Report of the Workshop on Crustaceans (Aristeus Antennatus, Aristaeomorpha Foliacea, Parapenaeus Longirostris, Nephrops Norvegicus) Maturity Stages (WKMSC)

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ICES WKMSC REPORT 2009

ICES ADVISORY COMMITTEE

ICES CM 2009/ACOM:46

REF. PGCCDBS, RCM MED, PGMED

Report of the Workshop on crustaceans (Aristeus antennatus, Aristaeomorpha foliacea, Parapenaeus longirostris, Nephrops norvegicus) maturity stages (WKMSC)

19-23 October 2009 Messina, Italy

International Council for the Exploration of the Sea Conseil International pour l’Exploration de la Mer

H. C. Andersens Boulevard 44–46 DK-1553 Copenhagen V Denmark Telephone (+45) 33 38 67 00 Telefax (+45) 33 93 42 15 www.ices.dk [email protected]

Recommended format for purposes of citation:

ICES. 2010. Report of the Workshop on crustaceans (Aristeus antennatus, Aristaeo- morpha foliacea, Parapenaeus longirostris, Nephrops norvegicus) maturity stages (WKMSC) , 19-23 October 2009, Messina, Italy. ICES CM 2009/ACOM:46. 77 pp.

For permission to reproduce material from this publication, please apply to the Gen- eral Secretary.

The document is a report of an Expert Group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council.

ICES WKMSC REPORT 20099 | i

Contents

Executive summary ...... 3

1 Opening of the meeting ...... 3 1.1 Opening of the meeting and adoption of the Agenda ...... 3 1.2 Scientific justification and aims ...... 3 1.3 Terms of Reference ...... 4 1.4 Data Collected before the Workshop ...... 5

2 Results of the workshop ...... 5 2.1 Available information on reproductive biology and ecology of species in the North-East Atlantic (ICES Division IXa, Nephrops Functional Units 28, 29 and 30) and the Mediterranean Sea (GFCM GSAs 1, 5, 6, 9, 10, 11, 15, 16 and 20) ...... 5 2.2 The gonad cycle according to macroscopic and microscopic approaches and images analyses ...... 9 2.3 Establishment of a standard histological procedure and scale...... 10 2.3.1 Protocol and terminology to classify the oogenic stages ...... 10 2.3.2 Descriptions of the proposed microscopic scale ...... 12 2.4 The proposal for a common macroscopic scale ...... 14 2.5 Comparison and conversion of previous maturity scales with the new one ...... 17

3 Conclusion and recommendations for new WK ...... 23

4 References ...... 23

Annex 1: List of participants...... 26

Annex 2: Agenda ...... 31

Annex 3: WGMSC terms of reference for the next meeting ...... 33

Annex 4: Recommendations ...... 33

Annex 5: Crustaceans gonad reference photos...... 34

Annex 6: The ICES (International Council for the Exploration of the Seas) ...... 73

Annex 7: The GFCM (General Fisheries Commission for the Mediterranean) Geographical Subareas (GSA) ...... 75

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Executive summary

During the meeting the participants reviewed the already employed and produced new scales of micro- and macroscopic maturity stages for the four crustacean species of main commercial values for the Eastern Atlantic and Mediterranean fisheries: (Aristeus antennatus, Aristaeomorpha foliacea, Parapenaeus longirostris, Nephrops norvegicus). These scales were proposed to be adopted by all Institutes which are involved in European DCF. A conversion table from both the historical and presently used scales to the new proposed common scales was also provided. Common problems in both micro- and macroscopic stage assignment were outlined and possible solutions were also provided. Results and comments are added to the present report. A reference image collection was built up thanks to the contribution of all the participants. Histological validations and stage descriptions were also illustrated and discussed. All participants felt that all the aims of the workshop were at- tained and suggested future activity/meetings in order to improve standardization among scientists who work in this field.

1 Opening of the meeting

1.1 Opening of the meeting and adoption of the Agenda The welcome and opening address of the meeting was pronounced by Fabio Fioren- tino (Chair) and Emilio De Domenico (Coordinator of the PhD Course on Environ- ment Science, Marine Environment and Resources of the University of Messina). During the opening, a video on “Beautiful sensations of the Straits of Messina“ was shown. A welcome cocktail was also offered by the Organisation. The Chair high- lighted the important role of EU, the Italian Ministry for Agricultural, Alimentary and Forestry Policies (MiPAAF) and ICES, in supporting the workshop. In this sense, a special acknowledgment was addressed to Paolo Carpentieri (MiPAAF delegate to the DCR Commission) and to the General Director of the MiPAAF - PEMAC VI, Francesco Saverio Abate, in charge to follow, for Italian Ministry, the workshop offi- cial permissions. Thanks were extended to the Italian Society of Marine Biology (SIBM), which supported the participation to the meeting, as well as to the team of the CNR-IAMC of Messina and of the University of Messina, who jointly organised the workshop. The Chair also underlined the important role of Helle Gjeding Jørgen- sen and Almuth Janisch as Assisting Secretary of the Advisory Programme of ICES. Finally many thanks went to all the participants who made their best effort in order to fulfil all the terms of references of the Workshop.

A complete list of participants is presented in Annex 1 of this report, whereas the Workshop Agenda is available in Annex 2.

1.2 Scientific justification and aims The gonads development pattern and maturity stage recognition are important biological items to be studied in fishery sciences. They allow to discriminate life phases or “stanzas” (recruits, juveniles, adults, hermaphrodites, neoteny occurrence, etc), to characterise the eggs development and emission modality (total or serial batch spawner), to set the spawning season of a species, monitoring of long‐term changes in the spawning cycle, and to estimate the achievement of sexual maturity (and the related computation of the Spawning Stock Biomass). Furthermore these studies are relevant for many other topics regarding the life cycle (for example, energy budget

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allocation and maturity-survival-longevity trade-offs) of any exploitable or already exploited marine living resource. For example, a comparison of the reproductive structure (as deduced by maturity stages), spawning occurrence, investment rate in the gonads (gonosomatic index) and size at maturity, among different geographical areas and under heterogeneous level of exploitation can be used to highlight the response of the investigated resources to both environmental and fishing variations. Any accurate comparison of the reproductive pattern, however, would require a higher coherence and consistence among the different methods employed to gather the basic data. Indeed, logistic and administrative constraints, the accumulation of new knowledge or the personal feelings of the scientists in charge have determined the use of different macroscopic maturity scales, even for the same species, often in the same geographical area in successive times. Such difficulties are enhanced in case of crustacean species, whose reproductive patterns and their links with the life cycle traits are less documented than with the more usual fish species. The need of a common and standardized system for identification and macroscopic classification of maturity stages in the assessment of the fishery resources by the laboratories collecting maturity data involved in WKMSC, has to be considered as an important priority to optimize DCR. Therefore, this workshop had the objectives of reviewing the previous methodologies and scales, defining objective criteria to classify the maturity stages both on micro and macro scale, reaching an agreement on a common scales to be used in the future, and figuring out conversion rules between the old and new scales. A collateral but still relevant goal of this Workshop was to build a collection of micro and macro descriptions (mainly photos) realised by the different laboratories to get an objective measure to what extent the criteria to classify maturity stages is coherent between technicians, and to pinpoint the major sources of disagreement . Finally, it is worth pointing out the fact that if the new standard micro- and macro- maturity scales are accepted, this would improve considerably the exchange of data between laboratories and hopefully the production of comparable reproductive esti- mates to be used in the assessments of such highly priced resources.

