Vladimir Laptikhovsky
email: vladimir.laptikhovsky@cefas.co.uk

PhD Gavan Cooke
email: gavan.cooke@anglia.ac.uk

PhD Christopher Barrett

PhD Sophie Lozach

Eleanor Macleod

PhD Daniel Oesterwind
email: daniel.oesterwind@thuenen.de

Edel Sheerin

Michael Petroni

Leigh Barnwall

PhD Jean-Paul Robin

PhD Louise Allcock

PhD Anne Marie Power
email: annemarie.power@nuigalway.ie

PhD Ángel F González

PhD Erica Vidal
email: ericavidal2000@yahoo.com.br

PhD Graham J Pierce
email: g.j.pierce@iim.csic.es

L Forbesii

DSc Angel Guerra
email: angelguerra@iim.csic.es

Identification of benthic egg masses and spawning grounds in commercial squid in the English Channel and Celtic Sea: Loligo

Keywords: Loligo vulgaris, Loligo forbesii, reproduction, egg masses, spawning grounds Loligo vulgaris, Loligo forbesii, reprodiction, egg masses, spawning grounds

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Introduction

Two commercial squid species genus Loligo, L. vulgaris (European squid) and L. forbesii (veined squid), with nearly coinciding species ranges inhabit waters around Europe from the east Mediterranean to the North Sea [START_REF] Jereb | Cephalopod biology and fisheries in Europe: II. Species Accounts[END_REF]. Both species potentially reproduce all year round but with distinctive peaks, mostly in the cold season, and mature females of both species are often captured together in the same hauls. In certain parts of their range (Celtic Sea/English Channel/North Sea), there is possible overlap of spawning grounds, however information to permit unambiguous identification of egg masses in these areas is absent. Spawning is extended in both species, but seasonal migrations are little studied. It is assumed that there is an important temporal and spatial overlap in occurrence of egg masses of both species in these European waters [START_REF] Martins | Biology of Pre-and Post-Hatching Stages of Loligo vulgaris Lamarck, 1798 and Loligo forbesi Steenstrup[END_REF], particularly in the Celtic Sea and English Channel, which are the most important areas for loliginid fisheries in Europe, accounting for about one third of annual landings in the Northeast Atlantic (Royer, 2002). L. forbesii and L. vulgaris are both annual species and spawn only once in their life and deposit their eggs on various substrates in relatively shallow waters where bottom fisheries (e.g. dredges and beam trawls) can also occur. As eggs might be destroyed by bottom fisheries, more complete knowledge of spatial and seasonal distribution of loliginid spawning grounds is needed for their protection and management to support a successful reproduction.

Loligo vulgaris reproduces in the English Channel between November and April, peaking in February-March [START_REF] Moreno | Biological variation of Loligo vulgaris (Cephalopoda: Loliginidae) in the eastern Atlantic and Mediterranean[END_REF] whereas L. forbesii lays eggs in this area in December-January and may continue well into the spring, with some mature animals found also in summer [START_REF] Holme | The biology of Loligo forbesi Steenstrup (Mollusca: Cephalopoda) in the Plymouth area[END_REF][START_REF] Jereb | Cephalopod biology and fisheries in Europe: II. Species Accounts[END_REF]. In the southern North Sea, L. vulgaris was found to lay eggs later in the year, from April to August with some mature animals being captured in winter (Tinbergen and Vervey, 1945;[START_REF] Oesterwind | Biology and meso-scale distribution patterns of North Sea cephalopods[END_REF]. Loligo forbesii reproduces around Scotland from December to June, peaking in March, but some mature specimens can be found there throughout the year, and egg masses are reported by fishermen up to September [START_REF] Lum-Kong | Timing of spawning and recruitment in Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Scottish waters[END_REF][START_REF] Pierce | The life history of Loligo forbesi (Cephalopoda: Loliginidae) in Scottish waters[END_REF][START_REF] Oesterwind | Biology and meso-scale distribution patterns of North Sea cephalopods[END_REF]. It seems that more recently the summer breeding population of L. forbesii has declined and that the winter breeding population is dominant in Scottish waters [START_REF] Pierce | Interannual variation in life-cycle characteristics of the veined squid (Loligo forbesi) in Scottish (UK) waters[END_REF]. In Irish waters spawning occurs mostly between November and April and developing egg masses were found in the north Celtic Sea in every month but August [START_REF] Collins | Recruitment, maturation and spawning of Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Irish waters[END_REF].

Loligo vulgaris lays eggs mostly at 20 -70 m, and occasionally down to depths of >100 m [START_REF] Moreno | Identification of cephalopod paralarvae from the NE Atlantic[END_REF][START_REF] Jereb | Cephalopod biology and fisheries in Europe: II. Species Accounts[END_REF]. Loligo forbesii spawns slightly further offshore -at 10-150 m [START_REF] Jereb | Cephalopod biology and fisheries in Europe: II. Species Accounts[END_REF], with gradually increasing depth during the winter reproductive season from inshore waters to ~ 140 m [START_REF] Smith | Habitat and distribution of post-recruit life stages of the squid Loligo forbesii[END_REF]. In fjord-like areas with extremely steep slopes this species may occasionally lay eggs as deep as deep as at >700 m (Salman and [START_REF] Laptikhovsky | First occurrence of egg masses of Loligo forbesi (Cephalopoda: Loliginidae) in deep waters of Aegean Sea[END_REF].

