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Egg development and fecundity estimation in deep-sea red crab, Chaceon affinis (Geryonidae), off the Canary Islands (NE Atlantic)
Fisheries Research 109 (2011) 373–378
Contents lists available at ScienceDirect
Fisheries Research journal homepage: www.elsevier.com/locate/fishres
Short communication
Egg development and fecundity estimation in deep-sea red crab, Chaceon affinis (Geryonidae), off the Canary Islands (NE Atlantic) Victor M. Tuset a,c,∗ , Domingo I. Espinosa b , Antonio García-Mederos a , José I. Santana a , José A. González a a Departamento de Biología Pesquera, Instituto Canario de Ciencias Marinas (ICCM-ACIISI), Grupo de Biología Pesquera, P.O. Box. 56, E-35200 Telde (Las Palmas), Canary Islands, Spain b Departamento de Biología Animal, Universidad de La Laguna, c/Astrofísico F. Sánchez s/n, La Laguna, Tenerife, Spain c Instituto de Ciéncias del Mar (ICM-CSIC), Passeig Marítim 37-49, 08003, Barcelona, Catalu˜ na, Spain
a r t i c l e
i n f o
Article history: Received 5 November 2010 Received in revised form 3 February 2011 Accepted 28 February 2011 Keywords: Egg development Fecundity Chaceon affinis NE Atlantic
a b s t r a c t The goal of this work was to elaborate on reproductive knowledge of the deep-sea red crab, Chaceon affinis, off the Canary Islands by providing information regarding its egg development and fecundity. Six stages for eggs were observed, from fully filled with yolk to the embryo occupying almost all the space inside the egg. A correlation was established between egg stage and the colour of the egg mass. Morphological analyses indicated that the eggs are spherical in shape and that they increase the 10.7% in maximum diameter from the initial to the final stage. Fecundity (AFE, annual fecundity estimation), defined as the number of eggs borne per females, was calculated with two methods, manual by hand counter and using an automated morphology system. No statistical differences for the relationships carapace width (CW)–AFE and total weight (TW)–AFE were detected between the two methods. The number of eggs ranged from 199,690 to 566,956 (105–160 mm CW). A positive correlation was obtained between AFE and CW (r2 = 0.672) and TW (r2 = 0.785). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Geryonid deep-sea red crabs occur widespread throughout the world and many of them have interest to fisheries such as, e.g., Chaceon quinquedens Smith, 1879 and Chaceon fenneri (Manning and Holthuis, 1984), Chaceon maritae (Manning and Holthuis, 1981), Chaceon bicolour (Manning and Holthuis, 1989) and Chaceon notialis Manning and Holthuis, 1989, Chaceon chilensis ChirinoGálvez and Manning, 1989 (Elner et al., 1987; Erdman and Blake, 1988; Melville-Smith, 1988; Lindberg and Wenner, 1990; Arana, 2000; Steimle et al., 2001; Smith et al., 2004) and Chaceon affinis (Manning and Holthuis, 1989). This last species inhabits the eastern Atlantic Ocean (64◦ N–15◦ N) from Iceland to Senegal, including the Azores, Madeira, and the Canary and Cape Verde Islands between 130 and 2047 m depth (Kjenerud, 1967; Samuelsen, 1975; Manning and Holthuis, 1981; Sánchez and Olaso, 1985; Dawson and Webber, 1991), and also know from the Mid-Atlantic Ridges i.e., Menez Gwen, Lost City (Biscoito and Saldanha, 2000; Biscoito, 2006). In the Canary Islands, several studies demonstrated the relative abundance of this species at depths between 600 and 1000 m (López Abellán et al., 1994; González, 1995) and some biological aspects
(reproduction, sizes at maturity, population structure) have been studied (Fernández-Vergaz et al., 2000; López Abellán et al., 2002). Models of population dynamic used in the management of fishery resources require, among other factors, an estimation of fecundity. Fecundity is a general term used to describe the number of eggs produced by individual females (Arshad et al., 2006). In the red crabs, eggs are laid and held attached to the female pleopods under the abdomen for up to nine months until the eggs hatch and the larvae are released into the water column (Haefner, 1978; Erdman et al., 1991). Studies suggest that these species may not spawn annually, due to long intermoult, period for adult females (5–7 years), although it is possible that sperm could be stored for intermoult spawning (Hines, 1982; Lux et al., 1982: Erdman et al., 1991; Biesiot and Perry, 1995). The goal of this paper is to increase our knowledge on the reproduction of C. affinis in the Canary Islands previous to its possible economic exploitation by describing egg development and by estimating its fecundity. In addition, the application of an automated image analysis system for estimation fecundity and for egg diameter measurements in crustaceans was tested. 2. Materials and methods
∗ Corresponding author at: Instituto de Ciéncias del Mar (ICM-CSIC), Passeig Marí˜ Spain. Tel.: +34 928 132 900; fax: +34 928 tim 37-49, 08003, Barcelona, Cataluna, 132 908. E-mail addresses: [email protected], [email protected] (V.M. Tuset). 0165-7836/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2011.02.021
2.1. Sampling A total of 41 berried females were captured from two experimental research cruises, made in February–March 2005 (n = 20) and
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V.M. Tuset et al. / Fisheries Research 109 (2011) 373–378
Fig. 1. Location of the study area.