1.3 Terms of Reference a ) Compare the macroscopic maturity scales for Aristeus antennatus, Aristaeo- morpha foliacea, Parapenaeus longirostris, Nephrops norvegicus used in the different laboratories; b ) Standardize criteria to classify each maturity stage to be used for DCR, and discuss on the existing maturity scales; c ) Formulate conversion rules to make possible the correspondence between the locally used scales and the common ones; d ) Validate the macroscopic maturity stages according to the common standardized scales possibly using histological confirmation; e ) Standardize the criteria to classify each maturity stage; f ) Propose a common scale, with common classification criteria, to be used by all laboratories

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1.4 Data Collected before the Workshop Before the Workshop, each participant laboratory/group was invited to preliminary gather information, by species and whenever possible for both males and females, to: a ) describe the sexual development and the maturity pattern accepted and used at the present; b ) assemble the different (both historical and presently used) macroscopic maturity scales; c ) assemble the different (both historical and presently used) microscoscopic maturity scales; d ) collect the validation(s) of the macroscopic maturity stages after histological, morphometric or gravimetric analyses; e ) indicate the estimators (mean, median, size at the onset of sexual, SOM, or size at 50%, Lm, of maturity etc) employed to discriminate the gonads development stages (immature, maturing, mature etc) or life phases (juveniles and adults); f ) collect sketches and digital photos of the different maturity stages at both microscopic (histological preparation) and, macroscopic level. Each participant was hence invited to communicate to the WS Scientific Committee the material that intended to be presented at the workshop and the willingness to provide a Working Document (WD) containing a synoptic presentation of available information for its own laboratory, operative unit or geographical area, sub area or division. In fact, it is worth recalling that the Laboratories involved in the WS operate in areas where different bodies are engaged in producing management advices: the Scientific Advisory Committee for GFCM for the Mediterranean, and ICES for the western European coasts; the two bodies adopted the geographical sub-area (GSA) or area-division, respectively, as basic geographical reference.

2 Results of the workshop

2.1 Available information on reproductive biology and ecology of species in the North-East Atlantic (ICES Division IXa, Nephrops Functional Units 28, 29 and 30) and the Mediterranean Sea (GFCM GSAs 1, 5, 6, 9, 10, 11, 15, 16 and 20) Up 24 Working Documents were presented at the Workshop. Each Laboratory team presented their Working Documents as resumed (by Author and species) in the following section. Full presentations are available on the ICES Share point web page: http://groupnet.ices.dk/WKMSC2009/default.aspx 1) Cristina Silva (INRB/L-IPIMAR) illustrated the maturity scale for target crustaceans used and size at onset maturity in Portuguese waters (ICES Division IXa). Aristeus antennatus: The 5-stage maturity scale used for females of Aristeus antennatus was presented, based on macroscopic characteristics and adapted from Ribeiro Cascalho and Arro- bas (1982). Maturity ogive and size at onset of sexual maturity for females were estimated using data from the crustacean surveys, in May-June, along the period 1997- 2008. A weighted non-linear regression between the proportions of mature females over carapace length was applied and the L50 estimated was ≈ 22.4 mm of carapace length.

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Aristaeomorpha foliacea: The characterization of the maturity cycle of females of Aristaeomoprpha foliacea was presented based on a macroscopic maturity scale composed by 5 stages: immature, developing, maturing, mature and spent. Parapenaeus longirostris: The 4-stage maturity scale for females of Parapenaeus longirostris was presented, based on macroscopic characteristics and adapted from Arrobas and Ribeiro Cas- calho (1982). Maturity ogive and size at onset maturity for females were estimated using data from the crustacean surveys, in May-June, along the period 1997-2008. A weighted non-linear regression between the proportions of mature females over carapace length was applied and the L50 estimated (≈ 24 mm) was close to the presented by Ribeiro Cascalho (1988). Nephrops norvegicus: The 5-stage maturity scale used for females of Nephrops norvegicus was presented, based on macroscopic characteristics and adapted from Farmer (1974) and Figueiredo (1982). Maturity ogive for females and size at onset maturity for males and females estimated in the Workshop on Nephrops Stocks (2006), for Functional Units 28 and 29 (Alentejo and Algarve, respectively), using data from the crustacean surveys, in May- June, along the period 2003-2005, were presented. A weighted log-log non-linear regression between the proportions of mature females over carapace length was applied and the L50 estimated was ≈ 30 mm of carapace length. For males, the macroscopic classification was based on the length of the appendix masculina, which was regressed to the corresponding carapace length. A L50 ≈ 28.4 mm was estimated as the inflexion point in the linear relation between the length of the appendix masculina and the carapace length. 2) Laura Pirrera and Anna Perdichizzi (IAMC-CNR, section of Messina) showed the maturity scale used and size at onset maturity in the Southern Tyrrhenian Sea (GFCM GSA 10). In particular Laura Pirrera (IAMC-CNR of Messina) showed photos of the ovary stages of Aristeus antennatus and Parapenaeus longirostris and compared their observations with the descriptions given by the MEDITS scale (2007), for each maturity stage. Results demonstrated a correspondence with the scale except for some points, such as colours of many stages, both for the purple and the pink shrimp. Anna Perdichizzi (IAMC-CNR of Messina) showed the comparison between the macroscopic and microscopic stages of the ovaries in Aristaeomorpha foliacea, in an at- tempt to validate the MEDITS macroscopic scale. The histological analysis showed a correspondence with the MEDITS stages but for stages 2c and 2d, no marked histological differences were observed, probably because these stages are very short-lived. Furthermore, no functional differences were found between stages 2a and 2b, but the proportion of the different cells differed. 3) Margarida Cristo (University of Algarve) illustrated the Nephrops norvegicus male maturity scale used in Portuguese waters (ICES Division IXa, FUs 28 and 29). Maturity in males is rarely mentioned in reproduction studies. In over exploited populations of Norway lobster, as in the Algarve (South Portugal), more males than females are caught and mating may be at stake if the male fraction of the population is near maturation size. In this work a male maturity scale for Nephrops norvegicus was presented, with three differentiation stages, based on the number and type of

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male reproductive cells. An immature stage is characterized by the presence of sper- matogonia and primary spermatocytes, showing large nucleolus. An intermediate stage is characterized by the presence of some spermatozoids concentrated in areas surrounded by primary and secondary spermatocytes. Finally, a mature stage is characterized by concentrations of spermatozoids showing a characteristic clusters disposition with all capsules oriented in the same direction and the spines oriented to the lumen of the seminiferous tubes. Size at onset of male maturation is 24 mm carapace length. Spermatogenesis occurs throughout the year with intensification during spring. 4) Maria Cristina Follesa and Cristina Porcu (Department of Animal Biology and Ecology - University of Cagliari) showed female maturity scales of Aristeus antennatus, Aristaeomorpha foliacea and Parapenaeus longirostris from Sardinian seas (GFCM GSA 11). Monthly samples were obtained from the bottom trawl survey (MEDITS) and landing data from March to October 2009. For each individual sampled carapace length (CL, mm), sex and maturity stage were recorded. The maturity scales, compared with the MEDITS ones (Anonymous, 2007), were validated through macroscopic and microscopic observations. For each stage a CL range of the sampled specimens was also defined. In general, in the red-shrimps A. foliacea and A. antennatus the macroscopic analysis showed only the colour of resting stage in disagreement with the MEDITS stage. In P. longirostris, the colour of maturing, mature and spent the ovaries did not correspond with that defined by the MEDITS scale; these differences have been also proved by histological analysis. For all species, some biological estimators (the mean values of Gonado-Somatic Index for each stage examined and size at first maturity) were also presented. 5) Francesca Gravino & Roberta Pace (Malta Centre For Fisheries Sciences) reported the maturity scales used in Maltese waters (GFCM GSA 15). Malta adopts a common maturity scale for the identification of crustacean maturity stages, the MEDITS scale. This scale is used both for fisheries dependent (on-board observations on commercial vessels) and independent (experimental) surveys. Up to 2008, however, the old (not detailed) two – three stages MEDITS scale (Anon. 1994) was used. However, from MEDITS 2009 to date, the detailed MEDITS scale is being used (Anon. 2007) both for MEDITS surveys and for other data collection purposes. Malta collects data on maturity stages for females only, and these are always determined macroscopically, without dissection. Malta presented the photos taken in the laboratories at MCFS (Malta Centre for Fisheries Sciences) for the maturity stages of the four species: Aris- taeomorpha foliacea, Aristeus antennatus, Nephrops norvegicus and Parapenaeus longirostris.