Despite this extensive knowledge, much of what we know about seasonal timings is historical and needs to be re-evaluated in the light of climate change and the known plasticity of squid life-history traits in response to environmental drivers [START_REF] Doubleday | Global proliferation of cephalopods[END_REF].

Egg masses consist of numerous finger-like capsules ("strings") that females attach to the ground one by one (Fig. 1). If egg capsules are already present on the spawning site, loliginid females tend to attach their spawn to already existing masses. Thus, egg capsules of the same egg mass might be at different stages of embryonic development because they were laid by different females at different times [START_REF] Drew | Sexual activities of the squid Loligo pealei (Les.). I. Copulation, egg laying and fertilization[END_REF][START_REF] Arkhipkin | Adaptations for the cold water spawning in squid of the family Loliginidae: Loligo gahi around the Falkland Islands[END_REF]. Because of this, some single large egg masses of L. vulgaris could contain as many as 39,760 eggs (568 capsules, mean egg count of 70) in the Mediterranean [START_REF] Bohadsch | De quibusdam anomalous marinis eorumque propietatibus[END_REF] and ~42,000 in the English Channel [START_REF] Lee | The octopus or the "devil-fish" of fiction and of facts[END_REF].

Throughout its distribution range L. vulgaris lays from 50 to 160 eggs in capsules of 60-170 mm [START_REF] Grimpe | Zur Kenntnis der Cephalopodenfauna der Nordsee[END_REF][START_REF] Mangold-Wirz | Biologie des céphalopodes bentiques et nectoniques de la Mer Catalane[END_REF][START_REF] Sen | A preliminary study on the effects of different light intensities on hatching of European squid (Loligo vulgaris Lamarck, 1798) eggs[END_REF], although in Portugal a 140 mm egg capsule was reported to contain ~ 174 eggs [START_REF] Moreno | Dissertação apresentada para acesso à categoria de Investigador Auxiliar. Unidade de Recursos Marinhos e Sustentabilidade[END_REF]. The respective values for L. forbesii are 36-100 eggs and 80-200 mm [START_REF] Grimpe | Zur Kenntnis der Cephalopodenfauna der Nordsee[END_REF][START_REF] Segawa | Illustrated embryonic stages of the eastern Atlantic squid Loligo forbesi[END_REF][START_REF] Hanlon | Laboratory culture and estimated life span of eastern Atlantic squid, Loligo forbesi Steenstrup, 1856 (Mollusca: Cephalopoda)[END_REF][START_REF] Porteiro | First finding of natural laid eggs from Loligo forbesi Steenstrup, 1856 (Mollusca: Cephalopoda) in the Azores[END_REF][START_REF] Orsi-Relini | First record of an egg mass of Loligo forbesi (Cephalopoda: Loliginidae) in the Ligurian Sea, with notes about egg laying[END_REF][START_REF] Pham | First description of spawning in a deep-water loliginid squid, Loligo forbesi (Cephalopoda: Myopsida)[END_REF]. Therefore, the evidence suggests that individual variation in egg numbers and capsule length is very high and is probably not useful for distinguishing species.

Loligo forbesii produce much larger eggs than L. vulgaris [START_REF] Grimpe | Zur Kenntnis der Cephalopodenfauna der Nordsee[END_REF], but during embryonic development the egg diameter, along with individual capsule length and width, increases several times due to increasing egg volume [START_REF] Boletzky | On egg and capsule dimensions in Loligo forbesi (Mollusca: Cephalopoda): a note[END_REF][START_REF] Martins | Biology of Pre-and Post-Hatching Stages of Loligo vulgaris Lamarck, 1798 and Loligo forbesi Steenstrup[END_REF][START_REF] Moreno | Dissertação apresentada para acesso à categoria de Investigador Auxiliar. Unidade de Recursos Marinhos e Sustentabilidade[END_REF]. Therefore, although several descriptions were published during the last century, there is still no unambiguous criterion to distinguish between spawn from L. vulgaris and L. forbesii.

This paper provides unambiguous criteria to distinguish between eggs of both species based on i) observation of egg masses of known species origin and ii) observation of genetically identified egg masses and iii) distribution and timing of mature females. The results will support the identification and mapping of Loligo spawning grounds in the NE Atlantic (Irish Sea, Celtic Sea, English Channel and North Sea). Results are provided in conjunction with the Cephalopod Citizen Science project (https://www.researchgate.net/project/The-Cephalopod-Citizen-Science-Project), which was launched in December 2017 to gather information on wild cephalopods via observations by the recreational diving community. By enabling identification of egg masses to species level, we aim to support efforts to map the spatio-temporal variability of spawning grounds of both species and help to mitigate the impact of fisheries on squid spawning grounds to support a sustainable use of this fishery resource.

