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onad plasticity and gametogenesis in the endangered Spanish toothcarpphanius iberus (Teleostei: Cyprinodontidae)
. García-Alonsoa,∗,1, A. Ruiz-Navarrob,1, E. Chaves-Pozoc, M. Torralvab, A. García-Ayalac
Department of Biological Sciences, University of Hull, HU6 7RX Hull, England, United KingdomDepartment of Zoology and Anthropology, Facultad de Biología, Universidad de Murcia, E-30100 Murcia, SpainDepartment of Cell Biology and Histology, Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain
r t i c l e i n f o
rticle history:eceived 16 July 2008eceived in revised form 23 October 2008ccepted 4 November 2008vailable online 27 December 2008
eywords:
a b s t r a c t
The Spanish toothcarp Aphanius iberus is an endangered species which inhabits small rivers, creeks,salt marshes and marine salt pans in the Mediterranean coast of Spain. No differences in weights wereobserved among females or males taken from different environments. Analyses of the morphology of thegonads and the gametogenesis were performed in fish taken from different environments by comparinggamete development in females and in males and gonadal cell proliferation in the testis. A high degree ofplasticity was observed in the gonad morphology of A. iberus. Females possess two ovaries which show
non-restricted oogenesis with all germ cell stages within the same ovigerous lamellae, while males pos-sess gonads without any clear division with the typical restricted pattern observed in cyprinodontid fish.Some females and males showed asymetrically developed gonads. Proliferation of germ cells in testis islocated only in the periphery of the gonads corresponding with primary and secondary spermatogonia.Salinity did not influence gonad plasticity or the appearance of mature germ cells in either females ormales. This is the first study to provide a microscopic description of oogenesis and spermatogenesis in A.
t env
iberus at extreme differen
. Introduction
There is an urgent need to increase our knowledge of the lifeistory and eco-physiological traits of fish species under threat, asnecessary tool for management and conservation programmes
o be undertaken (for review sees Wootton et al., 2000). Knowl-dge of reproduction is one of the bases of fish biology, and,herefore, for management and conservation (Meffe and Carroll,997). Cyprinodontiforms comprise species adapted to extremend fluctuating environmental conditions, such as high ranges ofemperature and salinity (e.g. Aphanius spp.; García-Berthou and
oreno-Amich, 1999), tidal salt marshes (e.g. Fundulus hetero-litus; Kneib, 1986) or temporal ponds (e.g. Austrolebias charrua;rezo et al., 2007). Survival in such diverse and extreme con-itions implies a high degree of plasticity in the life cycle andeneral physiology of these small teleosts. It has previously been
hown that the general metabolism of fish, especially in cyprin-dontids, is affected by several factors including salinity (Jordan etl., 1993; Plaut, 2000; Wuenschel et al., 2004). Moreover, analysesf ammonia excretion rates in this species demonstrate a corre-
lation between salinity and ammonia excretion, although it alsodepends on body size and reproduction stage (Oliva-Paterna et al.,2007). It might therefore be expected that salinity would inter-fere in reproduction. However, Oltra and Todolí (2000) observedno differences between the numbers of viable embryos when A.iberus was induced to spawn in different environmental condi-tions.
Aphanius iberus, an eurythermic and euryhaline cyprinodontidfish, is an endemic fish which inhabits a wide range of environmentsalong the SE Mediterranean coast of Spain, such as the brackishwaters of salt marshes, coastal lagoons and river-mouths (García-Berthou and Moreno-Amich, 1992; Oliva-Paterna et al., 2006). Thespecies is catalogued as endangered (EN A2ce; IUCN, 2007) andis one of the few Iberian fish species protected by national andinternational laws (Elvira, 1995; Doadrio, 2002). However, due tothe introduction of exotic fish (Elvira and Almodóvar, 2001; Caiolaand De Sostoa, 2005) and destructive human impacts on its naturalhabitats (Planelles, 1999), the geographical range of A. iberus con-tinues to shrink. Nowadays it is found mainly in small fragmentedsaline localities (Doadrio, 2002; Araguas et al., 2007). Many pre-
vious studies have focused on the ecology of A. iberus and someaspects of its life history (Vargas and De Sostoa, 1997; Planelles,1999). A. iberus show an extraordinary phenotypic plasticity andis well adapted to fluctuating environments (Oliva-Paterna et al.,2006; Alcáraz et al., 2008).