6) José Luis Pérez Gil (Instituto Español de Oceanografía. Centro Oceanografico de Fuengirola) showed the maturity scales used in Spanish Mediterranean waters. Deep-water rose shrimp (Parapenaeus longirostris) (Lucas, 1846) is one of the most important commercial crustacean in the south of the Spanish Mediterranean (GFCM GSA, 1, Alboran sea) and its abundance decreases towards the north of the Spanish area (GFCM GSAs 6 and 5). It is caught almost exclusively by trawl. The method used in all the Mediterranean Spanish Institutes for the females´ maturity description is that of Crosnier (Crosnier et al., 1970). According this method, the females´gonad maturity is based on their size and colors. Following this scale, three maturity stages exist (I Immature, II Developing and III Mature). Sobrino, 1998, dividing the previous III stage in two newer ones: III as mature and IV as advanced mature. This last scale has been used by the IEO, in the Gulf of Cadiz. The maturity of males, in the whole

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Spain, is based on the presence or absence of the petasma´s fusion, adopting, thus, a scale of two stages: I. Immature and 2: Mature. In the Spanish Mediterranean deep waters zone, the Violet shrimp (Aristeus antennatus) (Risso, 1816) is exploited mainly in the Ibiza Channel, Catalonia, Levante, Murcia and Almería. The maturity of this species is determined by a macroscopic observation. For females a scale of seven ovary maturity scales is used (Demestre and For- tuño, 1992): I, Undeveloped juvenile, II, Undeveloped adults, III, Developing, IV, Maturation, V, Advance Maturation, VI Spawning, VII Post-spawning. Male maturity is, also, macroscopically determined as a function of the shortening of the rostrum and the petasma´s fusion (Sardá and Demestre, 1989; Sardá and Cartes, 1997). Ac- cording them, two stages exist: I, Undeveloped (juveniles) males: Petasma not joined, long rostrum and II, Developed (adults): Petasma completely joined, short rostrum. 7) Kapiris Kostas (Hellenic Centre of Marine Research) reported the maturity scales used in Greek waters (GFCM GSA 20). Only in the last 15 years, it has been shown that both aristeids have the potential to support a viable fishery in the Greek Ionian Sea. The existed results demonstrated the high reproductive capacity of both red shrimps in the Ionian Sea. The main mating period (>50% mated females) of both aristeids lasts longer in the eastern Ionian than other areas of the Mediterranean. Ovarian maturity stages were recorded by macroscopic observation using the modified empirical scales of Levi and Vacchi (1998) for A. foliacea and Relini-Orsi, Relini (1979) for A. antennatus. according to these modified scales five stages of development were established based on the ovarian colour and size: stage I: undeveloped; stage II: developing; stage III: mature; stage IV: spawning and stage V (spent). thus, the spent individuals (sp) were separated from those belonging to stage I in both species, the ovarian maturation process (appearance of stages III and IV) seemed to be rapid, lasting one to two months. A. foliacea has a rather earlier and more extended spawning period than A. antennatus. The gonad weight and the gonadosomatic mean value of the spent individuals were between the stages I and II, for both species. The smallest mature female of A. foliacea and A. antennatus was 36 mm CL and 26 mm CL, respectively. The 50% size at first maturity (CL50mf) was 38.84 mm CL for A. foliacea and 29.45 mm CL for A. antennatus and the 50% of females bearing spermatophores (CL50sf) was 36.84 mm CL for A. foliacea, 26.03 mm CL for A. antennatus. Both aver- age absolute and relative fecundities estimated for A. antennatus were statistically higher than those estimated for A. foliacea. In conclusion, A. foliacea shows lower fecundity than A. antennatus, having a lower ovogenetic activity, but it produces larger oocytes. The mature males (presence of the spermatophores in the terminal ampoul- lae) in A. foliacea were consistently very high during the whole year but varied widely in A. antennatus. The smallest reproductive male measured 26 mm and 20 mm CL, in A. foliacea and A. antennatus, respectively. 8) Marina Sartini (Centro Interuniversitario di Biologia Marina ed Ecologia Applicata of Livorno - Italy) presented mean size according to maturity stage, mean size according to “mature” and “immature” specimens and maturity ogives for Aristeus antennatus, Parapenaeus longirostris and Nephrops norvegicus (GFCM GSA 9). Macroscopic maturity stages were assigned according to the MEDITS scale (2007) based on six stages (seven stages for N. norvegicus). Data used for the analysis came from biological sampling of commercial trawling collected in the context of DCR (Campbiol 2008) and “Medits 2008”. For A. antennatus the size at first maturity (L50) was 29.03 mm CL according to “Campbiol 2008” data and 25.94 mm CL for “Medits 2008” data. Con- sidering P. longirostris the L50 determined from the two sets of data was 20.95 mm CL and 19.94 mm CL respectively. Finally the maturity ogive estimated for N. norvegicus

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reported a L50 of 28.10 mm CL (Campbiol 2008) and 27.06 mm CL (Medits 2008). Photos of the macroscopic maturity stages taken from fresh dissected females of P. longirostris and N. norvegicus were presented. 9) Yolanda Vila and Marina Delgado (Instituto Español de Oceanografía. Centro Oceanográfico de Cádiz) presented macroscopic and microscopic maturity stages of Nephrops norvegicus from the Gulf of Cadiz (ICES Division IXa, FU 30). Monthly samples were obtained from the commercial bottom trawl fleet from May to August 2009. For each individual sampled carapace length (mm), total weight (g), sex and maturity were recorded. Macroscopic maturity stages were assigned according to a 5-stage scale based on Farmer (1974): Immature (stage I), developing (stage II), maturing (stage III), mature (stage IV) and post spawning (stage V). For the ovarian histological analysis, gonads were fixed in 4% buffered formaldehyde, dehydrated in increasing concentrations of ethanol, embedded in resin, cut in 3 µm slices and stained in Tolu- idine Blue. Ovarian histological staging was carried out according to the most advanced oocyte. Six types of cells were identified: oogonia, previtellogenic, early vitellogenic and late vitellogenic oocytes. Post ovulatory follicles and atretic oocytes were also observed. In order to validate the macroscopic stages, the annual reproductive cycle must be covered with histological analysis. 10) Sergio Ragonese (IAMC-CNR, section of Messina) showed some relevant results about reproduction of Parapenaeus longirostris in GFCM GSA 16 (Southern coast of Sicily). Standing stock and corresponding basic biological features of the species off the southern coasts of Sicily (Central Mediterranean Sea), are regularly monitored thru experimental trawl surveys (MEDITS and GRUND programs), respectively carried out in spring-summer and autumn-winter. A broad (all years combined) size at onset of sexual maturity (SOM, as carapace length; CLm) has been obtained by ana- lyzing the data on the female ovarian maturity stages gathered in the period 1994-2007. Immature/juvenile specimens represent the bulk of the catch in both kinds of survey, and result well discriminated from the adult components. The SOM has been computed with the logistic approach, with values slightly different for MEDITS (CLm= 22.4 mm) and GRUND (CLm= 20.6 mm). Although the more conservative MEDITS value might be precautionarily employed, the GRUND estimation, closer to the spawning peak, should be preferred as reference for assessment and management of the rose shrimp Sicilian stock.

2.2 The gonad cycle according to macroscopic and microscopic approaches and images analyses The participants examined the available maturity stages, comparing the photos by stage with macroscopic and microscopic description in order to describe the gonad cycle. The common analyses were mainly focused to females because maturity in male decapods cannot be readily determined from macroscopic examination of gonads and associated structures, and few such studies have been documented. A critical review of the examined materials is reported in Annex 4.

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Here is the list of Institutes involved in the review.