November–December 2006 (n = 21), coinciding with the spawning period October–May (López Abellán et al., 2002). Individuals were recollected using bottom traps at depths of 600–1000 m (Carvalho et al., 2007) to the south of Gran Canaria (Canary Islands, northeastern Atlantic) (Fig. 1). Only fresh samples, the last day of collecting, were taken to the laboratory for the posterior study: 14 females we used for the morphological study of eggs and 30 females for the fecundity estimation. Carapace width (CW in mm) and total weight (TW in g) were taken from each crab.
under a compound microscope with reflected light, to count the eggs using a hand counter. (b) In method 2 (November–December sample), fresh egg masses were stored in 4% buffered formalin, detached from the pleopod filaments using tweezers, placed on
2.2. Egg development The fresh egg mass was separated from the pleopods, placed on a mesh of 100 m, and washed with seawater under pressure to detach all the eggs. Then some eggs were placed in two Petri dishes with seawater under a compound microscope with reflected light. Four images for each Petri dish (one by quadrant) were taken in each female with a digital video camera (Basler A310, Vision Technologies, Basler AG, Ahrensburg) for the automated morphology analysis (Fig. 2). Approximately, 40 eggs were measured in each quadrant. Image-Pro Plus version 4.1.0 software (Media Cybernetics, Inc.) was used to calculate the following shape parameters and indices: projected area (A in mm2 ), maximum diameter (D in mm), minimum diameter (d in mm), roundness (4A/(D2 )), and aspect ratio (D/d) (Russ, 1990). Egg morphology was described and classified according to Arshad et al. (2006) and Walker et al. (2006) for other brachyuran crabs, and colour changes of the egg mass considering previous studies in Chaceon spp. (Wigley et al., 1975; Haefner, 1978). 2.3. Estimation of fecundity The gravimetric method was applied to determine fecundity (AFE, annual fecundity estimation), which was defined as annual egg production, considering that spawning might occur only once a year, using a subsample of 5% by wet weight of the egg mass (Lowerre-Barbieri and Barbieri, 1993). Two methodological procedures were applied: (a) in method 1 (February–March sample), the fresh egg mass was separated from the pleopods, placed on a mesh of 100 m, and washed with seawater under pressure to detach all the eggs. Then the eggs were placed in Petri dishes with seawater
Fig. 2. Digitalized image of eggs for morphological analysis: (A) original image; (B) modified binary image.
V.M. Tuset et al. / Fisheries Research 109 (2011) 373–378
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Fig. 3. Chromatic variation of eggs attached to pleopods until their hatching: (A) red-orange or light purple; (B) burgundy; (C) dark purple; (D) brownish; (E) brown; (F) grey-black. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Petri dishes, and counted using an automated imaging system (Leica QWin Standard, Leica Microsystems GmbH). Berried females in embryo stage (see results, stage VI) were eliminated from the analysis, because some embryos could have hatched already. In both methods, power regressions were fitted to fecundity versus carapace width and total weight (Haddon, 1994). Analysis of covariance (ANCOVA) was used to determine the effect of the method on the relationships CW–AFE and TW–AFE, in which the dependent variable was the natural logarithm of the fecundity, the fixed factor the method, and the covariate the natural logarithm of CW or TW. 3. Results Six development stages were observed and correlated with the colour of the egg mass (Figs. 3 and 4): stage I, egg still undivided, fully filled with yolk, red-orange or light purple colour; stage II, the free region of yolk is just visible, burgundy colour; stage III, a slight pigmentation of the eyes appears, dark purple colour; stage IV, pigmented eyes enlarging in moon shape, brownish colour; stage V, visible pigmented structures enlarging eyes, segmented appendages and abdomen appearing, brown colour; stage VI, eyes acquiring oval shape, embryo occupies almost all the space inside the egg, grey-black colour. After the last stage, larvae are present. Egg development seems to be not completely synchronous and sometimes two colour patterns can be observed simultaneously, one in the inner part, and another in the outer part of the egg mass