However, relatively little is known about the reproduction of A.berus and, to the best of our knowledge, no information about itsametogenesis exists. Macroscopic studies (gonadosomatic index,SI) were performed in A. iberus from the Ebro River (Vargas ande Sostoa, 1997) whose principal reproductive season is from May
o August. The population in the Guadalquivir basin (Fernández-elgado et al., 1988), which has recently been described as a
eparate species (A. baeticus, Doadrio et al., 2002), presents twoeaks in the GSI, the major one occurring in spring (April–May)nd a polymodal size-frequency distribution of oocytes during theest of the spawning period. This oogenesis process reflects season-lly synchronic group ovary maturation (Fernández-Delgado et al.,988). In the province of Murcia, the reproduction period occursn summer (July–August) when animals are at the end of theirametogenesis processes and beginning to spawn (Oliva-Paterna,006).
The aim of the present work was to provide the first descriptionf gonadal morphology, oogenesis and spermatogenesis in popu-ations from habitats which principally differed in the degree ofalinity. More specifically, we carried out a microscopic study of theonads and an immunocytochemical method to detect proliferatingells. Histological method which has proved successful in deter-ining the reproductive pattern of teleost fishes (Parenti and Grier,
004). In this way, we hoped to establish the influence of salinityfreshwater and high saline marine water) on gametogenesis.
. Materials and methods
.1. Fish
Twenty-eight wild mature specimens of A. iberus (Valenciennes,846) were captured in June and July (2006), the main reproduc-ive season (Oliva-Paterna, 2006), using minnow-traps from threeampling sites in the south of Spain with the following salin-ty ranges: Chícamo (freshwater creek, 0–2‰) n = 10; La Hita (salt
arsh, 35–40‰) n = 7 and Marchamalo (marine salt pans, 65–70‰)= 11. All specimens belong to the same geographical and geneticroup (sensu, Doadrio et al., 1996). Fish from all sites were keptn cooled aerated tanks and transported to the laboratory (Uni-ersity of Murcia), where they were anaesthetised with clove oilssence (Eugenia caryophyllata, Mon Deconatur S.L., Barcelona) at0 ppm in fresh or saltwater according to García-Gómez et al.2002), depending on the origin of the specimens. The animalsere then weighed and decapitated. The trunks of the speci-ens (including the whole peritoneal cavity) were removed and
rocessed for light microscopy, as described below, in order tonalyse the position of the gonads and to describe the gameto-enesis process. For cell proliferation analysis, some specimensere anaesthetised for 1 min and injected intraperitoneally with0 �g g−1 body weight of 5-bromo-2′-deoxyuridine (BrdU, Sigma,aint Louis, USA) 2 h before sampling, maintaining the speci-ens in natural photoperiod and temperature conditions. The
xperiments described comply with the Guidelines of the Euro-ean Union Council (86/609/EU) and the Bioethical Committeef the University of Murcia (Spain) for the use of laboratory ani-als.
.2. Light microscopy
The samples were fixed for 24 h in Bouin’s solution or 4%
araformaldehyde solution, embedded in paraffin (Paraplast Plus;herwood Medical, Athy, Ireland), and serially sectioned (horizon-al plane) at 5 �m. Some sections were stained with haematoxylinnd eosin to determine the reproductive stage of each specimennd the degree of development of the gonads based on previous
d Cell 41 (2009) 206–213 207
gametogenesis classifications in teleost fish (Jalabert, 2005; Meijideet al., 2005). Slides were examined with an Axiolab (Zeiss) lightmicroscope. In order to determine oocyte growth, oocyte cell diam-eters were drawn manually and measured by image analysis usingan Axiolab (Zeiss) light microscope, a CoolSNAP digital camera (RSPhotometrics) and SPOT Advance 3.3 software (Diagnostic Instru-ments, Inc.).