COUNTRY - CITY INSTITUTE - COMPANY

National Research Council (CNR) – Italy, Messina and Mazara del Vallo (Trapani) Institute for Coastal Marine Environment (IAMC) University of Algarve – Portugal, Faro CCMAR National Research Council (CNR) - Italy, Ancona Institute for Marine Sciences (ISMAR) University of Messina - Italy, Messina Department of Animal Biology and Marine Ecology Spain, Cádiz and Fuengirola Instituto Español de Oceanografía (IEO) Malta, Fort San Lugan Malta Centre for Fisheries Sciences Hellenic Centre of Marine Research - Greece, Anavissos (Athens) Institute of Marine Biological Resources University of Bari – Italy, Bari Department of Animal Biology and Environment University of Cagliari – Italy, Cagliari Department of Animal Biology And Ecology University of Genova – Italy, Genova Dip.Ter.Ris. Italy, Livorno C.I.B.M. Portugal, Lisbon INRB/L-IPIMAR Italy, Monterotondo Scalo (Rome) National Research Council (CNR) - IBAF Italy, Bari COISPA

2.3 Establishment of a standard histological procedure and scale.

2.3.1 Protocol and terminology to classify the oogenic stages

Sampling on board Monthly samples, or at least 4 times a year (seasonally) but covering the reproductive season.

Procedure In order to validate macroscopic scales, sampling should cover all gradients of gonad aspect, with photos of freshly dissected animals showing the gonads. These individuals should be fixed separately, and then photographed after fixation. From each macroscopic classification at least 10 individuals should be processed. For the reproductive cycles, sampling should cover the whole length distribution. Sampling should be done by 2 or 5mm carapace length classes, depending on the size range, collecting 10 individuals of each size class. For histology, at least 30 individuals should be processed covering all the stages by length class.

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Gonads processing The gonads (testes and ovaries) have to be buffered in formaldehyde 4%. Fixation on board, after cutting the integument, for at least 24 h and a maximum of 48h, thereaf- ter transferring to 70º alcohol for storage. The gonads have to be embedded in resin. The sections have to be stained with hema- toxylin and eosin.

This the terminology used in the microscopic scale description: Oogonia (OO): cells with large nucleus and hardly visible cytoplasm. Nucleus with one nucleolus and peripheral heterochromatin. Early Primary Oocytes (EPO): cells with large nucleus with no nucleolus, and chromatin arranged in thin threads. Hardly visible cytoplasm. Late Primary Oocytes (LPO): the nucleus present several nucleoli. The cytoplasm becomes very much visible with unstained vesicles scattered throughout. Early Vitellogenic Oocytes (EVO): quadrangular or polyhedral cells with a round nucleus containing several nucleoli flattened again the nuclear wall and the cytoplasm field with yolk granules coloured pink and unstained vesicles. Late Vitellogenic Oocytes (LVO): cells rectangular, tightly packed cells, piled up as coins in a single row, their nucleus presenting rounded nucleoli. Advanced Late Vitellogenic Oocytes (ALVO): in these cells the nucleus migrates to the periphery and cortical granules (CG) appear. These cells are considered as the most sure index of the imminent spawning. Atresic oocytes (AO): these cells are irregular in shape and decrease gradually in size.

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2.3.2 Descriptions of the proposed microscopic scale

Aristaeus antennatus Proposed Name Microscopic Description stages Presence of oogonia (OO) and of both early primary oocytes (EPO) and occasionaly late primary oocytes (LPO) (25-90 µm), scattered around the stroma with no special organization. Oogonia with large nucleus and hardly visible cytoplasm. Nucleus with one nucleolus and peripheral heterochromatin. EPO 1 Immature with large nucleus with no nucleolus, and chromatin arranged in thin threads. Hardly visible cytoplasm. LPO the nucleus present several nucleoli. The cytoplasm becomes very much visible with unstained vesicles scattered through out.

Developing/ The ovarian parenchima is now structured into germinative zone containing 2 /Recovering OO and EPO and maturative zones organized into lobuli containing LPO.

Besides all the previous cell types, early (EVO) (80-190 µm) and late (LVO) (175- 300µm) vitellogenic oocytes are visible in the maturative zone. EVO, as polyhedral cells with a round nucleus containing several nucleoli flattened 3 Maturing agains the nuclear wall and the cytoplasm field with yolk granules colored pink and unstained vesicles and LVO completely stuck to one another forming a mosaic pattern theire nucleus presenting rounded nucleoli.

Besides all the previous cell types advanced LVO (ALVO) cells may occur.In 4 Mature these cells the nucleus migrates to the periphery and cortical granules (CG) appear*. These cells can indicate an imminent spawning.

The ovary tissue appears empty . Residulas of fully mature oocytes can still be 5 Spent seen and they possibly will be resorbed. Atresic oocytes (AO) are also present.

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Aristaeomorpha foliacea Proposed Name Microscopic Description stages

Presence of oogonia (OO) and of both early primary oocytes (EPO) and occasionaly late primary oocytes (LPO), scattered around the stroma with no special organization. Oogonia (< 10 µm) with large nucleus and hardly visible cytoplasm. Nucleus with one nucleolus and peripheral heterochromatin. Early 1 Immature primary oocytes with (15-25 µm) large nucleus with no nucleolus, and chromatin arranged in thin threads. Hardly visible cytoplasm. Late primary oocytes (25-90 µm) the nucleus present several nucleoli. The cytoplasm becomes very much visible with unstained vesicles scattered through out.

The ovarian parenchima is now structured into germinative zone containing Developing/ 2 oogonia and primary oocytes and maturives zones organized into lobulous /Recovering containing late primary oocytes.

Besides all the previous cell types, early (EVO) and late (LVO) vitellogenic oocytes are visible in the maturative zone. EVO, as quadrangular cells (90-300 µm in major axies) with a round nucleus containing several nucleoli flattened 3 Maturing agains the nuclear wall and the cytoplasm field with yolk granules colored pink and unstained vesicles. LVO as rectangular ( > 300µm), tightly packed cells, pilled up as coins in a single row, their nucleus presenting rounded nucleoli.

Besides all the previous cell types advanced LVO (ALVO) cells may occur.In 4 Mature these cells the nucleus is not visible and a convoluted perivitelline space along the cell membrane is visible. These cells can indicate an imminent spawning.

The ovary tissue appears empty. Residulas of fully mature oocytes can still be 5 Spent seen and they possibly will be resorbed. Atresic cells (AO) are also present.

Parapenaeus longirostris Proposed Name Microscopic Description stages Presence of oogonia (OO) and of both early primary oocytes (EPO) and occasionaly late primary oocytes (LPO), scattered around the stroma with no special organization. Oogonia with large nucleus and hardly visible cytoplasm. 1 Immature Nucleus with one nucleolus and peripheral heterochromatin. EPO with large nucleus with no nucleolus, and chromatin arranged in thin threads. Hardly visible citoplasm. LPO the nucleus present several nucleoli. The cytoplasm becomes very much visible. The ovarian parenchima is now structured into germinative zone containing Developing/ 2 OO and EPO and maturative zones containing LPO in which nucleus presents /Recovering nucleoli flattened agains the periphery Besides all the previous cell types, early (EVO) and late (LVO) vitellogenic oocytes are visible in the maturative zone. EVO, as polyhedral cells with a 3 Maturing round nucleus containing several round nucleoli and the cytoplasm field with yolk granules colored pink and unstained vesicles. Besides all the previous cell types advanced LVO (ALVO) cells occur. In these 4 Mature cells the nucleus is not visible and cortical granules (CG)appear. These cells can indicate an iminent spawning. The ovary tissue appears empty . Residulas of fully mature oocytes can still be 5 Spent seen and they possibly will be resorbed.

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2.4 The proposal for a common macroscopic scale On the basis of agreed macroscopic sketch confirmed by the microscopic description the following maturity scale were proposed for the investigated species.

Aristeus antennatus

PROPOSED MACROSCOPIC NAME COLOUR REMARKS STAGES DESCRIPTION

Ovaries not visible without dissection. The ovaries are thin and translucent with a tubular 1 Immature Uncoloured appearance adherent to the laterals of the stomach, not extending to the abdomen.

The cut-size length Ovaries barely visible without between the first dissection. Cephalic lobes small Developing/ maturation and 2 but distinguishable. The gonad Ivory to pink /Recovering recovering must be extends to the full length of the defined by abdomen. microscopic analysis.