(Fig. 3A). The eggs present a spherical shape, the mean sizes (maximum diameter, minimum diameter, and area) increasing from newly spawned ones (e.g., 0.585 mm D) to hatching (0.655 mm D) (Table 1). Fecundity ranged between 264,492 (118 mm CW) and 566,956 eggs (143 mm CW) using method 1 (n = 10) and from 199,690 (105 mm CW) to 559,308 eggs (160 mm CW) with method 2 (n = 20). The ANCOVA test did not show significant differences in the relationships CW–AFE (F = 1.474, P = 0.235) and TW–AFE (F = 0.188, P = 0.668) between methods, therefore relationships were calculated for the whole set of data. Regression analysis showed a significant, positive correlation between fecundity and carapace width (AFE = 3.180 × CW2.407 , r2 = 0.681, P < 0.001) and fecundity and total weight (AFE = 684.486 × TW0.977 , r2 = 0.764, P < 0.001) (Fig. 5). 4. Discussion The egg development of C. affinis studied herein was very similar to the general embryonic pattern observed in many brachyuran crabs, including an increase in egg size from newly spawned ones to just prior to hatching (Arshad et al., 2006; Walker et al., 2006). However, changes in colour patterns differ greatly among these deep-sea red crabs, making it necessary to develop a macroscopic scale for each one separately. The eggs of C. fenneri are initially light purple or burgundy, becoming dark purple and purple-brown,
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Fig. 4. Phases of egg development. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
while in C. quinquedens they are red-orange, then brown, purple, and finally black (Wigley et al., 1975; Haefner, 1978; Erdman and Blake, 1988). Crabs of genus Chaceon inhabit deep waters and seem to have continuous reproduction along year (Elner et al., 1987; Erdman and Blake, 1988; Steimle et al., 2001; Pinho et al., 2001; Smith et al., 2004). Berried females migrate to shallower and warmer waters enhancing eggs that are swept up surface by the current (Haefner,
1978). The larvae phase is pelagic and consists in four zoeal stages and a final megalopa. In the first postmegalop instar stage the carapace width is relatively large, which may be an adaptation to slow post-settlement growth (see Steimle et al., 2001). The eggs are the largest known for crabs with planktonic development: 600 m in maximum diameter in C. fenneri (Erdman and Blake, 1988; Hines, 1988; Erdman et al., 1991) and C. bicolour (Smith et al., 2004), 680 m D in C. maritae (Melville-Smith, 1987), 789 m D in C.
Table 1 Morphological values (mean ± standard deviation) of stages of egg development. Stage
n
Maximum diameter (D, mm)
I II III IV V VI
302 2042 616 326 533 334
0.585 0.582 0.580 0.590 0.601 0.655
± ± ± ± ± ±
0.031 0.039 0.043 0.047 0.033 0.038
Minimum diameter (d, mm) 0.551 0.541 0.551 0.558 0.566 0.627
± ± ± ± ± ±
0.029 0.043 0.045 0.048 0.035 0.036
Area (A, mm2 ) 0.254 0.251 0.257 0.262 0.272 0.328
± ± ± ± ± ±
0.026 0.034 0.038 0.042 0.030 0.036
Roundness 1.075 1.124 1.078 1.077 1.081 1.083
± ± ± ± ± ±
0.007 0.081 0.009 0.008 0.009 0.008
Ratio aspect 1.097 1.157 1.115 1.126 1.131 1.109
± ± ± ± ± ±
0.031 0.062 0.039 0.043 0.050 0.040
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can lose eggs during incubation through disease, egg predation, or other kinds of natural failure of egg development (Perkins, 1971). The high variability may be due to the large time that females carry the eggs as the incubation period may last up to nine months (Haefner, 1978; Erdman et al., 1991). It can be hypothesized that the use of females with eggs in different stages of development could influence the fecundity estimates. Nevertheless, Erdman and Blake (1988) and Hines (1988) found a similar trend for Chaceon spp. selecting only embryos in early stage. Moreover, our results prove that the comparative study between automated system versus manual counting, and also between fresh versus fixed samples, were not causing this high variability. Consequently, this study provides relevant information for stock assessment of this species, moreover the analysis of the colour of the eggs and their development may be used for a sustainable fishing due to the quick observation. Acknowledgements ˜ The authors are indebted to José A. Pérez-Penalvo, Rosa Domínguez-Seoane and Eliseba García for skilful technical assistance. Financial support was received from the EU ERDF in the framework of the PIC INTERREG III B project PESCPROF-2 (03/MAC/4.2/M8) and from the Spanish Government in the framework of project REDECA (CTM2005-07712-C03/MAR). References
Fig. 5. Relationships between fecundity and carapace width (A) and total weight (B).