Materials and Methods

In total, 21 squid egg masses were collected and frozen during beam trawl surveys by RV Cefas Endeavour in March -April 2017-2019 in the English Channel and Celtic Sea (Fig. 2). Visual observations of 58 Loligo egg masses from this area were extracted from the web page of UK Cephalopod Reports (https://www.facebook.com/groups/1772714999700580/), which is a part of the Cephalopod Citizen Science Project. Another 26 egg masses were reported by French divers for Base pour l'inventaire des observations subaquatiques (https://bioobs.fr/les-especes/especes-recherchees/).

Collected egg masses were defrosted in the lab and studied under a binocular microscope. Three random egg capsules were measured with 1 mm accuracy, the numbers of eggs were counted, egg length and embryo length were measured along the major axis to 0.1 mm accuracy, and the stage of embryonic development assigned following [START_REF] Naef | Cephalopoda Embryology. Fauna and Flora of the Gulf of Naples. Translated from German[END_REF]. These data were combined with available data from the literature on egg size, size at embryonic stage, egg capsule length, and numbers of eggs per capsule in both species, for inclusion in the analysis.

Parts from seven egg capsules belonging to seven different egg masses collected in 2019 were transferred to 96% ethanol and stored for DNA sequencing. DNA was extracted using the Invitrogen™ PureLink™ Genomic DNA Mini Kit, following the Mammalian Tissue and Mouse/Rat Tail Lysate protocol. DNA was eluted in 100 μL Genomic Elution Buffer and stored at -20 °C.

DNA barcoding, which targets the COI gene, was performed using forward primer LCO1490: 5'ggtcaacaaatcataaagatattgg-3' and reverse primer HC02198: 5'-taaacttcagggtgaccaaaaaatca-3' [START_REF] Folmer | DNA primers for amplification of mitochondrial cyto-chrome c oxidase subunit from diverse metazoan invertebrates[END_REF]. Each PCR contained 12.5 µL of Thermo Scientific™ DreamTaq Green PCR Master Mix (2X), 0.5 µM of each primer, 2.5 µL of DNA and 9 µL H20 resulting in final reaction volume of 25 µL. A negative control was also included to ensure cross-contamination did not occur. The PCR conditions were 94°C for 2 min, 35 cycles of 94°C for 40s, 50°C for 40s and 72°C for 90s, followed by 72°C for 10 mins [START_REF] Allcock | A new species of Pareledone (Cephalopoda: Octopodidae) from Antarctic Peninsula Waters[END_REF]. PCR products were visualised on a 1% (w/v) agarose gel and bands of 650 bp were obtained. PCR products were cleaned using Invitrogen™ PureLink™ PCR Purification Kit according to the manufacturer's instructions. Purified PCR products were then standardized to 12 ng/µL in accordance with the DNA sequencing facility specifications. Samples were prepared for sequencing by adding 5 µL of each purified PCR product to 5 µM forward primer LCO1490 resulting in a 10 µL reaction volume. Samples were sent to Eurofins Genomics (Germany) for DNA Sequencing on an ABI 3730XL DNA Analyzer.

All sequences obtained were input into MEGA Software (Molecular Evolutionary Genetics Analysis) [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF] trimmed to 479 bp and aligned using the MUSCLE algorithm. New sequences were submitted to Genbank with Accession Numbers MW264497-MW264503. Additional loliginid sequences for the COI gene were obtained from Genbank [L. vulgaris, KM517926-KM517928; L. forbesii, KM517907, KM517911, KM517913 (Gebhardt & Knebelsberger, 2015); Alloteuthis subulata EU668098-EU668100; A. media EU668077, EU668083, EU668097 (Anderson et al., 2008)] and were included in the analysis. All sequences were analysed together by running a statistical parsimony network using TCS software [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF].

Presence and absence of mature adult females were combined from the following surveys: Irish Groundfish survey (IGFS) 2018, Irish Anglerfish and Megrim Survey (IAMS) 2019 and 2020, the South Western Beam Trawl Survey, Quarter 1 (Q1SWBEAM) in 2017 -2019. Fishing activity was only performed during daylight for IGFS and Q1SWBEAM, whereas on IAMS fishing was conducted all day around (24 hours). Sampling gear on IGFS was the IBTS standard 'Grande Overture Verticale' demersal trawl (GOV 36/47) with mesh sizes ranging from 100 mm at the trawl opening to a 20 mm liner in a 25 mm codend. Trawl tows were standardised to roughly 30 mins at 4 knots, as far as possible. IAMS used a standard commercial-derived Jackson trawl with mesh size that varied from 200 mm in the wings gradually reducing to 100 mm in the codend and trawl tows were standardised to roughly 1 hour at 4 knots, when possible. The Q1SWBEAM was carried out by commerciallyrigged 4 m steel beam trawl with extended codend supplied with 40 mm mesh size liner.