2.3. Immunohistochemical staining
An indirect immunological method was performed to determinecell proliferation, according to Chaves-Pozo et al. (2005). Briefly, 4%paraformaldehyde-fixed sample sections from BrdU-treated speci-mens were incubated for 40 min in peroxidase-quenching solution[H2O2 (commercial solution at 30%, Panreac, Barcelona, Spain) inmethanol, 1:9], for 30 min in 1% periodic acid at 60 ◦C and finally for30 min in 5% bovine serum albumin (BSA, Sigma, Saint Louis, USA)in phosphate buffered saline (PBS, pH 7.4). Subsequently, they wereincubated with a monoclonal antibody anti-BrdU (Becton Dick-inson, San Jose, USA) at the optimal dilution of 1:100 in 1% BSAin PBS for 2 h at room temperature. Subsequently, sections werewashed in PBS, and incubated with a peroxidase-conjugated rabbitanti-mouse IgG (whole molecule) at the optimal dilution of 1:100in 1% BSA in PBS for 1 h at room temperature. The sections werethen washed twice in PBS and in 0.05 M Tris–HCl buffer (pH 7.6)for 5 min each. The peroxidase activity was revealed by incubationwith 0.05% 3,3′-diaminobenzidine tetrahydrochloride (Fluka, Stein-heim, Switzerland) in Tris–HCl buffer (pH 7.6) containing 0.05%H2O2 for 15 min at room temperature. The sections were slightlycounterstained with Meyer’s haematoxylin and the specificity ofthe reactions was determined by omitting the first antiserum andby using tissue sections from fish that had not been injected withBrdU.
2.4. Measurements and statistical analyses
Total lengths (±1 mm) and weights (±0.1 mg) from fish betweenthe three sampling sites (Table 1) and oocyte diameters until andincluding stage V (see Table 2) of ooogenesis were recorded. Onlyoocytes in which the cut was at the equator of the cell and thenucleus was clearly visible at the centre of the oocytes were takenfor this analysis. Comparison of fish sizes and oocyte diameters wasperformed using one way ANOVA (analysis of variance). The homo-geneity of variance was previously checked and all the analyseswere performed using the SPSS v. 16.0 (SPSS Inc., Chicago) statisticalpackage. Significant differences were recorded at p < 0.05.
3. Results
No differences in weight were observed among females or malestaken from the three different environments. However, standardlengths in females from Chícamo River (freshwater) were sig-nificantly higher compared than of those from the Marchamaloand La Hita (saltwater) populations (ANOVA; F(2,9) = 4.472; p < 0.05,Table 1).
3.1. Plasticity of the gonads
Horizontal sections of the whole and inviolate peritoneal cavitywere performed from the trunk of the animals (Fig. 1). In general,
the kidneys were found in the anterior and dorsal parts of the cavi-ties (extra-peritoneal), the liver and gut covering the anterior partsof the peritoneal cavities, and the central parts were occupied bythe gonads and gut (Fig. 1e, female and Fig. 1f, male). In the poste-rior parts, the dorsal and distal parts were covered by the gonads,
208 J. García-Alonso et al. / Tissue and Cell 41 (2009) 206–213
Table 1Mean lengths (mm) and weights (g) of Aphanius iberus collected from three different locations: a freshwater creek, Chícamo (n = 10), an evaporation salt pan, Marchamalo(n = 11) and a salt marsh, La Hita (n = 7). ANOVA tests were employed. F-statistics (F), degrees of freedom (d.f.) and p-values (p) are presented. CH (Chícamo River), MCH(Marchamalo) and LH (La Hita).
Table 2Cell number (n) and diameter (�m, mean ± standard deviation, SD) of each oogenesis stage from 12 Aphanius iberus females collected in the three different localities (Chícamo,Marchamalo and La Hita) and classification of oogenesis stages in A. iberus. I, oogonia; II, pre-vitellogenic oocytes; III, early vitellogenic oocytes; IV, late vitellogenic oocytes;V, post-vitellogenic oocytes; VI, oocytes in maturation and VII, mature or hydrated oocytes. GVBD (germinal vesicle breakdown).
I 34 ± 17 (67) 25 ± 7 (16) 31 ± 12 (13) Slightly basophilic cytoplasm and large spherical nucleus withmarked nucleolus.
II 70 ± 24 (796) 63 ± 22 (199) 80 ± 29 (89) Slightly basophilic cytoplasm and nucleolus appeared in thenucleus.
III 143 ± 35 (22) 135 ± 21 (17) 158 ± 23 (12) Their nucleus contains multiple nucleoli closed to the nuclearenvelope. Yolk vesicles appear in the cytoplasm, with presenceof first lipid droplets.
IV 164 ± 32 (32) 185 ± 33 (22) 240 ± 57 (11) Secondary yolk vesicles appear in the cytoplasm and morelipid droplets are visible.
V 334 ± 87 (30) 330 ± 77 (26) 398 ± 78 (19) Central nucleus or germinal vesicle and almost all thecytoplasm covered by lipid droplets and yolk vesicles.
