Reproduction of Merluccius merluccius (Actinopterygii: Merlucciidae) from the northern Atlantic coasts of Morocco based on histological analysis of gonads

Biological material

The fish used in the present study were collected from the area between Asilah (35°28’10.387”N, 6°2’19.111”W) and El Jadida (33°15’14.321”N, 8°30›2.401»W) (Fig. 1), where the stock of M. merluccius is exploited (El Bouzidi et al. 2022El Bouzidi C., Abid N., Awadh H., et al. 2022. Growth and mortality of the European hake Merluccius merluccius (Linnaeus, 1758) from the North of Moroccan Atlantic coasts, Egypt. J. Aquat. Res. 48: 233-239. https://doi.org/10.1016/j.ejar.2021.12.003 ). The fishes were collected monthly from the catch of local trawler fisheries. A total of 1780 fishes were collected between 2017 and 2018 (23 months). For each individual, total length (TL) to the lowest half cm, total weight (TW) to 0.1 g and gonad and liver weight to 0.01 g were recorded. In addition, sex and stage of maturity were estimated from macroscopic examination of the gonads.

Histological techniques

374 gonads were fixed immediately after dissection. The gonads were fixed for at least 24 h in Davidson solution (Fournie et al. 2000Fournie J.W., Krol R.M., Hawkins W.E. 2000. Fixation of fish tissues. In The laboratory fish. Academic Press, 2000. pp. 569-578. https://doi.org/10.1016/B978-012529650-2/50043-3 ). Then, the tissues were dehydrated by a gradual series of ethanol (ethanol 70°, ethanol 95°, absolute ethanol), moved to xylene, and then embodied in low-melting kerosene (raised to 56°C-58°C). Two 2 µm sections were prepared from each gonad and stained with haematoxylin-eosin using an automated stainer of the brand MEDIZINTECHNIK MEDITE.

Histological examination of gonads

Histological slides were examined under an optical microscope. Readings were taken three times: twice by the same person followed by an independent examination by a second person.

Microscopic classification of Merluccius merluccius ovaries and testes

The germ cell stages and the general state of the gonadal maturity were classified using the criteria of Dietrich et al. (2009)Dietrich D., Krieger H., Rumpf S., et al. 2009. Histological Analysis of Endocrine Disruptive Effects in Small Laboratory Fish. Wiley-Interscience. 369pp. https://doi.org/10.1002/9780470431795 and Murua and Motos (2006)Murua H., Motos L. 2006. Reproductive strategy and spawning activity of the European hake Merluccius merluccius (L.) in the Bay of Biscay. J. Fish Biology. 69: 1288-1303. https://doi.org/10.1111/j.1095-8649.2006.01169.x .

M. merluccius is a partial spawner with asynchronous germ cell development (Murua 2010Murua H. 2010. The Biology and Fisheries of European Hake, Merluccius merluccius, in the North-East Atlantic. Adv. Mar. Biol. 58: 97-154. https://doi.org/10.1016/B978-0-12-381015-1.00002-2 ). This means that the gonads contain all germ cell maturation stages; only their relative frequency differs between the various gonad maturation stages (Table S1). In the ovaries, in addition to the maturing oocytes, postovulatory follicles and atretic oocytes can also be observed.

Macroscopic classification of Merluccius merluccius ovaries and testes

For the macroscopic staging of the gonads, the criteria established by Holden and Raitt (1974)Holden M.J., Raitt D.F.S. 1974. Manuel des sciences halieutiques. Deuxième partie. Méthodes et recherches sur les ressources et leur application. Doc. Tech. FAO, 223 pp., of fractional spawners were applied (Table S2). Since the resting stage is macroscopically similar to the maturing stage, the two stages were grouped together. From macroscopic stage 3 on, both sexes were considered to be mature.

Sex ratio

The sex ratio was calculated for the whole sample pool (n=1780) as well as per length class (1 cm). The variation of the sex ratio according to the length was defined as the proportion of males and females by length class in the whole sampled population and the monthly sex ratio was obtained by dividing the number of females and males by the total number of individuals per month.