Females were considered mature if they had reached at least stage 3a of the ICES WGCEPH scale (ICES, 2010) which corresponds to ovary containing a high proportion of large turgid amber-coloured oocytes (>=2mm), with plenty of oocytes in the oviducts. Females with maturity stage 3b were also included. These stages correspond to stages 4 and 5 on [START_REF] Lipinski | Universal maturity scale for the commercially important squids (Cephalopoda: Teuthoidea). The results of maturity classification of Illex illecebrosus (LeSueur 1821) population for years 1973-1977[END_REF] maturity scale for matured and spent respectively. The data were organised by month of observation i.e. November, December, February, March and April (no surveys carried out in this area during the month of January). Data collection spanned four years, 2017-2020.

Corresponding Oceanographic data (March-April 2017-2019) were downloaded from ICES Dataset collections (http://www.ices.dk/data/dataset-collections/Pages/default.aspx).

Results

Identification of egg masses collected in the West English Channel and Celtic Sea

The sampled egg masses (Fig. 2) had a capsule length which varied from 51 to 134 mm, and the number of eggs per capsule ranged from 40 to 138, which fits well into the range of values known for L. vulgaris in the Mediterranean Sea (Fig 3). Precise identification of the species based on these two characters would be doubtful, as egg size increases over development and this was not considered. Particularly, there was an important overlap between egg capsules containing ~80-110 eggs which had a similar length (115-140 mm) at early stages of development in L. forbesii and at stages close to hatching in L. vulgaris (Fig. 3).

Increase in egg and embryo size during development

Analysis of available data from the literature combined with our observations on genetically identified egg masses allowed reconstruction of curves of increase of both egg size and embryo size during development in both squid species (Fig. 4). Unfortunately, due to some damaged embryos because of freezing we were not able to assign embryonic stages in some egg masses the development of which was somewhere between stage VIII to XII. Dimensions of these eggs are shown on the Fig. 4 as stage 10.

At all stages of embryonic development, eggs of L. forbesii were much larger and contained larger embryos that those of L. vulgaris. Egg size at embryonic stage in all egg masses sampled in the present study fit the curve derived for L. vulgaris.

Finally, a statistical parsimony network analysis resulted in all of the seven egg samples falling out in one network, together with Loligo vulgaris sequences obtained from GenBank [see above]. Genbank sequences of L. forbesii, A. subulata, and A. media formed three separate networks, one per species, so all seven egg samples were considered to be L. vulgaris. Of the egg masses that were not genetically identified, egg and embryo size at the observed developmental stage fit well into information existing for the L. vulgaris of the West Mediterranean in all sampled capsules (Fig. 3).

Distribution of the Loligo squid egg masses in the Irish and Celtic Sea and the English Channel

Loliginid egg masses were reported across the entire Northeast Atlantic shores (Fig. 2) with the greatest number of observations around the English Channel. Despite intensive survey activity by Cefas in the Irish Sea by bottom trawls, no egg masses were collected there. Very few were found or seen in the central North Sea, and species identification of these masses remains uncertain.

All identified egg masses of L. vulgaris were collected during research surveys and were sampled at depths ranged from 12 to 115 m (mean depth of 66 m). Number of capsules varied from 8 to 64 (mean 37.7 mm). Capsule length varied from 51 to 134 (mean 85.1) mm, number of eggs -from 51 to 138 mm (mean 94.2). Distribution of egg masses coincided with bottom temperature range of 8.5-10°C and bottom salinities of 35-35.5 psu (Fig. 5,6).

Distribution of mature females

With one exception, mature females in the northern part of the study area in November/December were identified as L. forbesii. These were restricted spatially to a few stations located offshore to the west (November) and south (December) of Ireland, whereas the southern part of the Celtic Sea and English Channel remained unsampled. No observations were available in January. By February the mature females fished around Cornish peninsula were dominated by L. vulgaris and in March, mature L. forbesii were mostly northwest of Ireland, where their occurrence was very high later in April. The absence of mature females of both species in April (Fig. 6) shows that spawning in the Celtic Sea and west English Channel is generally over at this time