Ovaries are clearly visible through integument. Cephalic 3 Maturing Lilac and abdominal extensions are well developed and turgid.

Turgid ovaries occupying the whole dorsal area. Lobes and Violet or 4 Mature abdominal extensions well purple developed. Eggs well visible.

Ovaries large but flaccid with Ivory with 5 Spent purple spots. purple spots

ICES WKMSC REPORT 2009 | 15

Aristaeomorpha foliacea

PROPOSED MACROSCOPIC NAME COLOUR REMARKS STAGES DESCRIPTION

Ovaries not visible without dissection. The ovaries are thin and translucent with a 1 Immature tubular appearance Uncoloured adherent to the laterals of the stomach, not extending to the abdomen.

The cutsize Ovaries barely visible length between without dissection. the first Cephalic lobes small Cream- Developing/Resting maturation and 2 but distinguishable. orange or /Recovering recovering must The gonad generally reddish be defined by extends up to 3rd microscopic abdominal somite. analysis. Ovaries are clearly visible through integument. Ovaries developed and turgid, Light to 3 Maturing with evident cephalic dark grey lobes. The gonad generally extends to the 4th abdominal somite. Turgid ovaries extending to the whole dorsal area. Lobes well developed and Anthracite 4 Mature abdominal extensions to black may reach the 5th somite. Eggs well visible.

Ovaries large but 5 Spent flaccid with blackish Greyish spots.

16 | ICES WKMSC REPORT 2009

Parapenaeus longirostris

PROPOSED MACROSCOPIC NAME COLOUR REMARKS STAGES DESCRIPTION

Ovaries not visible without dissection. The ovaries are thin and translucent with a tubular 1 Immature Uncoloured appearance adherent to the dorsal portion of the stomach, not extending to the abdomen.

The cutsize length Ovaries are barely visible between the first Developing/ without dissection. The Whitish, pale maturation and 2 Resting/ cephalic lobes start to cover the yellow or recovering must be /Recovering sides while the abdominal cream orange defined by extensions occupy all somites. microscopic analysis.

Ovaries are clearly visible through integument. Ovaries developed and turgid, with Dark yellow, 3 Maturing cephalic lobes and abdominal cream orange extensions occupying the or light green. entirely the dorsal portion. The gonads appear granular.

Turgid ovaries extending to the whole dorsal area. Lobes Different tones 4 Mature and extensions well of dark green developed. Eggs well visible.

The colour Ovaries after spawning are classification was 5 Spent fully extended but loose Dark brown supported on turgidity becoming flaccid. histological analysis of 10 individuals.

ICES WKMSC REPORT 2009 | 17

Nephrops norvegicus

PROPOSED NAME MACROSCOPIC DESCRIPTION COLOUR REMARKS STAGES

Ovaries not visible without dissection. The ovaries are 1 Immature Uncoloured No picture available translucent, thin and threadlike. The histological analyses do Ovaries barely visible not differentiate the without dissection. The developing gonads in the Developing/ gonads extends up to the White to first maturation from the 2 /Recovering 1st somite of the abdomen cream recovering ones. The cut-size and have a granular length shall be defined by appearance. area/stock, after analyses of lengths vs. maturity data. Ovaries are clearly visible through integument. The gonad occupies one third 3 Maturing of the cephalotoracic Light green space. The gonads extends up to the 1st somite of the abdomen. Turgid ovaries occupying Reabsortion of the ovarian the whole dorsal material can be observed in 4 Mature cephalotoracic space and Dark green any of the stages 3 to 5. extending up to the 2nd somite. Eggs visible. Ovaries flaccid with green spots. Re-absorption of Ivory with 5 Spent ovarian material. Most green spots likely with green eggs on pleopods.

Berried female shall be Berried females recorded as a remark.

2.5 Comparison and conversion of previous maturity scales with the new one The new proposed scale was compared with the existing maturity scales derived from the participants’ presentations and from literature and when possible the stage numbers were assigned as for the new scale. Any discrepancies and remarks were pointed out and the group also made some suggestions for the new proposed scale, to be discussed with the whole group.

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Aristeus antennatus

MEDITS (1994) MEDITS (2007) PROPOSED RIBEIRO CASCALHO ET AL., 1982 KAPIRIS MACROSCOPIC DESCRIPTION RELINI E ORSI-RELINI, 1979 DEMESTRE ET AL., 1992 IN ANON. IN ANON. STAGES AS MODIFIED BY SILVA, 2009 (MODIFIED) 1998 2007 Ovaries not visible without dissection. The ovaries are thin and translucent with a 1 tubular appearance adherent I 1 1 1 1 I to the laterals of the stomach, not extending to the abdomen.

Ovaries barely visible without dissection. Cephalic lobes 2 small but distinguishable. The II 2 2+3 2a+2b II gonad extends to the full length of the abdomen.

Ovaries are clearly visible through integument. Cephalic 3 III 3 4+5 2c III and abdominal extensions are 2 well developed and turgid.

Turgid ovaries occupying the whole dorsal area. Lobes and 4 IV 4 6 2d IV abdominal extensions well developed. Eggs well visible.

Ovaries large but flaccid with 5 I 5 7 2e SP purple spots.

ICES WKMSC REPORT 2009 | 19

Aristaeomorpha foliacea

MEDITS MEDITS PROPOSED LEVI AND VACCHI, KAO ET AL., (1994) (2007) KAPIRIS IPIMAR MACROSCOPIC DESCRIPTION STAGES 1998 1999 IN ANON. IN ANON. (MODIFIED) (SILVA) 1998 2007 Ovaries not visible without dissection. The ovaries are thin and translucent with a 1 tubular appearance adherent to the laterals 1st I 1 1 I 1 of the stomach, not extending to the abdomen.

Ovaries barely visible without dissection. Cephalic lobes small but distinguishable. 2 2nd II 2a+2b II 2 The gonad generally extends up to 3rd abdominal somite.

Ovaries are clearly visible through integument. Ovaries developed and turgid, 3 with evident cephalic lobes. The gonad 3rd III 2c III 3 generally extends to the 4th abdominal 2 somite.

Turgid ovaries extending to the whole dorsal area. Lobes well developed and 4 4th IV 2d IV 4 abdominal extensions may reach the 5th somite. Eggs well visible.

Ovaries large but flaccid with blackish 5 / 2e spots. SP 5

20 | ICES WKMSC REPORT 2009

Nephrops norvegicus MEDITS (1994) MEDITS (2007) PROPOSED STAGES MACROSCOPIC DESCRIPTION FARMER (MODIFIED) IN ANON. IN ANON. 2007 1998

Ovaries not visible without dissection. The ovaries are 1 1 1 1 translucent, thin and threadlike.

Ovaries barely visible without dissection. The gonads extends 2 up to the 1st somite of the 2 2a+2b abdomen and have a granular appearance.

Ovaries are clearly visible through integument. The gonad occupies one third of the 3 3 2c cephalotoracic space. The gonads extends up to the 1st somite of the abdomen. 2

Turgid ovaries occupying the whole dorsal cephalotoracic 4 4 2d space and extending up to the 2nd somite. Eggs visible.

Ovaries flaccid with green spots. Re-absorption of ovarian 5 5 2e material. Most likely with green eggs on pleopods.

Berried females Ovigerous 3 3

| 21

Parapenaeus longirostris

PROPOSED MACROSCOPIC DESCRIPTION CROSNIER ET AL., 1970 FROGLIA, 1982 NOUAR, 1985 DE RANIERI ET AL., 1986 ARCULEO ET AL., 1992 DE RANIERI ET AL., 1998 STAGES

Ovaries not visible without dissection. The ovaries are thin and translucent with 1 a tubular appearance adherent to the 1 1 1 1 1 1 dorsal portion of the stomach, not extending to the abdomen.

Ovaries are barely visible without dissection. The cephalic lobes start to 2 2 2 2 2 cover the sides while the abdominal extensions occupy all somites.