Gonadosomatic index (GSI)

The gonadosomatic index of the whole samples (n=1780), was calculated as a percentage of the gonad weight in relation to the TW of the fish, $GSI=\left[\frac{GW}{TW}\right]\times 100$ (Barber and Blake 2006Barber B.J., Blake N.J. 2006. Reproductive physiology. In: Shumway S. E., Parsons G. J. (eds): Scallops: biology, ecology, and aquaculture. Elsevier. Amsterdam, pp. 357-406. https://doi.org/10.1016/S0167-9309(06)80033-5 ).

Body length at first maturity

To estimate body length at 50% maturity (L₅₀) (i.e. the length at which 50% of the individuals are mature), the observed proportion of mature specimens was adapted to a logistic curve (Roa et al. 1999Roa R., Ernst B., Tapia. F. 1999. Estimation of size at sexual maturity: an evaluation of analytical and resampling procedures, Fish. Bull. 97: 570-580.). The proportion of mature individuals in each length class was calculated by setting the maturity threshold as follows; (i) stage 3 and higher (advanced maturation) in the macroscopic scale; (ii) stage ⅡC and higher (late development) of the microscopic staging of the female gonads; and (iii) stage ⅡB (mid-spermatogenic) and higher of the microscopic staging of the males gonads. We opted for a linear regression method to estimate the coefficient of the regression line β₁ and the constant β₀ (intercept). These parameters are necessary for the calculation of the equations $PCS=\frac{1}{1+e^{-\left({\beta }_0+{\beta }_1\bullet X\right)}}$ and L₅₀ = -β₀/β₁, where PCS is the probability for an individual to be mature at a given length X (Roa et al. 1999Roa R., Ernst B., Tapia. F. 1999. Estimation of size at sexual maturity: an evaluation of analytical and resampling procedures, Fish. Bull. 97: 570-580.).

Season- and length-related variation of sex ratio and gonadosomatic index

Fishes displaying gonads that were macroscopically classified as ovaries ranged between 16 and 73 cm TL. Fishes macroscopically classified as males ranged between 16 and 44 cm TL. Females were generally predominant in the larger size groups (>27 cm), and the length groups greater than 44 cm were composed exclusively of females. Males dominated in the length groups smaller than 28 cm TL (Fig. 2A). Accordingly, there was a significant difference in sex ratio between size groups (t-test=12.8; p<0.05). The overall sex ratio of the total sample displayed a significantly higher percentage of males (59%) than females (41%) (p=0.002).

The GSI values of females varied over the 23 months (Fig. 2B). In 2017 the highest values were recorded in January-February, then again in May-July and in September. In 2018, the variation of the female GSI was less strongly expressed than in 2017, but January and midsummer were still the periods with high GSI values. The GSI values of males generally showed less variation.

Gonad histological status of male and female European hake

The stages of germ cell development of females and males are listed in Table S1 and in Figure 3. A large number of females had immature ovaries (n=94). Females with developing ovaries were the second most frequent group (n=70); their ovaries were dominated by perinuclear and cortical alveolar oocytes (stage IIa, Table S1) Females with mature ovaries accounted for only 4% (n=7).

Among the males, a high number (n=68) of individuals showed immature testes, while males with mature gonads were rare (only 10% of the 176 males examined).

Season- and length-related variation of the gonad histological status

The annual variation of ovarian maturation stages is shown in Figure 4A: Females with immature ovaries were most abundant in September 2017 (75%), February 2018 (92%) and June 2018 (75%). Mature females were found only in July, January and March and accounted for a small percentage.

Immature males were abundant in February 2018 (86%), December 2017 (67%) and June 2018 (63%). Males with mature testes were observed mainly in January 2018, August 2017 and October 2017 (Fig. 4B).

All females smaller than 21 cm were immature, while mature females were observed only in fish of ³34 cm (Fig. 4C). Small males (≤18 cm) were immature, while maturing individuals appeared at 19 cm TL and mature males at 23 cm and more. At a body length of 34 cm, all males displayed spent testes (Fig. 4D). Pearson’s correlation tests showed a significant correlation between TL and gonad maturity stage for both sexes (p value <0.01, <0.05, Table 1A).