affinis (present study), and 846 m D in C. quinquedens (Haefner, 1978; Elner et al., 1987; Hines, 1988; Erdman et al., 1991). However, large egg size has reproductive implications by decreasing the number of eggs (Hines, 1982, 1988). Comparison of fecundity estimates in deep-sea red crabs does not reveal great differences among species, although it can be concluded that there is a positive correlation female size and the number of eggs: 36,000–226,000 eggs (90–118 mm CW) in C. quinquedens (Hines, 1988; Steimle et al., 2001), 15,592–288,312 eggs (98–133 mm CW) in C. bicolor (Smith et al., 2004), 107,000–350,000 in C. maritae (MelvilleSmith, 1987), 160,000–375,000 eggs (110–143 mm CW) in C. fenneri (Erdman and Blake, 1988; Hines, 1988), and 199,690–566,956 eggs (105–160 mm CW) in C. affinis (present study). In all these species it has been found that fecundity increased with the increase of carapace width or wet weight of the individuals (Melville-Smith, 1987; Erdman and Blake, 1988; Hines, 1988; Steimle et al., 2001; Smith et al., 2004; present study), as also occurs in other brachyurans (Haddon, 1994; Mantelatto and Fransozo, 1997; Litulo, 2004, 2005; Arshad et al., 2006). However, in deep-sea red crabs it has been noted that these variables are not good predictors of fecundity, reaching variability values of 45–65% (C. quinquedens, C. fenneri, and C. affinis) or at least 13–26% (C. bicolor). Also, it is known crabs
Arana, P.M., 2000. Estimación de abundancia y biomasa del cangrejo dorado (Chaceon chilensis), en el archipiélago de Juan Fernández, Chile. Inv. Mar. 28, 53–68. Arshad, A., Kamarudin, M.S., Saad, C.R., 2006. Study on fecundity and larval development of blue swimming crab Portunus pelagicus (Linnaeus, 1758) under laboratory conditions. Res. J. Fish. Hydrobiol. 1, 35–44. Biesiot, P.M., Perry, H.M., 1995. Biochemical composition of the deep-sea red crab Chaceon quinquedens (Geryonidae): organic reserves of developing embryos and adults. Mar. Biol. 124, 407–416. Biscoito, M., 2006. Chaceon affinis. In: Desbruyères, D., Segonzac, M., Bright, M. (Eds.), Handbook of Deep-sea Hydrothermal Vent Fauna, 2nd ed. Denisia, Linz, p. 458. Biscoito, M.J., Saldanha, L., 2000. Occurrence of Chaceon affinis (Decapoda: Geryonidae) in the vicinity of a hydrothermal vent site on the mid-Atlantic ridge. J. Crust. Biol. 20, 128–131. Carvalho, D., Delgado, J., Biscoito, M., Frietas, M., González, J.A., Santana, J.I., Tuset, V.M., Isidro, E., Pinho, M.R., Consorcio Pescprof. 2007. Recursos Pesqueros de Aguas Profundas del Atlántico Centro-Oriental: alternativas a la pesca en la Macaronesia. Memoria científico-técnica final del Proyecto PESCPROF-2 (PIC Interreg III B, 03MAC/4.2/M8). European Union, Regional Policy, FEDER. Telde (Las Palmas), p. 154. Dawson, E.W., Webber, W.R., 1991. The deep-sea red crab Chaceon (“Geryon”): a guide to information and a reference list of the family Geryonidae. Nat. Mus. New Zealand Miscell. Ser. 24, 1–83. Elner, R.W., Koshio, S., Hurley, G.V., 1987. Mating behavior of the deep-sea red crab, Geryon quinquedens Smith (Decapod, Brachyura, Geryonidae). Crustaceana 52, 194–201. Erdman, R.B., Blake, N.J., 1988. Reproductive biology of female golden crabs Geryon fenneri Manning and Holthuis, from southeastern Florida. J. Crust. Biol. 8, 392–400. Erdman, R.B., Blake, N.J., Lockhart, F.D., Lindberg, W.J., Perry, H.M., Waller, R.S., 1991. Comparative reproduction of the deep-sea crabs Chaceon fenneri and C. quinquedens (Brachyura: Geryonidae) from the northeast Gulf of Mexico. Invertebr. Reprod. Dev. 19, 175–184. Fernández-Vergaz, V., López Abellán, L.J., Balguerías, E., 2000. Morphometric, functional and sexual maturity of the deep-sea red crab Chaceon affinis inhabiting Canary Islands waters: chronology of maturation. Mar. Ecol. Prog. Ser. 204, 169–178. González, J.A., 1995. Catálogo de los Crustáceos Decápodos de las islas Canarias. Publicaciones Turquesa, Santa Cruz de Tenerife. Haddon, M., 1994. Size-fecundity relationships, mating behaviour, and larval release in the New Zealand paddle crab, Ovalipes catharus (White, 1843) (Brachyura: Portunidae). N. Z. J. Mar. Freshw. Res. 28, 329–334. Haefner Jr., P.A., 1978. Seasonal aspects of the biology, distribution and relative abundance of the deep-sea red crab Geryon quinquedens Smith, in the vicinity of the Norfolk Canyon, Western North Atlantic. Proc. Natl. Shellfish Assoc. 68, 49–62. Hines, A.H., 1982. Allometric constraints and variables of reproductive effort in brachyuran crabs. Mar. Biol. 69, 309–320. Hines, A.H., 1988. Fecundity and reproductive output of two species of deep-sea crabs, Geryon fenneri and G. quinquedens (Decapoda, Brachyura). J. Crust. Biol. 8, 557–562.