Ovaries are clearly visible through 2 2 integument. Ovaries developed and turgid, with cephalic lobes and 3 3 3 3 abdominal extensions occupying the entirely the dorsal portion. The gonads 3 appear granular. Turgid ovaries extending to the whole 4 dorsal area. Lobes and extensions well 3 3 4 4 4 developed. Eggs well visible. Ovaries after spawning are fully 5 extended but loose turgidity becoming / 1 / 5 / 1 flaccid.

22 |

Parapenaeus longirostris

SOBRINO 1998 ARROBAS AND MEDITS MODIFIED BY RIBEIRO PROPOSED (1994) MEDITS (2007) BIANCHINI AND MACROSCOPIC DESCRIPTION SOBRINO, 1998 SIBM, 2005 GARCIA- CASCALHO, STAGES IN ANON. IN ANON. 2007 RAGONESE, 2009 RODRIGUEZ ET 1982, MODIFIED 1998) AL., 2009 BY SILVA, 2009

Ovaries not visible without dissection. The ovaries are thin and translucent with a tubular 1 1 1 1 1 1 1 1 appearance adherent to the dorsal portion of the stomach, not extending to the abdomen. Ovaries are barely visible without dissection. The cephalic lobes 2 start to cover the sides while the 2 2 2a+2b 2 2 2 abdominal extensions occupy all somites. Ovaries are clearly visible through integument. Ovaries developed and turgid, with 3 cephalic lobes and abdominal 3 3 2c 3 extensions occupying the entirely 2 the dorsal portion. The gonads 3 3 appear granular. Turgid ovaries extending to the whole dorsal area. Lobes and 4 4 4 2d 4 extensions well developed. Eggs well visible. Ovaries after spawning are fully 5 extended but loose turgidity / 2e / / 4 becoming flaccid.

ICES WKMSC REPORT 20099 | 23

3 Conclusion and recommendations for new WK

Conclusions On the basis of the knowledge and experiences gained by the research groups attend- ing the WK in the last decades, new maturity scales were proposed during the present meeting and a comparison with the previous existing maturity scales was also detect in order to give a common tool for exchanging data and scientific information also referred to the previous studies. The most important change to the previously adopted scales was related to developing and recovering stages. In fact, according to the experience and knowledge of the involved teams, it is impossible to distinguish at present the developing and recovering specimens from only a macroscopic point of view. Moreover, although not concerning the ovary maturity stages, it is recommended to record always information on the occurrence of both berried females in N. norvegicus and spermatophora in A. antennatus and A. foliacea females. Finally, taking into account the difficulties to detail maturity condition of gonads in males at macroscopic level, no maturity scales for males were presented in this report. However, it is to remark the possibility to distinguish the juveniles from the adult specimens. In N. norvegicus, this distinction is possible when the morphometric relationship between the appendix masculina and carapace length is available.

Recommendations for the new WK

• Collect information on condition of males (Juveniles and Adults). • Collect data on length of the appendix masculina for N. norvegicus and the corresponding carapace length. • Complete eggs measures for P. longirostis. • Perform histological analyses for N. norvegicus. • Compare colour of fresh and stored specimens. • Improve methods for maturity ogive estimation solving the problems of differentiating the recovering from developing in first maturation.

4 References

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Annex 1: List of participants

NAME INSTITUTION/ADDRESS PHONE/FAX E-MAIL

Institute for Coastal Marine Phone : FIORENTINO, Environment – CNR +39 0923948966 Fabio Via L. Vaccara 61 [email protected] Fax : (Chair) 91026 Mazara del Vallo +39 0923906634 (Italy) IBAF - CNR BIANCHINI, Via Salaria, Km 29 Phone : [email protected] Marco 00015 Monterotondo Scalo (Rome) +39 0690672539 (Italy) University of Messina Department of Animal Biology Phone: BONFIGLIO, and Marine Ecology +39 0906765527 [email protected] Rossella Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) Institute for Coastal Marine Phone : Environment – CNR BOTTARI, +39 090711263 Spianata San Raineri 86 [email protected] Teresa Fax : 98122 Messina +39 090669007 (Italy) Institute for Coastal Marine Phone : Environment – CNR BUSALACCHI, +39 090711263 Spianata San Raineri 86 [email protected] Barbara Fax : 98122 Messina +39 090669007 (Italy) University of Messina Department of Animal Biology Phone: CONTI, and Marine Ecology +39 0906765527 [email protected] Fabio Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) University of Messina Department of Animal Biology Phone: COSENTINO, and Marine Ecology +39 0906765540 [email protected] Andrea Salita Sperone 31 Fax: [email protected] 98167 S. Agata (ME) +39 090393409 (Italy) CCMAR - University of Algarve Phone : CRISTO, Campus de Gambelas +35 1289800978 [email protected] Margarida 8005-139 Faro Fax : (Portugal) +35 1289800069 University of Messina Department of Animal Biology Phone: D’AGATA, and Marine Ecology +39 090391435 [email protected] Alessia Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765556 (Italy)

ICES WKMSC REPORT 20099 | 27

NAME INSTITUTION/ADDRESS PHONE/FAX E-MAIL

University of Messina Department of Animal Biology Phone: DE DOMENICO, and Marine Ecology +39 090391435 [email protected] Elena Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765556 (Italy) University of Messina Department of Animal Biology Phone: DE DOMENICO, and Marine Ecology +39 0906765527 [email protected] Emilio Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) University of Messina Department of Animal Biology Phone: DE DOMENICO, and Marine Ecology +39 0906765541 [email protected] Francesca Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) University of Cagliari Department of Animal Biology Phone: FOLLESA, and Ecology +39 0706758014 [email protected] Maria Cristina Viale T. Fiorelli 1 Fax: 09126 Cagliari +39 0706758022 (Italy) COISPA Phone : GAUDIO, Via dei Trulli 18/20 +39 0805433596 [email protected] Palma 70100 Torre a Mare (BA) Fax : (Italy) +39 0805433586 Institute for Coastal Marine Phone : Environment – CNR GIORDANO, +39 090711263 Spianata San Raineri 86 [email protected] Daniela Fax : 98122 Messina +39 090669007 (Italy) Malta Centre for Fisheries Sciences Fort San Lugan GRAVINO, Phone : Marsaxlokk [email protected] Francesca +356 22293326 BBG 1283 (Malta) University of Messina Department of Animal Biology Phone: IACONO, and Marine Ecology +39 090391435 [email protected] Francesco Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765556 (Italy) Oloturia SUB IARIA, Via Consolare Pompea 253-255 Phone : www.oloturiasub.it Gianmichele 98168 Messina +39 090312526 (Italy)

28 | ICES WKMSC REPORT 20099

NAME INSTITUTION/ADDRESS PHONE/FAX E-MAIL

University of Messina Department of Animal Biology Phone: IARRERA, and Marine Ecology +39 0906765541 [email protected] Samuela Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) University of Bari Department of Animal Biology Phone : INDENNIDATE, and Environmental +39 0805443350 [email protected] Antonella Via Orabona 4 Fax : 70100 Bari +39 0805442495 (Italy) Institute of Marine Biological Resources - Hellenic Centre of Phone : KAPIRIS, Marine Research +21 09856712 [email protected] Kostas 47 Km Athens-Sounio Fax : 19013, P.O. 713 Anavissos, Athens +21 09811713 (Greece) University of Messina Department of Animal Biology Phone: LIGUORI, and Marine Ecology +39 0906765527 [email protected] Marilia Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) University of Messina Department of Animal Biology Phone: LO BUE, and Marine Ecology +39 0906765527 [email protected] Andrea Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) Institute for Coastal Marine Phone Environment – CNR MANCUSO, +39 090669003 Spianata San Raineri 86 [email protected] Monique Fax 98122 Messina +39 090669007 (Italy) University of Messina Department of Animal Biology Phone: MANGANO, and Marine Ecology +39 0906765541 [email protected] Maria Cristina Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) Institute for Coastal Marine Phone Environment – CNR MICALE, +39 090669003 Spianata San Raineri 86 [email protected] Valeria Fax 98122 Messina +39 090669007 (Italy)