Difference between macroscopic and histological gonad staging

Histologically, 66% of the investigated fishes were classified to be immature. Macroscopically, however, only 34% of the examined gonads were classified as immature. This difference might be due to the fact that individuals classified macroscopically as mature were sometimes classified as immature individuals in the histological analysis. Differences between the microscopic and macroscopic gonad classifications were also found for spent gonads: while according to the histological examination, individuals with spent gonads were found in January, March and August 2018 and November 2017, macroscopically, fish with spent gonads were observed only in November 2017. Overall, the difference between macroscopic and microscopic staging was significant (Table 2B).

Length at first maturity (L₅₀)

The length at first sexual maturity (L₅₀) based on histological data was estimated to be 34.7 cm for females and 28.6 cm for males. Based on the macroscopic data the L₅₀ was found to be 30.6 cm for females and 26.8 cm for males (Fig. 5B, D).

Intersex cases

A small number of the histologically examined fish (4 cases) displayed intersex gonads, i.e. the gonads appeared either as testes but also contained a small number of female germ cells, or the gonads displayed an ovarian histology but also contained a small fraction of male germ cells.

In addition, we observed a single intersex case in which the gonads were composed of well-differentiated but anatomically separated ovarian and testicular tissues within the same individual (Fig. 6).

Question 1: Does the sex ratio of M. merluccius change with body length?

The sex ratio varied with body length: males were predominant in size classes below 27 cm (TL), while females were predominant among the larger individuals. Similar findings have been reported for M. merluccius populations from other regions: Khoufi et al. (2014)Khoufi W., Ferreri R., Jaziri H., et al. 2014. Reproductive traits and seasonal variability of Merluccius merluccius from the Tunisian coast. J. Mar. Biol. Ass. UK. 94: 1545-1556. https://doi.org/10.1017/S0025315414000356 observed for the Mediterranean Sea that males were predominant in size classes of less than 40 cm (TL) and females in the larger size classes. For hake populations of Atlantic Ocean, the critical body length for the sex ratio shift was 45 and 50 cm (El Habouz et al. 2011El Habouz H., Recasens L., Kifani S., et al. 2011. Maturity and batch fecundity of the European hake (Merluccius merluccius, Linnaeus, 1758) in the eastern central Atlantic. Sci. Mar. 75: 447-454. https://doi.org/10.3989/scimar.2011.75n3447 , Costa 2013Costa A.M. 2013. Somatic condition, growth and reproduction of hake, Merluccius merluccius L., in the Portuguese coast. Open J. Mar. Sci. 13: 12-30. https://doi.org/10.4236/ojms.2013.31002 ). On the Portuguese coasts, a dominance of males in small size classes and of females in large size classes of hake populations has also been reported (ICES 1988ICES. 1988. Working Group on Assessment of Hake Stocks. Copenaghen, 51 pp.).

This size-related shift of the sex ratio could have two explanations: protandric sexual development (Todd et al. 2016Todd E.V., Liu H., Muncaster S., Gemmell N.J. 2016. Bending Genders: The Biology of Natural Sex Change in Fish. Sex Dev. 10: 223-241. https://doi.org/10.1159/000449297 ); or sex-specific differences in growth, as occurs in a number of fish species (Cervino 2014Cervino S. 2014. Estimating growth from sex ratio-at-length data in species with sexual size dimorphism Fish. Res. 160: 112-119. https://doi.org/10.1016/j.fishres.2013.11.010 ). If the European hake were a protandric species, one might expect the occurrence of transition stages that display intersex gonads with morphological characteristics of males and females. This was not observed in the present study. What we found was a very low number of sexually mixed gonads (<1%). The morphology of these gonads does not resemble the gonadal morphologies of protandric transition stages as described in the literature (e.g. Shihab et al. 2017Shihab I., Gopalakrishnan A., Vinesh N., et al. 2017. Histological profiling of gonads depicting protandrous hermaphrodistism in Eleutheronema tetradactylus. J. Fish. Biol. 90: 202-211 https://doi.org/10.1111/jfb.13324 ). Thus, the second possible explanation, i.e. the differential growth of male and female fish, appears to be responsible for the size-related shift of the sex ratio. The available literature reports a clear difference in growth rate and L infinity between males and females (Murua 2010Murua H. 2010. The Biology and Fisheries of European Hake, Merluccius merluccius, in the North-East Atlantic. Adv. Mar. Biol. 58: 97-154. https://doi.org/10.1016/B978-0-12-381015-1.00002-2 , El Bouzidi et al. 2022El Bouzidi C., Abid N., Awadh H., et al. 2022. Growth and mortality of the European hake Merluccius merluccius (Linnaeus, 1758) from the North of Moroccan Atlantic coasts, Egypt. J. Aquat. Res. 48: 233-239. https://doi.org/10.1016/j.ejar.2021.12.003 ). Overall, our findings argue against protandric development and in favour of gonochoristic development of hake in Moroccan waters, with a low level of natural or environmental intersex cases.