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Kjenerud, J., 1967. A find of Geryon affinis Milne-Edwards and Bouvier, 1984 (Crustacea Decapoda) off the coast of Norway. Sarsia 29, 193–198. Lindberg, W.J., Wenner, E.L., 1990. Geryonid crabs and associated continental slope fauna: a research workshop report. Florida Sea Grant College Tech. Pap. 58: 1–61. Litulo, C., 2004. Fecundity of the pantropical fiddler crab Uca annulipes (H. Milne Edwards, 1837) (Brachyura: Ocypodidae) at Costa do Sol Mangrove, Maputo Bay, southern Mozambique. Western Indian Ocean J. Mar. Sci. 3, 87–91. Litulo, C., 2005. Fecundity and size at sexual maturity of the fiddler crab Uca vocans (Linnaeus, 1758) (Brachyura: Ocypodidae). Thalassa 21, 59–65. ˜ López Abellán, L.J., Santamaría, M.T.G., Balguerías, E., 1994. Resultados de la campana experimental de pesca, realizada en aguas del suroeste de la isla de Tenerife, Canarias 9206. Inf. Téc. Inst. Esp. Oceanogr. 147, 1–62. López Abellán, L.J., Balguerías, E., Fernández-Vergaz, V., 2002. Life history characteristics of the deep-sea crab Chaceon affinis population off Tenerife (Canary Islands). Fish. Res. 58, 231–239. Lowerre-Barbieri, S.K., Barbieri, L.R., 1993. A new method of oocytes separation and preservation for fish reproduction studies. Fish. Bull. 91, 165– 170. Lux, F.E., Ganz, A.R., Rathjen, W.F., 1982. Marking studies on the red crab Geryon quinquedens Smith off southern New England. J. Shellfish Res. 2, 71–80. Manning, R.B., Holthuis, L.B., 1981. West African brachyuran crabs. Smithsonian Contrib. Zool. 306, 1–379. Melville-Smith, R., 1987. The reproductive biology of Geryon maritae (Decapoda Brachyura) off southwest Africa/Namibia. Crustaceana 53, 11–27. Melville-Smith, R., 1988. Comparative population size estimates for a portion of the red crab Geryon maritae stock off southwest African coast. South African J. Mar. Sci. 6, 23–31.
Mantelatto, F.L.M., Fransozo, A., 1997. Fecundity of the crab Callinectes ornatus Ordway, 1863 (Decapoda, Brachyura, Portunidae) from the Ubatuba region, São Paulo, Brazil. Crustaceana 70, 214–224. Perkins, H.C., 1971. Egg loss during incubation from off-shore northern lobster (Decapoda: Homaridae). Fish. Bull. 69, 451–453. Pinho, M.R., Gonc¸alves, J.M., Martins, H.R., Menezes, G.M., 2001. Some aspects of the biology of the deep-water crab, Chaceon affinis (Milne-Edwards and Bouvier, 1894) off the Azores. Fish. Res. 51, 283–295. Russ, J.C., 1990. Computer-assisted Microscopy: the measurement and analysis of images. New York, Plenum Press. Samuelsen, T.J., 1975. The third record of Geryon affinis, A. Milne-Edwards and Bouvier (Crustacea, Decapoda) from western Norway. Sarsia 59, 47– 48. Sánchez, F., Olaso, I., 1985. Presencia de Geryon affinis Milne-Edwards y Bouvier, 1894, en el golfo de Vizcaya (Decapoda, Brachyura). Bol. Inst. Esp. Oceanogr. 2, 155–157. Smith, K.D., Potter, I.C., Hall, N.G., 2004. Biological and fisheries data for managing the deep-sea crabs Hypothalassia acerba and Chaceon bicolor in Western Australia. Projects No. 1999/154 and 2001/055. FRDC Australia. Steimle, F.W., Zetlin, C.A., Chang, S., 2001. Essential Fish Habitat Source Document: Red Deepsea Crab, Chaceon (Geryon) quinquedens, Life History and Habitat Characteristics. NOAA Tech. Memo. NMFS NE 163, 1–27. Walker, A., Ando, S., Smith, G.D., Lee, R.F., 2006. The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis. Comp. Bioch. Physiol. Part B 143, 201–208. Wigley, R.L., Theroux, R.B., Murray, H.E., 1975. Deep-sea red crab, Geryon quinquedens, survey off northeastern United States. Mar. Fish. Rev. 37, 1–27.