ICES WKMSC REPORT 20099 | 29

NAME INSTITUTION/ADDRESS PHONE/FAX E-MAIL

University of Messina Department of Animal Biology Phone: NATALOTTO, and Marine Ecology +39 090391435 [email protected] Antonino Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765556 (Italy) Malta Centre for Fisheries Sciences Fort San Lugan PACE, Phone : Marsaxlokk [email protected] Roberta +35 622293315 BBG 1283 (Malta) Institute for Coastal Marine Phone : Environment – CNR PERDICHIZZI , +39 090711263 Spianata San Raineri 86 [email protected] Anna Fax : 98122 Messina +39 090669007 (Italy) Institute for Coastal Marine Phone : Environment – CNR PERDICHIZZI, +39 090711263 Spianata San Raineri 86 [email protected] Francesco Fax : 98122 Messina +39 090669007 (Italy)

Instituto Español de Oceanografia Phone : PEREZ GIL, Puerto Pesquero S/N +34 952197100 [email protected] Josè Luis 29640 Fuertegirola Fax : (Spain) +34 952463808

Institute for Coastal Marine Phone : Environment – CNR PIRRERA, +39 090711263 Spianata San Raineri 86 [email protected] Laura Fax : 98122 Messina +39 090669007 (Italy) University of Cagliari Department of Animal Biology Phone: PODDA, and Ecology +39 0706758014 [email protected] Benedetta Viale T. Fiorelli 1 Fax: 09126 Cagliari +39 0706758022 (Italy) University of Cagliari Department of Animal Biology Phone: PORCU, and Ecology +39 0706758042 [email protected] Cristina Viale T. Fiorelli 1 Fax: 09126 Cagliari +39 0706758022 (Italy) University of Messina Department of Animal Biology Phone: PORPORATO, and Marine Ecology +39 0906765541 [email protected] Erika Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy)

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NAME INSTITUTION/ADDRESS PHONE/FAX E-MAIL

Institute for Coastal Marine Phone : Environment – CNR PROFETA, +39 090711263 Spianata San Raineri 86 [email protected] Adriana Fax : 98122 Messina +39 090669007 (Italy) Institute for Coastal Marine Phone : Environment – CNR RAGONESE, +39 0923948966 Via L. Vaccara 61 [email protected] Sergio Fax : 91026 Mazara del Vallo +39 0923906634 (Italy) Institute for Coastal Marine Phone : Environment – CNR RINELLI, +39 090711263 Spianata San Raineri 86 [email protected] Paola Fax : 98122 Messina +39 090669007 (Italy) University of Messina Department of Animal Biology Phone: RUGGERI, and Marine Ecology +39 0906765541 [email protected] Gioacchino Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) Centro Interuniversitario di Biologia Phone : Marina ed Ecologia Applicata SARTINI, +39 0586260723 Viale N. Sauro 4 [email protected] Marina Fax : 57126 Livorno +39 0536260723 (Italy) Phone : INRB/L-IPIMAR SILVA, +35 1 213027096 Av. Brasilia , 1449-006 Lisbon [email protected] Cristina Fax: (Portugal) +351 213015948 University of Messina Department of Animal Biology Phone: SPANO’, and Marine Ecology +39 0906765541 [email protected] Nunziacarla Salita Sperone 31 Fax: 98167 S. Agata (ME) +39 0906765526 (Italy) Instituto Español de Oceanografia Centro Oceanográfico de Cádiz VILA, Puerto Pesquero, Muelle de Levante Phone: [email protected] Yolanda S/N +34 956294189 11006 Cádiz (Spain)

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Annex 2: Agenda

Monday, 19 October 17:00 – 18:30: participants’ registration, local arrangements, computer and network arrangements 19:00 – 19:30: Screening of the video “Beautiful sensations of the Straits of Messina” – Gianmichele Iaria, Oloturia sub 19:30 – 20:30 Participants’ welcome cocktail at Villa Pace.

Tuesday, 20 October 09:00 Opening of meeting; Introduction, short overview of ToRs, adoption of the Agenda, adoption of template 09:30 Participants presentation of own reference collection of gonads (macroscopic and microscopic) pictures and drawn, maturity scales and estimators employed about Aristeus antennatus 10:30 Coffee break 10:45 Reviewing, comparing and discussing differences/similarities on the existing maturity scales and criteria used for Aristeus antennatus 12.00 Producing maturity stages sampling protocols, adoption and standardisation of the criteria for classifying each maturity stage of Aristeus antennatus to be used by every laboratory for the EU Data Collection Regulation (DCR) 13:30 Lunch break 15:00 Participants presentation of own reference collection of gonads (macroscopic and microscopic) pictures and drawn, maturity scales and estimators employed about Aristaeomorpha foliacea 16:00 Reviewing, comparing and discussing differences/similarities on the existing maturity scales and criteria used for Aristaeomorpha foliacea 17:00 Coffee break 17:15 Producing maturity stages sampling protocols, adoption and standardisation of the criteria for classifying each maturity stage of Aristaeomorpha foliacea to be used by every laboratory for the EU Data Collection Regulation (DCR) 18:30 End of session

Wednesday, 21 October 09:00 Participants presentation of own reference collection of gonads (macroscopic and microscopic) pictures and drawn, maturity scales and estimators employed about Parapenaeus longirostris 10:30 Coffee break 10:45 Reviewing, comparing and discussing differences/similarities on the existing maturity scales and criteria used for Parapenaeus longirostris 12.00 Producing maturity stages sampling protocols, adoption and standardisation of the criteria for classifying each maturity stage of Parapenaeus longirostris to be used by every laboratory for the EU Data Collection Regulation (DCR)

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13:30 Lunch break 15:00 Participants presentation of own reference collection of gonads (macroscopic and microscopic) pictures and drawn, maturity scales and estimators employed about Nephrops norvegicus 16:00 Reviewing, comparing and discussing differences/similarities on the existing maturity scales and criteria used for Nephrops norvegicus 17:00 Coffee break 17:15 Producing maturity stages sampling protocols, adoption and standardisation of the criteria for classifying each maturity stage of Nephrops norvegicus to be used by every laboratory for the EU Data Collection Regulation (DCR) 18:30 End of session 21:00 Social Dinner offered by PhD Course on Environmental Sciences: “Marine Environment and Resources” - University of Messina

Thursday, 22 October 9:00 Harmonising the different sections and proposing a follow-up for problems and uncertainties 10:30 Coffee break 10:45 Elaborating and discussing the final report 13:30 Lunch break 15:00 Elaborating and discussing the final report 17:00 Coffee break 17:15 Elaborating and discussing the final report 18:00 End of session

Friday, 23 October 09:00 Adoption of the report 14:00 End of meeting

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Annex 3: WGMSC terms of reference for the next meeting

The terms of reference for the next Working Group on crustaceans (Aristeus antennatus, Aristaeomorpha foliacea, Parapenaeus longirostris, Nephrops norvegicus) maturity stages (WKMCS) are:

a ) Complete histological analyses for P. longirostis b ) Perform histological analyses for N. norvegicus c ) Compare colour of fresh and stored specimens for the target species d ) Collect information on sexual condition of males (Juveniles and Adults). e ) Improve methods for maturity ogive estimation solving the problems of differentiating the recovering from developing in first maturation

Annex 4: Recommendations

The WKMSC advices to scientists involved in researches carried out within the framework of the EU Data Collection Regulation, covering both the ICES Divisions and the GFCM Geographical Subareas the following recommendations:

RECOMMENDATION

1 Collect more information and develop methods to distinguish developing from recovering specimens

2. Collect always information on occurrence of berried females in N. norvegicus and of spermatophora in A. antennatus and A. foliacea females .

3. Distinguish males of deep water pink shrimp, giant red shrimp and violet shrimp in juveniles and adult on the basis of petasma status.

4. Separate males of Norway lobsters in juvenile and adult on the basis of morphometric relationships between the appendix masculina and carapace length if available.

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Annex 5: Crustaceans gonad reference photos

Reference macroscopic photos of the ovarian maturity stages proposed for Aristeus antennatus

Stage 1 – Immature

(a) (b) (c)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), IPIMAR (Portugal) (b) and University of Cagliari (Italy) (c).