The occurrence of a low number of individuals with intersex gonads has been reported for various gonochoristic fish species, such as European whitefish (Bittner et al. 2009Bittner D., Bernet D., Wahli T., et al. 2009. How normal is abnormal? Discrimination between deformations and natural variation in gonad morphology of European whitefish Coregonus lavaretus. J. Fish Biol. 74: 1594-1614. https://doi.org/10.1111/j.1095-8649.2009.02233.x ) and Japanese Medaka (Grim et al. 2007Grim K.C., Wolfe M., Hawkins W., et al. 2007. Intersex in Japanese medaka (Corsivo) used as negative controls in toxicologic bioassays: a review of 54 cases from 41 studies. Environ. Toxicol. Chem. 26: 1636-1643. https://doi.org/10.1897/06-494R.1 ). These gonadal abnormalities are usually interpreted as a spontaneous natural phenomenon (Bahamonde 2013Bahamonde P.A., Munkittrick K.R., Martyniuk C.J. 2013. Intersex in teleost fish: are we distinguishing endocrine disruption from natural phenomena? Gen. Comp. Endocr. 192: 25-35. https://doi.org/10.1016/j.ygcen.2013.04.005 ). Intersex gonads in fish can also be environmentally induced, for instance, by exposure to endocrine-disrupting compounds (Jobling et al. 1998Jobling S., Nolan M., Tyler CR., et al. 1998. Widespread sexual disruption in wild fish. Environ Sci Tech. 32: 2498-2506. https://doi.org/10.1021/es9710870 ).

Apart from the sexual size dimorphism, both male and female gonad maturity increased with body length: the longer the fish was, the more mature the gonad was. This is indicated from the significant correlation between the stages of maturity and the body of European hake (Pearson test, p<0.01) (Table 1). Importantly, the size maturity relationship applies for both male and female hake. Congruent findings were recorded by Murua (2010)Murua H. 2010. The Biology and Fisheries of European Hake, Merluccius merluccius, in the North-East Atlantic. Adv. Mar. Biol. 58: 97-154. https://doi.org/10.1016/B978-0-12-381015-1.00002-2 .

Question 2: Is there a seasonal variation in European hake maturity?

The variation in gonad maturity stages of M. merluccius over the years observed in this study shows limited interannual variation. Although values differed slightly between years [for instance, the percentage of immature females accounted for 50% in August 2017 compared with 60% in August 2018 (Fig. 4A)], the overall pattern appears similar between the two study years. This is in line with the reports in the literature (see El Habouz et al. 2011El Habouz H., Recasens L., Kifani S., et al. 2011. Maturity and batch fecundity of the European hake (Merluccius merluccius, Linnaeus, 1758) in the eastern central Atlantic. Sci. Mar. 75: 447-454. https://doi.org/10.3989/scimar.2011.75n3447 ).