Contents lists available at ScienceDirect
Fisheries Research journal homepage: www.elsevier.com/locate/fishres
Short communication
Egg development and fecundity estimation in deep-sea red crab, Chaceon affinis (Geryonidae), off the Canary Islands (NE Atlantic) Victor M. Tuset a,c,∗ , Domingo I. Espinosa b , Antonio García-Mederos a , José I. Santana a , José A. González a a Departamento de Biología Pesquera, Instituto Canario de Ciencias Marinas (ICCM-ACIISI), Grupo de Biología Pesquera, P.O. Box. 56, E-35200 Telde (Las Palmas), Canary Islands, Spain b Departamento de Biología Animal, Universidad de La Laguna, c/Astrofísico F. Sánchez s/n, La Laguna, Tenerife, Spain c Instituto de Ciéncias del Mar (ICM-CSIC), Passeig Marítim 37-49, 08003, Barcelona, Catalu˜ na, Spain
a r t i c l e
i n f o
Article history: Received 5 November 2010 Received in revised form 3 February 2011 Accepted 28 February 2011 Keywords: Egg development Fecundity Chaceon affinis NE Atlantic
a b s t r a c t The goal of this work was to elaborate on reproductive knowledge of the deep-sea red crab, Chaceon affinis, off the Canary Islands by providing information regarding its egg development and fecundity. Six stages for eggs were observed, from fully filled with yolk to the embryo occupying almost all the space inside the egg. A correlation was established between egg stage and the colour of the egg mass. Morphological analyses indicated that the eggs are spherical in shape and that they increase the 10.7% in maximum diameter from the initial to the final stage. Fecundity (AFE, annual fecundity estimation), defined as the number of eggs borne per females, was calculated with two methods, manual by hand counter and using an automated morphology system. No statistical differences for the relationships carapace width (CW)–AFE and total weight (TW)–AFE were detected between the two methods. The number of eggs ranged from 199,690 to 566,956 (105–160 mm CW). A positive correlation was obtained between AFE and CW (r2 = 0.672) and TW (r2 = 0.785). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Geryonid deep-sea red crabs occur widespread throughout the world and many of them have interest to fisheries such as, e.g., Chaceon quinquedens Smith, 1879 and Chaceon fenneri (Manning and Holthuis, 1984), Chaceon maritae (Manning and Holthuis, 1981), Chaceon bicolour (Manning and Holthuis, 1989) and Chaceon notialis Manning and Holthuis, 1989, Chaceon chilensis ChirinoGálvez and Manning, 1989 (Elner et al., 1987; Erdman and Blake, 1988; Melville-Smith, 1988; Lindberg and Wenner, 1990; Arana, 2000; Steimle et al., 2001; Smith et al., 2004) and Chaceon affinis (Manning and Holthuis, 1989). This last species inhabits the eastern Atlantic Ocean (64◦ N–15◦ N) from Iceland to Senegal, including the Azores, Madeira, and the Canary and Cape Verde Islands between 130 and 2047 m depth (Kjenerud, 1967; Samuelsen, 1975; Manning and Holthuis, 1981; Sánchez and Olaso, 1985; Dawson and Webber, 1991), and also know from the Mid-Atlantic Ridges i.e., Menez Gwen, Lost City (Biscoito and Saldanha, 2000; Biscoito, 2006). In the Canary Islands, several studies demonstrated the relative abundance of this species at depths between 600 and 1000 m (López Abellán et al., 1994; González, 1995) and some biological aspects
(reproduction, sizes at maturity, population structure) have been studied (Fernández-Vergaz et al., 2000; López Abellán et al., 2002). Models of population dynamic used in the management of fishery resources require, among other factors, an estimation of fecundity. Fecundity is a general term used to describe the number of eggs produced by individual females (Arshad et al., 2006). In the red crabs, eggs are laid and held attached to the female pleopods under the abdomen for up to nine months until the eggs hatch and the larvae are released into the water column (Haefner, 1978; Erdman et al., 1991). Studies suggest that these species may not spawn annually, due to long intermoult, period for adult females (5–7 years), although it is possible that sperm could be stored for intermoult spawning (Hines, 1982; Lux et al., 1982: Erdman et al., 1991; Biesiot and Perry, 1995). The goal of this paper is to increase our knowledge on the reproduction of C. affinis in the Canary Islands previous to its possible economic exploitation by describing egg development and by estimating its fecundity. In addition, the application of an automated image analysis system for estimation fecundity and for egg diameter measurements in crustaceans was tested. 2. Materials and methods
∗ Corresponding author at: Instituto de Ciéncias del Mar (ICM-CSIC), Passeig Marí˜ Spain. Tel.: +34 928 132 900; fax: +34 928 tim 37-49, 08003, Barcelona, Cataluna, 132 908. E-mail addresses: [email protected], [email protected] (V.M. Tuset). 0165-7836/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2011.02.021
2.1. Sampling A total of 41 berried females were captured from two experimental research cruises, made in February–March 2005 (n = 20) and
374
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Fig. 1. Location of the study area.