Discussion

The results confirm the assumption that eggs and embryos of L. vulgaris are smaller than those of L. forbesii. While this has been suggested for individual developmental stages in the past, the present study adds a size comparison that is stage-specific, to allow eggs/embryos of the two species to be identified once the stage of development is known. Mature eggs in the oviduct of L. vulgaris from warm waters off Morocco and the Mediterranean are of 2.0-2.2 mm along their major axis [START_REF] Mangold-Wirz | Biologie des céphalopodes bentiques et nectoniques de la Mer Catalane[END_REF][START_REF] Laptikhovsky | Fecundity of the squid Loligo vulgaris Lamarck, 1798 (Myopsida, Loliginidae) off Northwest Africa[END_REF], and 1.6-2.7 mm off Portugal [START_REF] Coelho | Population structure, maturation pattern and fecundity of the squid Loligo vulgaris from southern Portugal[END_REF]. Recently laid eggs are just marginally larger: 2.3-2.8 mm along their major axis [START_REF] Mangold-Wirz | Biologie des céphalopodes bentiques et nectoniques de la Mer Catalane[END_REF][START_REF] Sen | A preliminary study on the effects of different light intensities on hatching of European squid (Loligo vulgaris Lamarck, 1798) eggs[END_REF]. [START_REF] Grimpe | Zur Kenntnis der Cephalopodenfauna der Nordsee[END_REF] described freshly deposited eggs as being 3.5 mm in length and 3.1 mm in width, which is consistent with our observations from the English Channel (2.7-3.6 mm along the major axis). It may indicate that eggs in the northern periphery of the species range are larger as in many boreal fish and squid including the Channel population of common cuttlefish [START_REF] Laptikhovsky | Latitudinal and bathymetric trends in egg size variation: a new look at Thorson's and Rass's rules[END_REF]Laptikhovsky et al., 2020). By the end of embryonic development, egg size in L. vulgaris increases to some 5-7 mm [START_REF] Jecklin | Beitrag zur Kenntnis der Laichgallerten und der Biologie der Embryonen decapoder Cephalopoden[END_REF][START_REF] Boletzky | On egg and capsule dimensions in Loligo forbesi (Mollusca: Cephalopoda): a note[END_REF]this study).

The size of unlaid eggs in L. forbesii is 2.6-3.2 mm from Bay of Biscay to Irish waters [START_REF] Guerra | The life history of Loligo vulgaris and Loligo forbesi (Cephalopoda: Loliginidae) in Galician waters (NW Spain)[END_REF][START_REF] Collins | Recruitment, maturation and spawning of Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Irish waters[END_REF], becoming 3.0-3.1 mm when just laid, quickly increasing to ~4 mm at the stage when the blastoderm covers 2/3 of yolk, and attaining 8-12 mm at hatching [START_REF] Boletzky | On egg and capsule dimensions in Loligo forbesi (Mollusca: Cephalopoda): a note[END_REF][START_REF] Segawa | Illustrated embryonic stages of the eastern Atlantic squid Loligo forbesi[END_REF][START_REF] Moreno | Dissertação apresentada para acesso à categoria de Investigador Auxiliar. Unidade de Recursos Marinhos e Sustentabilidade[END_REF]. Therefore neither egg size on its own, nor a combination of egg cluster length with number of eggs, provides a definite tool for egg mass identification, which is only possible after embryonic stage has been determined and egg length (or embryo length) measured along their major axis. The Fig. 4 of this paper might be used as a solution of this problem, which is necessary for mapping of the species-specific spawning grounds, and their temporal and spatial variation. Without exact knowledge of which species reproduces where and when is impossible to understand its foraging and reproductive migrations and to introduce necessary measures for fisheries management if such measures would be deemed necessary.

Measurement and counts of egg clusters collected in this study and their unambiguous genetic identification as belonging to L. vulgaris confirm previous doubts of [START_REF] Martins | Biology of Pre-and Post-Hatching Stages of Loligo vulgaris Lamarck, 1798 and Loligo forbesi Steenstrup[END_REF] who found a description of egg masses of "L. forbesii" collected off Plymouth [START_REF] Holme | The biology of Loligo forbesi Steenstrup (Mollusca: Cephalopoda) in the Plymouth area[END_REF] more similar to L. vulgaris from Portugal than to L. forbesii from Scotland, and therefore suspected mis-identification. Such a mistake is explainable as both species occur off Plymouth from October to May with L. forbesii being more abundant with some females maturing or mature, and egg masses were collected in November -August [START_REF] Holme | The biology of Loligo forbesi Steenstrup (Mollusca: Cephalopoda) in the Plymouth area[END_REF]. Therefore, these egg masses potentially could belong to either species. Historically this area might be a common spawning ground for both species, though nowadays only both mature females and egg/embryo masses of L. vulgaris were observed there in spring (Fig. 2,5,6). Thus, the spawning of L. vulgaris in the western English Channel seems to take place in late winter and to be completed by April as no mature females were seen by then (Fig 6). No indication of spawning by L. forbesii was found there recently: neither egg masses nor mature females apart from a single specimen off Brittany. This could be attributed to two alternatives.

The first is that L. forbesii spawning takes place in the eastern Celtic Sea/English Channel outside of our survey months (i.e. in summer/autumn), which is highly unlikely as no major reproduction occurs in these months [START_REF] Collins | Recruitment, maturation and spawning of Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Irish waters[END_REF]. The second, more likely, alternative is that this species nowadays spawns outside the study area, although it might have used this area half a century ago, together with L.vulgaris.