The results must be interpreted with caution because several factors influence the seasonal variation. Both the timing and duration of the maturation and spawning cycle can be seasonally variable due to changing environmental conditions, notably temperature and food access, which influence available energy resources for the reproductive investment (Khoufi et al. 2014Khoufi W., Ferreri R., Jaziri H., et al. 2014. Reproductive traits and seasonal variability of Merluccius merluccius from the Tunisian coast. J. Mar. Biol. Ass. UK. 94: 1545-1556. https://doi.org/10.1017/S0025315414000356 ). Also, sample size and sample composition can influence the annual variation of reproductive parameters. Nevertheless, with all necessary caution, it appears that around January and in midsummer, a high percentage of fish with mature gonads are present, suggesting that these are periods of enhanced spawning activity. This interpretation is in line with the published literature: El Habouz et al. (2011)El Habouz H., Recasens L., Kifani S., et al. 2011. Maturity and batch fecundity of the European hake (Merluccius merluccius, Linnaeus, 1758) in the eastern central Atlantic. Sci. Mar. 75: 447-454. https://doi.org/10.3989/scimar.2011.75n3447 found that males and females were involved in the spawning process all year around, but a peak occurred in winter (January-February) and a secondary one in summer (July-August). In contrast, for hake population on the Portuguese coast, Costa (2013)Costa A.M. 2013. Somatic condition, growth and reproduction of hake, Merluccius merluccius L., in the Portuguese coast. Open J. Mar. Sci. 13: 12-30. https://doi.org/10.4236/ojms.2013.31002 reported three breeding peaks: in January-March, May-June and August. On the Mediterranean coast, Khoufi et al. (2014)Khoufi W., Ferreri R., Jaziri H., et al. 2014. Reproductive traits and seasonal variability of Merluccius merluccius from the Tunisian coast. J. Mar. Biol. Ass. UK. 94: 1545-1556. https://doi.org/10.1017/S0025315414000356 also observed three spawning peaks in January, April and August. Murua (2010)Murua H. 2010. The Biology and Fisheries of European Hake, Merluccius merluccius, in the North-East Atlantic. Adv. Mar. Biol. 58: 97-154. https://doi.org/10.1016/B978-0-12-381015-1.00002-2 in the Atlantic Ocean, showed a definite seasonal peak of GSI or gonad maturity for M. merluccius. The disparity in spawning periods in the European hake populations across the regions may be explained by the latitude factor (Khoufi et al. 2014Khoufi W., Ferreri R., Jaziri H., et al. 2014. Reproductive traits and seasonal variability of Merluccius merluccius from the Tunisian coast. J. Mar. Biol. Ass. UK. 94: 1545-1556. https://doi.org/10.1017/S0025315414000356 ) or by biological and environmental effects (Murua and Moto 2006Murua H., Motos L. 2006. Reproductive strategy and spawning activity of the European hake Merluccius merluccius (L.) in the Bay of Biscay. J. Fish Biology. 69: 1288-1303. https://doi.org/10.1111/j.1095-8649.2006.01169.x ).

Question 3: What are the differences between the macroscopical or histological staging of the gonads?

The classification of maturation stages differed between the two methods. This may be partly explained by the asynchronous oocyte development, which can be detected only by histology and not by macroscopy. Additionally, at the macroscopic scale, it is not possible to distinguish between immature/maturing and resting gonads or between spent and immature ones (Costa and Carmo Silva 2016Costa A.M., Silva M.C. 2016. Why do we need histology of hake (Merluccius merluccius) gonads? The Portuguese case. Relat. Cient. Téc. Portugal, 14 pp.). With such differences in resolution between the two methods, the macroscopic method appears to be less reliable than the histological one. Congruent conclusions were drawn by (Costa and Carmo Silva 2016Costa A.M., Silva M.C. 2016. Why do we need histology of hake (Merluccius merluccius) gonads? The Portuguese case. Relat. Cient. Téc. Portugal, 14 pp.).

Question 4: Is there any difference between the L₅₀ obtained macroscopically and microscopically?

The L₅₀ is an important parameter in fisheries science. It provides indications for the minimum size for sustainable management of fish populations, although the choice of the legal minimum size for commercial fishing depends on other factors as well. The information needed for the L₅₀ is the gonad maturity stage: at what size does the fish reach gonad maturity for the first-time? In the present study the L₅₀ analysis yielded different values when based on microscopic or macroscopic gonad examination of the same samples (n=369). The L₅₀ was 35 cm (histology-based) and 31 cm (macroscopy-based) for females, while for males it was 29 cm (histology-based) and 27 cm (macroscopy-based). Thus, the L₅₀ increased by almost 4 cm for females and by 2 cm for males when assessed by histology rather than by macroscopy. Given the difference between microscopical and macroscopical gonad classifications, as discussed above, it is not surprising that the two methods also yield different L₅₀ values.