VI 574 ± 130 (5) 421 ± 92 (17) 445 ± 110 (9) Migration of the nucleus or germinal vesicle to the peripheryof the cytoplasm. Proteolysis of yolk protein, coalescence oflipid droplets and hydration process.
VII 669 (1) 609 ± 109 (4) 614 ± 80 (
Fig. 1. Schematic representation and pictures of female and male Aphanius iberus.(a) and (b) Schemes of a female and a male showing the coronal plane of the sections.(c) and (d) Schemes showing the distribution of gonads in an horizontal section. (e)and (f) Low magnification pictures of horizontal (coronal) plane of a female and amale. Bars indicate 4 mm (e) and 2 mm (f). K, kidney; Ov, oviduct; Oo, oocytes; L,liver; T, testis; G, gut.
3) Homogenous cytoplasm with some lipid droplets andperipheral cortical alveoli. GVBD occurs.
while the most ventral parts, until the anus, were occupied by thegut.
The gonads were located dorsally to the gut and sometimes cov-ering a considerable space in the peritoneal cavity (Figs. 1 and 2).In females both ovaries showed a similar development and do notappeared at lateral positions. The ovaries were in the central partof the cavity and separated by a distinguishable layer of connectivetissue (Fig. 2a). Both ovaries are caudally connected in a commonoviduct. In males, testes were located centrally with no clear divi-sion (Fig. 2b). However, some females (Fig. 2c) and males (Fig. 2d)showed asymmetrically developed gonads, where the right ovaryand testis, respectively, were considerably larger than their coun-terparts on the left side, which could even be very small and difficultto distinguish and separate from the bigger one.
No differences in weight were observed among females or males
taken from the three different environments. However, standardlengths in females from Chícamo River (freshwater) were sig-nificantly higher compared than of those from the Marchamaloand La Hita (saltwater) populations (ANOVA; F(2,9) = 4.472; p < 0.05,Table 1).
J. García-Alonso et al. / Tissue and Cell 41 (2009) 206–213 209
Fig. 2. Gonads of Aphanius iberus. (a) and (c) Two adult females showing different ovary size and location, with oocytes at different stages of gamete development. L, lumen;O f adulm e gon
3
elnoaictasloovcEbfi
v, oviduct; V, post-vitellogenic oocyte. (b) and (d) One central and lateral testis ouscle; G, gut; VD, vas deferens; asterisk indicates the connective tissue between th
.2. Oogenesis
The ovary of A. iberus was formed by folds of the germinalpithelium, named ovigerous lamellae, which surround an ovarianumen or cavity (Fig. 2a), in which all germ cell stages of ooge-esis were present. These ovigerous lamellae contained nests ofogonia, oocytes and follicles in various stages of developmentnd growth, embedded in a smooth connective tissue and delim-ted by epithelial cells (Fig. 3). In order to define the germinalell populations, the morphology and diameters of the cells wereaken into account. Table 2 shows the oogenesis classificationnd the average of oocyte diameters at each stage. The earliesttep in oogenesis is represented by oogonia (stage I), which wereocated through out the ovary and appeared among more devel-ped oocytes (Fig. 3a and b). Oogonia develop into perinucleolarr pre-vitellogenic oocytes (stage II). At the end of this stage, pre-
itellogenic oocytes were surrounded by a continuous follicularell layer (the granulose layer) formed by flattened cells (Fig. 3a).arly vitellogenic oocytes (stage III) were characterized by a weakerasophilic cytoplasm, with yolk granules randomly distributed andrst lipid droplets forming a ring (Fig. 3a). Surrounding the late
t males, respectively. Pictures are in Posterior-Anterior direction (left to right). M,ads. Bar indicates: 300 �m.
