction of the whole secondary tube: scales shape and arrangement and the free apex (asterisk) are visible (SEM). g, h Close-up of the dorsal posterior end showing the primary and the secondary tube of one furcal branch (g light microscopy; h SEM). Note the very thin scales (arrow) tightly adhering to the primary tube and its free apex (asterisk). i, j TEM images showing the duo-gland adhesive system (arrows) of the furcal tubes. pt Primary tube, st secondary tube. Scale bars a-e, g = 16 lm; f, h = 8 lm; i, j = 3 lm

Fig. 3

 3 Fig. 3 Ultrastructure of the body cuticle and of the scales. a The cuticle is made of two layers, a thin exocuticle (arrow) and a thicker endocuticle (asterisk). The scales are solid and homogeneous in structure (TEM). b Shape and arrangement of the dorsal scales (SEM). c Cross section of a pedunculate scale (TEM). d, e Cross and longitudinal sections of the trunk showing the arrangement of the scales. Note the sequence of scales fully adhering to the cuticle (arrow) and others linked to the cuticle through a peduncle (asterisk) (TEM). f Longitudinal section of the trunk of Musellifer delamarei. The ultrastructure of the cuticle and scales look like in Diuronotus aspetos. All the scales are fully adhering to the cuticle (TEM). g Single cilium surrounding the mouth ring (TEM). h Sensory cephalic receptor with about five short cilia (TEM). i A cilium lying under the dorsal body scales (TEM). j An epidermal multiciliated cell (TEM). k Basal ciliary structures: a single basal body and two striated fibres are present (TEM). bb Basal body, c cilium, cr cephalic receptors, ds dorsal scales, s scales, sf striated fibres. Scale bars a, c, g-k = 1 lm; b, d, e, f = 10 lm

Helgol Mar Res (2010) 64:27-34

Acknowledgments We gratefully acknowledge the staff and facilities of Arctic Station at Qeqertarsuaq, Disko, West Greenland. Funds for this study were provided to R.M.K. by the Danish Natural Science Research Council . The research benefitted from a M.I.U.R. grant to MB (PRIN 2007-Sexuality, reproduction and specific identification of Gastrotrichs and Placozoans).

Balsamo M (1992) Hermaphroditism and parthenogenesis in lower bilateria: Gnathostomulida and Gastrotricha. In: Dallai R (ed) Sex origin and evolution. Selected Symposia and monographs. UZI Mucchi, Modena, pp 309-327 Ehrhardt C, Svendsen HK (1994) Marine Gastrotricha og Rotifera ved Qaamassoq-Disko. Arktisk Biologisk Feltkursus Qeqertarsuaq/ Godhavn 1994. University of Copenhagen, pp 53-72 Ferraguti M, Balsamo M, Fregni E (1995) The spermatozoa of three species of Xenotrichulidae (Gastrotricha, Chaetonotida): the two ''du ¨nne Nebengeisseln'' of spermatozoa in Heteroxenotrichula squamosa are peculiar para-acrosomial bodies. Zoomorphology 115:151-159 Franze ´n A ˚(1955) On spermiogenesis, morphology of the spermatozoon, and biology of fertilization among invertebrates. Zool Bidr Uppsala 31:355-482 Guidi L, Marotta R, Pierboni L, Ferraguti M, Todaro MA, Balsamo M (2003) Comparative sperm ultrastructure of Neodasys ciritus and Musellifer delamarei, two species considered to be basal among Chaetonotida (Gastrotricha). Zoomorphology 122:135-143 Hochberg R (2001) A special form of sensory cilia in Lepidodermella squamata (Gastrotricha Chaetonotida). Ophelia 55(2):137-139 Hochberg R, Litvaitis MK (2000) Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships. Biol Bull 198:299-305 Hummon WD (1969) Musellifer sublittoralis, a new genus and species of Gastrotricha from San Juan Archipelago, Washington. Trans Am Microsc Soc 88:282-286 Hummon MR (1984) Reproduction and sexual development in a freshwater gastrotrich. 2. Kinetics and fine structure of postparthenogenic sperm formation. Cell Tissue Res 236:619-628 Kristensen RM, Niilonen T (1982) Structural studies on Diurodrilus Remane (Diurodrilidae fam. n.), with description of Diurodrilus westheidei sp. n. from the Arctic interstitial meiobenthos, West Greenland. Zool Scr 11:1-12 Kristensen RM, Nørrevang A (1982) Description of Psammodrilus aedificator sp. n. (Polychaeta) with notes on the Arctic interstitial fauna of Disko Island, West Greenland. Zool Scr 11:267-279 Leasi F, Todaro MA (2008) The muscular system of Musellifer delamarei (Renaud-Mornant 1968) and other chetonotidans with implication for the phylogeny and systematization of the Paucitubulatina (Gastrotricha). Biol J Linn Soc 94:379-398 Marotta R, Guidi L, Pierboni L, Ferraguti M, Todaro MA, Balsamo M (2005) Sperm ultrastructure of Macrodasys caudatus (Gastrotricha: Macrodasyida) and a sperm based phylogenetic analysis of Gastrotricha. Meiofauna Mar 14:9-21 Remane A (1961) Neodasys uchidai nov. spec., eine zweite Neodasys-Art (Gastrotrich Chaetonotoidea). Kieler Meeresforsch 17:85-88 Rieger RM (1976) Monociliated epidermal cells in Gastrotricha: significance for concepts of early metazoan evolution. Zeitsch Zool Syst Evolutionsforsch 14:198-226 Rieger GE, Rieger RM (1977) Comparative fine structure study of the gastrotrich cuticle and aspects of cuticle evolution within the Aschelminthes. Zeitsch Zool Syst Evolutionsforsch 15:81-124 Ruppert EE (1991) Gastrotricha. In: Harrison FW, Ruppert EE (eds) Microscopic anatomy of invertebrates, 4, Aschelminthes. Wiley, New York, pp 41-109 Todaro MA, Balsamo M, Kristensen RM (2005) A new genus of marine chaetonotids (Gastrotricha) with a description of two new species from Greenland and Denmark. J Mar Biol Assoc UK 85:1391-1400