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Stage 2 – Developing/recovering

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c)

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(d) (e) (f)

Photos produced by IAMC – CNR UOS OF Messina (Italy) (e), IPIMAR (Portugal) (f) and University of Cagliari (Italy) (a - d).

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Stage 3 – Maturing

Photo produced by C.I.B.M (Italy).

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c)

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(d) (e) (f)

Photos produced by C.I.B.M. (Italy) (d), IAMC – CNR UOS of Messina (Italy) (c), IEO (Spain) (f), IPIMAR, (Portugal) (e) and University of Cagliari (Italy) (a, b).

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Stage 4 – Mature

Photo produced by C.I.B.M (Italy).

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c)

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(d) (e)

Photos produced by IAMC – CNR UOS of Messina (Italy) (c), IEO (Spain) (a), IPI- MAR (Portugal) (b) and University of Cagliari (Italy) (d, e).

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Stage 5 – Spent

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c) (d)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), IPIMAR (Portugal) (d) and University of Cagliari (Italy) (b, c).

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Reference microscopic photos of the ovarian maturity stages proposed for Aristeus antennatus

Stage 1 – Immature

(a)

Photos produced by University of Cagliari (Italy) (a).

Stage 2 – Developing/recovering

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IEO (Spain) (b).

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Stage 3 – Maturing

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IEO (Spain) (b).

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Stage 4 – Mature

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IEO (Spain) (b).

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Stage 5 – Spent

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IEO (Spain) (b).

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Reference macroscopic photos of the ovarian maturity stages proposed for Aristaeomorpha foliacea

Stage 1 – Immature

Photo produced by MCFS (Malta)

(a) (b)

Photos produced by University of Cagliari (Italy) (a) and IAMC – CNR UOS of Messina (Italy) (b).

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Stage 2 – Developing/recovering

Photo produced by IAMC – CNR UOS of Messina (Italy)

Photo produced by MCFS (Malta)

(a) (b) (c) (d) (e)

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(f) (g) (h) (i)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a, e, i), IPIMAR (Portugal) (b, g) and University of Cagliari (Italy) (c, d, f, h).

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Stage 3 – Maturing

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c) (d)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), IPIMAR (Portugal) (b) and University of Cagliari (Italy) (c, d).

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Stage 4 – Mature

Photos produced by IAMC – CNR UOS of Messina (Italy)

Photos produced by MCFS (Malta)

(a) (b) (c) (d)

Photos produced by IAMC – CNR UOS of Messina (Italy) (d), IPIMAR (Portugal) (c) and University of Cagliari (Italy) (a, b).

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Stage 5 – Spent

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c) (d)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), University of Cagliari (Italy) (b, c) and IPIMAR (d).

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Reference microscopic photos of the ovarian maturity stages proposed for Aristaeomorpha foliacea

Stage 1 – Immature

(a)

(b)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), and University of Cagliari (Italy) (b).

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Stage 2 – Developing/recovering

(a)

(b)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), and University of Cagliari (Italy) (b).

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Stage 3 – Maturing

(a)

(b)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), and University of Cagliari (Italy) (b).

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Stage 4 – Mature

(a)

(b)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), and University of Cagliari (Italy) (b).

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Stage 5 – Spent

Photos produced by University of Cagliari (Italy).

Reference macroscopic photos of the ovarian maturity stages proposed for Parapenaeus longirostris

Stage 1 – Immature

Photo produced by IEO – Malaga (Spain).

Photo produced by IAMC – CNR UOS of Messina (Italy).

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(a) (b) (c)

Photos produced by University of Cagliari (Italy) (a), IPIMAR (Portugal) (b) and IAMC – CNR UOS of Messina (Italy) (c).

Stage 2 – Developing/recovering

Photo produced by University of Cagliari (Italy).

Photo produced by IAMC – CNR UOS of Messina (Italy).

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(a) (b) (c) (d)

(e) (f)

Photos produced by University of Cagliari (Italy) (a, b), CIBM of Livorno (Italy) (c), IPIMAR (Portugal) (d) and IAMC – CNR UOS of Messina (Italy) (e, f).

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Stage 3 – Maturing

Photo produced by CIBM of Livorno (Italy)

Photo produced by IEO – Malaga (Spain).

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c) (d)

Photos produced by IAMC – CNR UOS of Messina (Italy) (a), IPIMAR (Portugal) (b), University of Cagliari (Italy) (c) and CIBM of Livorno (Italy) (d).

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Stage 4 – Mature

Photo produced by CIBM of Livorno (Italy)

Photo produced by IEO – Malaga (Spain)

Photo produced by IAMC – CNR UOS of Messina (Italy).

(a) (b) (c)

Photos produced by CIBM of Livorno (Italy) (a) and University of Cagliari (Italy) (b, c)

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(d) (e)

Photos produced by IPIMAR (Portugal) (d) and IAMC – CNR UOS of Messina (Italy) (e)

Stage 5 – Spent

Photo produced by University of Cagliari (Italy).

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Reference microscopic photos of the ovarian maturity stages proposed for Parapenaeus longirostris

Stage 1 – Immature

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IAMC – CNR UOS of Mazara del Vallo (Italy) (b).

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Stage 2 – Developing/recovering

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IAMC – CNR UOS of Mazara del Vallo (Italy) (b).

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Stage 3 – Maturing

(b)

Photos produced by University of Cagliari (Italy) (a) and by IAMC – CNR UOS of Mazara del Vallo (Italy) (b).

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Stage 4 – Mature

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IAMC – CNR UOS of Mazara del Vallo (Italy) (b).

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Stage 5 – Spent

(a)

(b)

Photos produced by University of Cagliari (Italy) (a) and by IAMC – CNR UOS of Mazara del Vallo (Italy) (b).

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Reference macroscopic photos of the ovarian maturity stages proposed for Nephrops norvegicus

Stage 1 - Immature No pictures available

Stage 2 - Developing or Recovering

Photo produced by MCFS (Malta)

(a) (b) (c)

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(d) (e)

Photos produced by C.I.B.M. (Italy) (a), IEO (Spain) (b), IPIMAR (Portugal) (c) and MSS (Scotland) (d and e).

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Stage 3 – Maturing

(a) (b) (c)

(d) (e)

Photos produced by IEO (Spain) (a and b), IPIMAR (Portugal) (c) and MSS (Scot- land)(d and e).

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Stage 4: Mature

Photo produced by C.I.B.M. (Italy).

(a) (b) (c)

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(d) (e)

Photos produced by MCFS (Malta)(a), IPIMAR (Portugal) (b), IEO (Spain)(c), MSS (Scotland)(d) and C.I.B.M. (Italy) (e).

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Stage 5: Spent No picture of the gonad in this stage was available.

Observations:

Ovigerous female

Photo produced by IEO (Spain).

Re-absorption

(a) (b) (c)

Photos produced by MSS (Scotland)(a and b) and C.I.B.M. (Italy) (c). Note for all images made available by MSS: © Crown copyright. Reproduced with permission of Marine Scotland.

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Annex 6: The ICES (International Council for the Exploration of the Seas)

ICES management divisions in the Northeast Atlantic.

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Smaller divisions, the Functional Units (FU), are used for the assessment and management of Nephrops stocks.

48 VIIIa

47 23

46 VIIIe VIIId 24

45 VIIIb VIIIc 44 25 31

43 N ) º ( 26 d e 42 t u t i L a 41 27

40 IXb

38 28

37 29 30 IXa 36 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 Longitude (º W)

The ICES Division IXa comprises 5 FUs: West Galicia (FU 26), North Portugal (FU 27), Southwest Portugal or Alentejo(FU 28, Alentejo), South Portugal or Algarve (FU 29) and Cadiz (FU 30). This report covers mainly the FUs 28, 29 and 30.

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Annex 7: The GFCM (General Fisheries Commission for the Mediterra- nean) Geographical Subareas (GSA)

Establishment of Geographical Sub-Areas in the GFCM area amending the Resolution GFCM/31/2007/2.

GFCM Geographical Sub-Areas (GSAs) map