Loligo forbesii was initially thought to spawn only inshore [START_REF] Lum-Kong | Timing of spawning and recruitment in Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Scottish waters[END_REF][START_REF] Collins | Recruitment, maturation and spawning of Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Irish waters[END_REF]. Spawning of Loligo-type eggs, presumed to be L. forbesii, was subsequently shown offshore by [START_REF] Lordan | The first evidence of offshore spawning in the squid species Loligo forbesi[END_REF] in the western Celtic Sea, in the west of Ireland and north Biscay (all were some distance offshore). These eggs were identified as L. forbesii on the basis that eggs were coincident in the trawl with mature males and females of this species, or, on the basis of identification to species of well-developed embryos (stage 28+) after [START_REF] Segawa | Illustrated embryonic stages of the eastern Atlantic squid Loligo forbesi[END_REF]. The eggs were obtained in water depths of 135-507 m, which is at the deeper end of the range of previous reports. They were captured in March and April west of Ireland and west of Brittany, which is consistent with our observations on occurrence of mature females. The authors point out that egg masses may have been dragged along for some distance in trawls, but such a distance might not be significant as these masses do not show unavoidable damage in a bottom trawl. Thus L. forbesii probably spawns to the west of our study area. In addition to offshore spawning, egg masses have also been reported inshore in the same area, on static fishing gear off the coast of county Cork (10 -50 m depth) in the western Celtic Sea. These identifications seem assured as egg masses were cultured, hatched and identified as L. forbesii on the basis of [START_REF] Segawa | Illustrated embryonic stages of the eastern Atlantic squid Loligo forbesi[END_REF]) (reported in Collins et al. 1995). Most were seen between Nov-April with a dip in January. "L. forbesii" egg masses have also been reported by fishermen from the west coast of Ireland (county Kerry) in September and October, which was suggested to be the site of spawning for the Rockall population. To the north of the range, e.g. in Scotland, identification of Loligo egg masses is unambiguous because only L. forbesii is present there. Here, spawning females and eggs masses (inshore, attached to creels, but also in deeper waters) were present from December to June, but also in August and September [START_REF] Lum-Kong | Timing of spawning and recruitment in Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Scottish waters[END_REF]. Overall, the spawning area of L. forbesii spans northern waters (Scottish coast, northern North Sea) and almost certainly further offshore in the Celtic Sea [START_REF] Lordan | The first evidence of offshore spawning in the squid species Loligo forbesi[END_REF] and western Celtic Sea [START_REF] Collins | Recruitment, maturation and spawning of Loligo forbesi Steenstrup (Cephalopoda: Loliginidae) in Irish waters[END_REF]. The precise extent and boundary of L. forbesii spawning grounds in the western Celtic Sea requires more work, ideally using genetic barcoding, and should also investigate possible annual variability. Meanwhile, although L. vulgaris is rare in the bottom trawl surveys in Quarter 1 in the northern North Sea, some mature females are present occasionally [START_REF] Oesterwind | Biology and meso-scale distribution patterns of North Sea cephalopods[END_REF] so the possibility of spawning activity there cannot be entirely ruled-out, particularly taking into account recent climate change. This is supported by the historical observation on summer spawning grounds of L. vulgaris off Netherlands [START_REF] Tinbergen | List of figures Fig. 1 A large egg mass of Loligo vulgaris probably laid by several females, Portland, English Channel[END_REF]. Hence, like the western Celtic Sea, this is an area where further research identifying egg masses to species level is necessary to get a complete understanding of the life cycle, including possible climate-related shifts.

In conclusion, the importance of the eastern Celtic Sea / English Channel as a spawning ground for L. vulgaris, particularly in spring months, is shown. Existing data suggest that this area in spring is used by L. vulgaris as spawning grounds, while we could not provide any spawning evidence for L. forbesii in our study area during this season. Given a lack of egg masses, reproduction of either species in the Irish Sea is probably not very intensive. Further work is needed to discover the boundaries of important spawning grounds of L. forbesii off the Irish west coast, in the western and southern Celtic Sea. Fig. 2. Occurrence of egg masses of Loligo spp. Samples used for genetic analysis are marked by cross.

Fig. 3 Existing data on relationship between egg capsule length and the number of eggs with respect to developmental stage. Egg masses shortly after fertilisation are shown by small symbols, and close to hatchingby large symbols. Egg clusters of L. vulgaris from Portugal were identified "on the basis of species distribution and abundance" [START_REF] Martins | Biology of Pre-and Post-Hatching Stages of Loligo vulgaris Lamarck, 1798 and Loligo forbesi Steenstrup[END_REF] so their identification requires validation. Identification of L. forbesii from NW Scotland is not in doubt as there is no reproduction of L. vulgaris there (our data).

Fig. 4

 4 Fig. 4 Increase of egg and embryo size during development of L. vulgaris and L. forbesii. Egg masses with uncertain stages between VIII and XII are shown as Stage 10.

Fig. 5 .

 5 Fig. 5. Averaged distribution of bottom temperature in March-April 2017-2019.

Fig. 6 .

 6 Fig. 6. Averaged distribution of bottom salinity in March-April 2017-2019.

Fig. 7

 7 Fig. 7 Occurrence of mature female L.forbesii (circles) and L.vulgaris (rectangles) in the studied area. Position of hauls where no mature squid were found are shown by crosses.