November–December 2006 (n = 21), coinciding with the spawning period October–May (López Abellán et al., 2002). Individuals were recollected using bottom traps at depths of 600–1000 m (Carvalho et al., 2007) to the south of Gran Canaria (Canary Islands, northeastern Atlantic) (Fig. 1). Only fresh samples, the last day of collecting, were taken to the laboratory for the posterior study: 14 females we used for the morphological study of eggs and 30 females for the fecundity estimation. Carapace width (CW in mm) and total weight (TW in g) were taken from each crab.
under a compound microscope with reflected light, to count the eggs using a hand counter. (b) In method 2 (November–December sample), fresh egg masses were stored in 4% buffered formalin, detached from the pleopod filaments using tweezers, placed on
2.2. Egg development The fresh egg mass was separated from the pleopods, placed on a mesh of 100 m, and washed with seawater under pressure to detach all the eggs. Then some eggs were placed in two Petri dishes with seawater under a compound microscope with reflected light. Four images for each Petri dish (one by quadrant) were taken in each female with a digital video camera (Basler A310, Vision Technologies, Basler AG, Ahrensburg) for the automated morphology analysis (Fig. 2). Approximately, 40 eggs were measured in each quadrant. Image-Pro Plus version 4.1.0 software (Media Cybernetics, Inc.) was used to calculate the following shape parameters and indices: projected area (A in mm2 ), maximum diameter (D in mm), minimum diameter (d in mm), roundness (4A/(D2 )), and aspect ratio (D/d) (Russ, 1990). Egg morphology was described and classified according to Arshad et al. (2006) and Walker et al. (2006) for other brachyuran crabs, and colour changes of the egg mass considering previous studies in Chaceon spp. (Wigley et al., 1975; Haefner, 1978). 2.3. Estimation of fecundity The gravimetric method was applied to determine fecundity (AFE, annual fecundity estimation), which was defined as annual egg production, considering that spawning might occur only once a year, using a subsample of 5% by wet weight of the egg mass (Lowerre-Barbieri and Barbieri, 1993). Two methodological procedures were applied: (a) in method 1 (February–March sample), the fresh egg mass was separated from the pleopods, placed on a mesh of 100 m, and washed with seawater under pressure to detach all the eggs. Then the eggs were placed in Petri dishes with seawater
Fig. 2. Digitalized image of eggs for morphological analysis: (A) original image; (B) modified binary image.
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Fig. 3. Chromatic variation of eggs attached to pleopods until their hatching: (A) red-orange or light purple; (B) burgundy; (C) dark purple; (D) brownish; (E) brown; (F) grey-black. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Petri dishes, and counted using an automated imaging system (Leica QWin Standard, Leica Microsystems GmbH). Berried females in embryo stage (see results, stage VI) were eliminated from the analysis, because some embryos could have hatched already. In both methods, power regressions were fitted to fecundity versus carapace width and total weight (Haddon, 1994). Analysis of covariance (ANCOVA) was used to determine the effect of the method on the relationships CW–AFE and TW–AFE, in which the dependent variable was the natural logarithm of the fecundity, the fixed factor the method, and the covariate the natural logarithm of CW or TW. 3. Results Six development stages were observed and correlated with the colour of the egg mass (Figs. 3 and 4): stage I, egg still undivided, fully filled with yolk, red-orange or light purple colour; stage II, the free region of yolk is just visible, burgundy colour; stage III, a slight pigmentation of the eyes appears, dark purple colour; stage IV, pigmented eyes enlarging in moon shape, brownish colour; stage V, visible pigmented structures enlarging eyes, segmented appendages and abdomen appearing, brown colour; stage VI, eyes acquiring oval shape, embryo occupies almost all the space inside the egg, grey-black colour. After the last stage, larvae are present. Egg development seems to be not completely synchronous and sometimes two colour patterns can be observed simultaneously, one in the inner part, and another in the outer part of the egg mass
(Fig. 3A). The eggs present a spherical shape, the mean sizes (maximum diameter, minimum diameter, and area) increasing from newly spawned ones (e.g., 0.585 mm D) to hatching (0.655 mm D) (Table 1). Fecundity ranged between 264,492 (118 mm CW) and 566,956 eggs (143 mm CW) using method 1 (n = 10) and from 199,690 (105 mm CW) to 559,308 eggs (160 mm CW) with method 2 (n = 20). The ANCOVA test did not show significant differences in the relationships CW–AFE (F = 1.474, P = 0.235) and TW–AFE (F = 0.188, P = 0.668) between methods, therefore relationships were calculated for the whole set of data. Regression analysis showed a significant, positive correlation between fecundity and carapace width (AFE = 3.180 × CW2.407 , r2 = 0.681, P < 0.001) and fecundity and total weight (AFE = 684.486 × TW0.977 , r2 = 0.764, P < 0.001) (Fig. 5). 4. Discussion The egg development of C. affinis studied herein was very similar to the general embryonic pattern observed in many brachyuran crabs, including an increase in egg size from newly spawned ones to just prior to hatching (Arshad et al., 2006; Walker et al., 2006). However, changes in colour patterns differ greatly among these deep-sea red crabs, making it necessary to develop a macroscopic scale for each one separately. The eggs of C. fenneri are initially light purple or burgundy, becoming dark purple and purple-brown,
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Fig. 4. Phases of egg development. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
while in C. quinquedens they are red-orange, then brown, purple, and finally black (Wigley et al., 1975; Haefner, 1978; Erdman and Blake, 1988). Crabs of genus Chaceon inhabit deep waters and seem to have continuous reproduction along year (Elner et al., 1987; Erdman and Blake, 1988; Steimle et al., 2001; Pinho et al., 2001; Smith et al., 2004). Berried females migrate to shallower and warmer waters enhancing eggs that are swept up surface by the current (Haefner,
1978). The larvae phase is pelagic and consists in four zoeal stages and a final megalopa. In the first postmegalop instar stage the carapace width is relatively large, which may be an adaptation to slow post-settlement growth (see Steimle et al., 2001). The eggs are the largest known for crabs with planktonic development: 600 m in maximum diameter in C. fenneri (Erdman and Blake, 1988; Hines, 1988; Erdman et al., 1991) and C. bicolour (Smith et al., 2004), 680 m D in C. maritae (Melville-Smith, 1987), 789 m D in C.