vitellogenic oocytes (stage IV), the second follicular cell layer (thetheca layer) became apparent. The cytoplasm of late vitellogenicoocytes was filled up with lipid vacuoles and yolk granules (Fig. 3b).Post-vitellogenic oocytes (stage V) (Fig. 3b) still presented the ger-minal vesicle or nucleus in a central position, and all the cytoplasmwas homogenously covered by yolk granules and lipid droplets.Once the meiosis process, which had stopped in prophase I, reas-sumed, the maturation stage (stage VI) involved a rearrangementof the cytoplasm and migration of the nucleus (Fig. 3c). Finally,mature (hydrated) oocytes (stage VII) are ready to be ovulatedand be released for external fertilization. In this stage germi-nal vesicle or nucleus break-down (GVBD). Degenerated oocytes(atresic) occasionally occur and had an irregular shape and showeda highly condensed and basophilic nucleus surrounded by cyto-plasm filled with acidophilic and some basophilic granules (datanot shown). No indicators of masculinisation (presence of testis
in the ovaries) were observed. Furthermore, oocytes in stage IIwere the most abundant cells in the gonad of all the fish anal-ysed (Table 2). Because it was expected that salinity could influenceoocyte diameter, the diameters of oocytes from the three differ-ent environments were compared. No differences were observed
210 J. García-Alonso et al. / Tissue and Cell 41 (2009) 206–213
Fig. 3. Oogenesis in Aphanius iberus. (a) Ovary showing oocytes at early different stages of gamete development. Oogonia (I), pre-vitellogenic oocyte (II), early-vitellogenico ogenic( enved
is
3
cotmbiasgsi4ost
ocyte (III). (b) Oogonia (I); pre-vitellogenic (II), early vitellogenic (III) and late vitellV). (c) Ovary showing oocytes in maturation (VI) with a clearly developed vitellineroplets; Y, yolk. Bars indicate: 50 �m (a); 150 �m (b, c and d).
n oogenesis for the different salinity conditions, p > 0.05 (data nothown).
.3. Spermatogenesis
A. iberus males developed an asynchronous spermatogenesisharacterized by the presence of spermatogonia stem cells and cystsf all germ cell types in the tubules of the testis (Fig. 2b). Thus, theubules were formed by spermatogonia stem cells, and cysts of pri-
ary and secondary spermatogonia and spermatocytes (Fig. 4a and). As spermatogenesis proceeded, the amount of free spermatozoa
n the tubular lumen increased, at the same time as the cyst vari-bility decreased. Both fresh- and saltwater specimens showed aimilar degree of maturation and were in middle and late spermato-enesis, when most of the tubules are formed by spermatocytes andpermatid cysts and high amount of free spermatozoa are present
n the lumen of the tubules and in the efferent duct (Figs. 2b andd). Surrounded by germinal epithelia, cysts of spermatogonia arebserved in the testicular periphery, and successive stage of thepermatogenesis occurs in the tubules in direction to the centre ofhe testis (Fig. 2b).
(IV) oocytes are observed in the same ovigerous lamellae; post-vitellogenic oocytelope (see asterisk); (d) Mature oocyte (VII). F, follicle cell layer; N, nucleus; L, lipid
3.4. Cell proliferation
In males, the cysts located at the periphery of the gonad, whichcorrespond to primary and secondary spermatogonia cysts, pro-liferated during the spawning period (Fig. 5). The BrdU positivecells were localized at the periphery of the testis, corroboratingthe restricted spermatogonia-type testis. No immunostaining wasobserved in the control sections (Fig. 5d). In the case of females,they did not show clear immuno-positive reactions, indicating a lowor absence of proliferative rate in the ovaries during the spawningperiod (data not shown).
4. Discussion
Histological examination showed a high degree of plasticity inthe morphology and positioning of the gonads in the peritoneal
cavity of A. iberus. The present data demonstrate that A. iberuspossess two ovaries, both bound in the central part of their con-nective tissue, although each ovary has an independent lumen.This morphology differs from the regular elongated ovaries of othercyprinodontiforms, such as A. charrua (Arezo et al., 2007).
J. García-Alonso et al. / Tissue and Cell 41 (2009) 206–213 211
F e testS indica
sefdlwteo
ctgaehomtisn
ig. 4. Spermatogenesis in Aphanius iberus. (a) Spermatogonia in the periphery of thPG, spermatogonia; SPC, spermatocytes; SPD, spermatids; SPZ, spermatozoa. Bars
The oogenesis process appears to be non-restricted. All oocytetages appeared homogenously distributed along the ovary. Asxpected, pre-vitellogenic oocytes were the most abundant stageound, followed by vitellogenic oocytes, which increase in sizeue to the accumulation and storage of lipids and proteins (vitel-
ogenins), carbohydrates and phosphate groups. Very few oocytesere found undergoing maturation or in a mature stage, indicating
hat ovulation and spawning occurs frequently but in few oocytesach time. However, post-ovulatory follicles were rarely or notbserved, suggesting a rapid re-absorption of follicle tissues.