Acknowledgements

LB, AMP, MP, ES, LA were funded by the Cephs&Chefs project (EAPA_282/2016) of the European Regional Development Fund through the Interreg Atlantic Area Programme. We thank the staff and crew of the RV Celtic Explorer, and Cefas Endeavour for their help,

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


S Zouggar
email: salah.zouggar@ims-bordeaux.fr

M Zolghadri

P Girard

From customer needs to partners network specifications: (Design-Manufacturing-Supply) needs matrix

Keywords: Early design phase, NPD project, Co-design, Network specification

New Product Design (NPD) projects go sequentially through various phases of a new product design project: customer needs clarification, product specifications, design process, partners selection. Such sequencing ignores possible impacts of partners on the way that the product will be designed and realised. This paper explores early consideration of partners network for building up relevant partnerships in NPD projects. We attempt to highlight the gain of visibility through a suggested Design-Manufacturing-Supply Needs matrix. The new product is decomposed into three parts and by using needs matrix early in the design process, it will be possible to define design roles and profiles of needed partnerships and therefore of needed partners.

I. Introduction

Charles Fine in (Fine, 2003) proposes a method, called Double Helix, to define clearly the firm' strategy based on its relationships with suppliers, customers and its market position. The design process starts and progresses until the partners' selection phase; partners with whom the focal company should collaborate. The focal company is that studied company within a complex network linked with its direct customers and suppliers. Vonderembsea resumes the design of supply chain as an issue of product design: "Supply chain design should be, in part, a function of the product characteristics and expectations of the final customer", in [START_REF] Vonderembsea | Designing supply chains: Towards theory development[END_REF].

Recently some considerations of network of partners emerged in the scientific literature. NPD project improvement through partners consideration seems to be quite promising either for academics or for organizations. It has been shown that the nature and type of interactions between different actors during the product design process often affects the nature of the final product and its subsequent manufacturing [START_REF] Sharifi | Virtually co-located product design teams[END_REF]. We observe a gradual shift from intra organizational to inter organizational consideration of the design function in order to enhance organizational and product performance. A product design process is an iterative process. Performing design activities means redefining and redesigning. Customer's requirements, stakeholder's interpretations might be reviewed and redefined because of some appearing incompatibilities. Obviously coordination and communication are essentials to cope with these iterative process executions and they represent a confirmed mean of NPD improvement. In [START_REF] Zolghadri | Modelling mutual dependencies between products architecture and network of partners[END_REF] the parallel modelling of product and network development has been initiated. Authors outlined the necessity of parallel consideration and evolution of product specification and network characterization for enhancing NPD project performance.

However it still remains some lack of effective analysis tools, helping managers to get visibility on how the partners' profile impacts its future product. It is necessary to be aware of partners' impact on product, but it is necessary to have tools allowing to build effective network of partners in parallel with the product design. The aim of this research reported partly in this paper is to analyse progressively the way that customer needs lead to network specifications enabling the focal company to make right choices in terms of partners. The article is structured as follows. Next section discusses the relationships between the product design and network design. Section three presents the Design-Manufacturing-Supply needs matrix and discusses the way that this matrix supports decision-makers. Some conclusions will end the paper.

II. Network of Partners Development & New Product Development (NPD) projects

The effectiveness of new product introduction is a key competitive capability of companies. Of outmost importance to this capability is to focus on its core competence and delegate other functionalities to other members of the network thanks to their specific know how. Partners are selected for their specific skill and know-how. These partners may be consulting companies, sub-contractors, suppliers, research centres and universities. [START_REF] Booz | New Product Management for the 1980's[END_REF]

Improvable part

Undefined components to design, not yet elaborate, without precise specifications, thus requires a consequent investment for its design company or in collaboration with partners or entirely externalized.

Innovative part

Defined as … Product parts

adaptation and dimensioning.

Standard part

Known components, but not corresponding to the current needs or not enough, because of organizational, human or technological developments, leading to an Improvable part and its control, which according to the situation will be supported either by the company or in

Innovative part

Defined as … Product parts ernal resources

Table 1: Definition of product parts

In a NPD project, it will be then necessary to design and develop the network step-by-step according to the class of components required for the global product. NPD project consists in different phases that we match with network design and development project. The target is to initiate the build up of network simultaneously with the product.

Our first analysis was to observe and deduce the parallelism during product specification phase. (Ulrich et al.,2003) define six phases of the generic development process: planning, concept development, system-level design, detail design, testing and refinement, production rump-up. During the concept development phase "alternative product concepts are generated and evaluated and one or more concepts are selected for further development and testing". The system-level design "includes the definition of the product architecture and the decomposition of the product into subsystems and components". Finally, the detail design phase "includes the complete specification of the geometry, materials, tolerances, of all of the unique parts in the product and the identification of the standard parts to be purchased from suppliers".