The length at first maturity reported by Lahrizi (1996)Lahrizi H. 1996. Etude de la biologie de croissance et de reproduction du merlu blanc Merluccius merluccius (L. 1758) débarqué par les chalutiers au port de Casablanca. PhD thesis, Mohamed 7. Univ. Rabat. (41.1 cm for females; 37.8 cm for males) in the northern Atlantic coast of Morocco and by El Habouz et al. (2011)El Habouz H., Recasens L., Kifani S., et al. 2011. Maturity and batch fecundity of the European hake (Merluccius merluccius, Linnaeus, 1758) in the eastern central Atlantic. Sci. Mar. 75: 447-454. https://doi.org/10.3989/scimar.2011.75n3447 (33.8 cm for females; 28.6 for males) in the southern Atlantic coast of Morocco showed higher values than the macroscopical estimation obtained in the present study (31 cm for females; 27 cm for males). This may result from overexploitation of the European hake in the northern Atlantic coast of Morocco (El Bouzidi et al. 2022El Bouzidi C., Abid N., Awadh H., et al. 2022. Growth and mortality of the European hake Merluccius merluccius (Linnaeus, 1758) from the North of Moroccan Atlantic coasts, Egypt. J. Aquat. Res. 48: 233-239. https://doi.org/10.1016/j.ejar.2021.12.003 ). Given that fishing pressure can lead to the elimination of large and older hake, this could decrease the size at maturity as a compensatory response for the recovery of the stock (Domínguez-Petit et al. 2008Dominguez-Petit R., Alonso-Fernández A., Saborido-Rey F. 2008. Reproductive strategy and oocyte recruitment process of European hake (Merluccius merluccius) in Galician shelf waters. Cybium. 32: 317-318.). However, overexploitation may not be the only explanation for size changes at maturity; environmental conditions such as temperature, upwelling and North Atlantic Oscillation could play an important role in influencing size at maturity (Domínguez-Petit et al. 2008Dominguez-Petit R., Alonso-Fernández A., Saborido-Rey F. 2008. Reproductive strategy and oocyte recruitment process of European hake (Merluccius merluccius) in Galician shelf waters. Cybium. 32: 317-318.). For example, the histological L₅₀ found in this study was higher than that reported by El Habouz et al. (2011). This difference could be due to the upwelling activity that characterizes the southern Atlantic coast of Morocco (INRH 2019Institut National de Recherche Halieutique (INRH). 2019. Rapport annuel d’Etat des stocks et des pêcheries Marocaines au Maroc, Depmt. Des Péches, Maroc, Casablanca, ISSN : 2509-1727, 565pp.).

With respect to the histology-based L50, we found slightly higher values than Kahraman et al. (2017)Kahraman A.E., Yıldız T., Uzer U., Karakulak F.S. 2017. Age composition, growth and mortality of European hake Merluccius merluccius (Linnaeus, 1758) from the Sea of Marmara, Turkey. Acta Zool. Bulg. 69: 377-384. in their study on hake: 28.6 cm and 33.8 cm for males and females from the Sea of Marmara in Turkey. For females hake sampled along the Tunisian coast, the L₅₀ was 29.0 cm (Khoufi et al. 2014Khoufi W., Ferreri R., Jaziri H., et al. 2014. Reproductive traits and seasonal variability of Merluccius merluccius from the Tunisian coast. J. Mar. Biol. Ass. UK. 94: 1545-1556. https://doi.org/10.1017/S0025315414000356 ) while on the Galician Shelf the values obtained from L₅₀ females sampled in 2003 and 2004 were 44.45 cm and 42.97 cm, respectively. The differences in size at maturity reported from different regions may depend on parameters such as fishing pressure, temperature, food, genetic factors (Engelhard et Heino 2006Engelhard G. H., Heino M. 2006. Climate change and condition of herring (Clupea harengus) explain long- term trends in extent of skipped reproduction. Oecologia 149: 593-603. https://doi.org/10.1007/s00442-006-0483-3 ), overexploitation, latitudinal variation (Khoufi 2014Khoufi W., Ferreri R., Jaziri H., et al. 2014. Reproductive traits and seasonal variability of Merluccius merluccius from the Tunisian coast. J. Mar. Biol. Ass. UK. 94: 1545-1556. https://doi.org/10.1017/S0025315414000356 ), fishing mortality and upwelling (Domínguez-Petit et al. 2008Dominguez-Petit R., Alonso-Fernández A., Saborido-Rey F. 2008. Reproductive strategy and oocyte recruitment process of European hake (Merluccius merluccius) in Galician shelf waters. Cybium. 32: 317-318.).