Table 1 Morphological values (mean ± standard deviation) of stages of egg development. Stage
n
Maximum diameter (D, mm)
I II III IV V VI
302 2042 616 326 533 334
0.585 0.582 0.580 0.590 0.601 0.655
± ± ± ± ± ±
0.031 0.039 0.043 0.047 0.033 0.038
Minimum diameter (d, mm) 0.551 0.541 0.551 0.558 0.566 0.627
± ± ± ± ± ±
0.029 0.043 0.045 0.048 0.035 0.036
Area (A, mm2 ) 0.254 0.251 0.257 0.262 0.272 0.328
± ± ± ± ± ±
0.026 0.034 0.038 0.042 0.030 0.036
Roundness 1.075 1.124 1.078 1.077 1.081 1.083
± ± ± ± ± ±
0.007 0.081 0.009 0.008 0.009 0.008
Ratio aspect 1.097 1.157 1.115 1.126 1.131 1.109
± ± ± ± ± ±
0.031 0.062 0.039 0.043 0.050 0.040
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can lose eggs during incubation through disease, egg predation, or other kinds of natural failure of egg development (Perkins, 1971). The high variability may be due to the large time that females carry the eggs as the incubation period may last up to nine months (Haefner, 1978; Erdman et al., 1991). It can be hypothesized that the use of females with eggs in different stages of development could influence the fecundity estimates. Nevertheless, Erdman and Blake (1988) and Hines (1988) found a similar trend for Chaceon spp. selecting only embryos in early stage. Moreover, our results prove that the comparative study between automated system versus manual counting, and also between fresh versus fixed samples, were not causing this high variability. Consequently, this study provides relevant information for stock assessment of this species, moreover the analysis of the colour of the eggs and their development may be used for a sustainable fishing due to the quick observation. Acknowledgements ˜ The authors are indebted to José A. Pérez-Penalvo, Rosa Domínguez-Seoane and Eliseba García for skilful technical assistance. Financial support was received from the EU ERDF in the framework of the PIC INTERREG III B project PESCPROF-2 (03/MAC/4.2/M8) and from the Spanish Government in the framework of project REDECA (CTM2005-07712-C03/MAR). References
Fig. 5. Relationships between fecundity and carapace width (A) and total weight (B).
affinis (present study), and 846 m D in C. quinquedens (Haefner, 1978; Elner et al., 1987; Hines, 1988; Erdman et al., 1991). However, large egg size has reproductive implications by decreasing the number of eggs (Hines, 1982, 1988). Comparison of fecundity estimates in deep-sea red crabs does not reveal great differences among species, although it can be concluded that there is a positive correlation female size and the number of eggs: 36,000–226,000 eggs (90–118 mm CW) in C. quinquedens (Hines, 1988; Steimle et al., 2001), 15,592–288,312 eggs (98–133 mm CW) in C. bicolor (Smith et al., 2004), 107,000–350,000 in C. maritae (MelvilleSmith, 1987), 160,000–375,000 eggs (110–143 mm CW) in C. fenneri (Erdman and Blake, 1988; Hines, 1988), and 199,690–566,956 eggs (105–160 mm CW) in C. affinis (present study). In all these species it has been found that fecundity increased with the increase of carapace width or wet weight of the individuals (Melville-Smith, 1987; Erdman and Blake, 1988; Hines, 1988; Steimle et al., 2001; Smith et al., 2004; present study), as also occurs in other brachyurans (Haddon, 1994; Mantelatto and Fransozo, 1997; Litulo, 2004, 2005; Arshad et al., 2006). However, in deep-sea red crabs it has been noted that these variables are not good predictors of fecundity, reaching variability values of 45–65% (C. quinquedens, C. fenneri, and C. affinis) or at least 13–26% (C. bicolor). Also, it is known crabs
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