We focus in NPD phase called: concept development (see figure 1). Customer needs obtained by interviews with potential purchasers and by observing similar products in use. They include hidden needs, those that customers are not aware of or accept them without question and explicit needs, those generally expressed and reported by potential purchasers. The customer needs are then depicted into wished product specifications. Product specifications are established through product benchmarking of concurrent products. Target specifications are essentially a wish-list tempered by technical constraints. We suggest parallel analysis carried out to check whether these specifications imply collaboration, if yes we must know at which ERIMA08' Proceedings level. This additional step will be called: Identify expected collaboration. At this step the focal company would be able to define its partnership strategy according to its internal expertise and external required one, allowing more precise specification based on market analysis. Generate product concepts: development of a various product concepts to identify what types of products are possible in term of technicality and best matching to the announced target specifications. Simultaneously, it becomes possible to identify collaboration needs in design, manufacturing and supply. Select product concept: Through assessment and comparison between attributes the experts finalizes the choice and adopts one of the suggested concepts concluding hence on set of final specifications. Set partners technical profiles: once the final specifications established ensuring technical feasibility, the implied collaboration needs would be also identified by the Design, Manufacturing, Supply needs approach introduced hereafter.

III. Design-Manufacturing-Supply Needs Matrix

Concepts are generated from different kind of specifications; we quote Functional (Forces involved, Energy need, and used materials), manufacturing (production of components, purchase of components, assembly) Economic (design costs, development costs manufacturing costs) and Aesthetic: (Fashion, Future expectations). In order to satisfy that specification several know-how and skills are required in various fields such as: Marketing, Industrial design, Mechanical engineering, Electronic engineering, manufacturing processes, Graphic arts Packaging design. The main idea of suggested approach is to situate the contribution of these competencies and know-how in the activities of NPD project phases, namely Design, Manufacturing and Supply. For that, we propose to build for each product concept a DMS needs Matrix (see figure 2).

  suggest 6 major classes of products: New to the World, New to the Company, Additions to Existing Product Lines, Improvements in/Revisions to Existing Products, Repositionings, Cost Reductions. Nevertheless this classification does not consider the way that collaboration with partners could be necessary. According to the kind of innovation undergone in the product, it induces a specific behaviour of the focal company regarding its partners. That is the reason why, we suggest decomposing the product into three parts according to the degree of the possessed knowledge about them: Innovative part, Improvable part, Standard part (see Table.1).

	Standard part	Known component, whose design is well known, optimized requires a work of
		adaptation and dimensioning.

Known components, but not corresponding to the current needs or not enough, because of organizational, human or technological developments, leading to an effort of adaptation and design , with a possibility of calling up ext to Focal Company.

Parallel launching of NPD projects

		Product Product
	Establish Establish	Generate Generate	Select Select	Set Set
	target target	product product	product product	final final
	specifications specifications	concepts concepts	concepts concepts	specifications specifications
	Customer needs Customer needs			Product & network development Product & network development
	Identify Identify	Identify needs of Identify needs of	Set partners Set partners
	expected expected	design, manufacturing design, manufacturing	technical technical
	collaboration collaboration	and supply and supply	profiles profiles
		Network Network
		NPD Projects NPD Projects
	Figure 1:

Innovative part Design partner technical profile

Strategic decision on partners selection

Partners dependency links for each project phase

Innovative part Design partner technical profile

Strategic decision on partners selection

Partners dependency links for each project phase

ERIMA07' Proceedings

From customer needs to partners network specifications

ERIMA08' Proceedings

The columns represent the various activities (Design, Manufacturing, Supply) for each part (Innovative, Improvable, Standard) of the product. Various lines correspond to Knowledge or Know-How (K/K-H from 1 to n). The same K/K-H can be necessary for various parts and with various activities. An activity can require several K/K-H.

Such needs matrix reveal by a fusion of the corresponding matrices of selected concepts:

• Cumulated competencies by phase constitute the technical profile and will be used in the following phase of partner selection to identify the relevant partner for a specific identified need.

• Once technical needs are identified, strategic decisions can be made for each need based on the total or partial involvement of a partner regarding to internal expertise and capability, and based on the choice between a partner who has all the K/K-H needed or one partner for each K/K-H need.

• For each phase we can build the dependency links between the potential partners during the selection phase to ensure a functional and structural compatibility among partners directly or indirectly linked.

IV. Conclusion:

To succeed in NPD projects, early network specification identification from customer needs is relevant for enhancing the performance of the whole project thanks to the anticipating reasoning about the technical profiles of required future collaboration. We suggest the Design, Manufacturing, Supply needs matrix. Once filled, these matrixes allow detecting essential knowledge/know-how and technical profiles of partners. This analysis can be done in a recursive manner from the highest-level concepts into lower-level ones.

The main purpose of this matrix is to offer an extended visibility of what is really needed in term of collaboration and potential partners' Knowledge/Know-How. It aims at guaranteeing an overall coherence of the way that the network of partners will collaborate with the focal company. One starts by identifying technical compatibility necessary to the project required during the selection phase. This identified compatibility added to others (time, costs, flexibility, geographic location, etc.) represent a new crucial indicator for managers. In the final paper, the described approach will be applied to a simplified illustrative case taken from bicycle manufacturing.