Final oocyte maturation involves proteolysis of the yolk andlarification, GVBD and a rapid increase in oocyte volume dueo water entering into the oocytes, after osmotic disequilibriumenerated by the proteolysis (Jalabert, 2005). This process differsccording to whether fish produce marine pelagic, marine dem-rsal or freshwater eggs. It has been suspected that the differentabitats inhabitated by A. iberus might influence the maturationf the oocytes, resulting in a variation in oocyte diameter. Pelagic
arine eggs show a greater increase in oocyte size, while freshwa-
er eggs such as those from rainbow trout show a small increasen size (Jalabert, 2005). However, the results did not show anyignificant difference in oogenesis, suggesting that salinity doesot modify the growth of oocytes during the maturation process
is. (b) Primary and secondary spermatocytes, (c) spermatids, and (d) spermatozoa.te: 50 �m (a, b and d); 100 �m (c).
in A. iberus. García-Marín et al. (1990) described a genetic varia-tion among the Mediterranean populations of A. iberus and nuclear(allozyme) and mitochondrial (cytochrome b) DNA revealed highgenetic divergence in A. iberus from the Iberian Peninsula (Perdiceset al., 2001). However, a unique geographical and genetic group ofA. iberus was previously described from the three sampling sites(Doadrio et al., 1996). More studies at molecular level are beingcarried out in the same population analysed here, as a complementto this morphological approach.
In several males, a large and centrally positioned testis wasobserved with lateral extension to both sides and no morpho-logical evidence for the presence of two clearly separated testeswas obtained. However, in other specimens, the results suggestedasymmetric testes and the testis shape varied among individu-als indicating extreme plasticity. Polarization of spermatogenesiswas not unexpected. The confinement of spermatogonia to the dis-tal end of the lobules in restricted lobular patterned testis is anarrangement typically found in cyprinodontid fishes (Grier, 1981;
Parenti and Grier, 2004). The localization of germ cells in the periph-ery of the testis has also been observed in other cyprinodontiformssuch as Fundulus heteroclitus (Selman and Wallace, 1986). Cyprin-odontiforms belong to Atherinomorpha, the only teleost group withspermatogonia cells confined to the most distal borders of the testis
212 J. García-Alonso et al. / Tissue and Cell 41 (2009) 206–213
F immus to c). (B
(puod
ipecp
fawme
tleag
ig. 5. Proliferative cells in the testis of Aphanius iberus. Germinal cells showingpecifically located in the spermatogonia area in the edge of the gonads (pictures aars indicate: 100 �m (a and d); and 50 �m (b and c).
Parenti and Grier, 2004). Moreover, the same authors pointed to thehylogenetic relevance of the restricted spermatogonial pattern as anique derived character for Atherinomorpha. No differences werebserved in the spermatogenesis process of males from the threeifferent environments.
The presence of cell proliferation in fish gonads is not surpris-ng. In contrast to mammals’ gonads, several works described theroliferation of cells taking place in gonads during fish gametogen-sis (Grier, 2000; Chaves-Pozo et al., 2005). A. iberus showed a clearell proliferation in the testis, a process that basically occurs in theeriphery and was restricted to spermatogonia germ cell type.
Finally, A. iberus specimens were for the first time success-ully anaesthetised with clove oil essence (Eugenia caryophyllata)n extract used for marine teleosts (García-Gómez et al., 2002)ith effective anaesthetic properties and very low cost. The ani-als showed excellent recovery, indicating the potential use of this
ssential oil for anaesthetising cyprinodontid fishes.In conclusion, A. iberus showed large morphological diversity of
he gonads, with females and males presenting central or unequalateral gonads. The gonads showed wide phenotypic plasticity, asxpected for animals adapted to extreme conditions including highnd low salinity levels. Interestingly, the final process of gameto-enesis was quite similar in specimens inhabiting very different
no-reaction to anti-BrdU (counterstained with haematoxiline). The reaction ared) Control section without first antibody. SPG, spermatogonia; SPC, spermatocytes.
salinity conditions and no differences were observed during theoogenesis process. Decreases in population size of this endangeredspecies in Chícamo River and La Hita saltmarsh do not seem to bea consequence of a dysfunction at gonad level. No feminization ormasculinisation of the gonads was observed. More studies shouldbe carried out in order to understand the reproductive mechanismsof this species, the advantage of such gonad plasticity and its adap-tation to extreme environments.
Acknowledgments
We are very grateful to FJ Oliva-Paterna and A Andreu (Universityof Murcia) for help in field sampling. This work was supported byLIFE-Nature Project LIFE04 NAT/ES/000035 Conservation of Apha-nius iberus genetic stocks (Murcia).
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