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Draft Sexual Differentiation in Juvenile American Lobster (Homarus americanus) Journal: Canadian Journal of Zoology Manuscript ID cjz-2018-0297.R1 Manuscript Type: Note Date Submitted by the Author: 07-Mar-2019 Complete List of Authors: Comeau, Michel; Fisheries and Oceans Canada Gulf Region, Benhalima, Kadra; Fisheries and Oceans Canada Gulf Region Is your manuscript invited for consideration in a Special Issue?: Not applicable (regular submission) Keyword: vasa deferentia, ovaries, pleopods, HISTOLOGY < Discipline, Homarus americanus, American lobster https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology

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Page 1: vasa deferentia, ovaries, pleopods, HISTOLOGY < Discipline ... · differentiation of the vasa deferentia was only observed two molts later for vagile juveniles at stage XIII (for

Draft

Sexual Differentiation in Juvenile American Lobster (Homarus americanus)

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2018-0297.R1

Manuscript Type: Note

Date Submitted by the Author: 07-Mar-2019

Complete List of Authors: Comeau, Michel; Fisheries and Oceans Canada Gulf Region, Benhalima, Kadra; Fisheries and Oceans Canada Gulf Region

Is your manuscript invited for consideration in a Special

Issue?:Not applicable (regular submission)

Keyword: vasa deferentia, ovaries, pleopods, HISTOLOGY < Discipline, Homarus americanus, American lobster

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

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Sexual Differentiation in Juvenile American Lobster (Homarus americanus)

Michel Comeau and Kadra Benhalima

Gulf Fisheries Centre, Department of Fisheries and Oceans, 343 avenue de l’Université,

Moncton, New Brunswick, E1C 9B6, Canada

(corresponding author: Michel Comeau: [email protected];

tel (506) 851-6136)

Short title: lobster sex differentiation

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Michel Comeau and Kadra Benhalima

Sexual Differentiation in Early Benthic-Phase American Lobster (Homarus americanus)

Abstract

American lobsters (Homarus americanus (H. Milne Edwards, 1837)) settling to the bottom after

their pelagic larval stage are sexually undifferentiated. Based on stereomicroscope observations,

the female’s gonopore first appeared for lobsters about 13 mm carapace length (CL), and the

dimorphism of the first pair of male pleopods was first observed at about 15 mm CL. Based on

histological observations, the internal reproductive system for both males and females began

differentiating at sizes ≥ 20 mm CL. The vasa deferentia were observed for males > 30 mm CL

indicating that the male reproductive system is fully formed. For females, the ovaries were

observed at sizes ≥ 20 mm CL but the oviducts were not detected in animals < 36 mm CL. The

male reproductive system is morphologically complete at a substantially smaller CL than for

females.

Key Words: Homarus americanus, American lobster, vasa deferentia, ovaries, pleopods,

histology

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INTRODUCTION

American lobsters (Homarus americanus (H. Milne Edwards, 1837)) are reported to be sexually

undifferentiated at hatching and little is known about gonad differentiation (Talbot and Helluy

1995). The main external characteristic used to differentiate lobster sexes is the first pair of

pleopods, which are modified in males to form the gonopod for copulation and are reduced in

females to prevent attachment of eggs (Herrick 1909). This dimorphism is observed for lobsters

>10 mm carapace length (CL) (Herrick 1909). Species within the class Malacostraca, which

includes lobster, are the only crustaceans with an androgenic gland and with the sexual

differentiation under hormonal control (Charniaux-Cotton 1954, 1962; Charniaux-Cotton and

Payen 1985). The androgenic gland, located near the distal vas deferens, secretes the hormone

responsible for the differentiation of the primary (testes and vasa deferentia) and secondary

(pleopod dimorphism) sexual characters for male lobsters (Gilgan and Idler 1967). The absence

of this gland in females results in the development of ovaries (Charniaux-Cotton 1954;

Hasegawa et al. 1993).

Detailed descriptions of the anatomy and morphology of the reproductive system in both male

and female American lobsters were recently reported by Comeau and Benhalima (2018a, 2018b)

using macroscopic examinations and microscopic (light microscopy, histochemistry, and

scanning electron microscopy) techniques. However, their descriptions are based on larger adult

animals with already differentiated gonads; thus, the sexual differentiation process for lobster is

still unknown. In this study, juvenile lobsters < 37 mm CL were collected and examined to

establish their sexual-development status. Stereomicroscopy was used to establish the onset of

pleopod dimorphism and histology was used to document gonad differentiation.

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MATERIALS AND METHODS

Juvenile lobsters (n = 40; 12.0–36.0 mm CL) were collected by scuba diving in Caraquet New

Brunswick, Canada (47°50’W:64°05’N), on 10 July 2013, which was preceding the settlement of

pelagic larvae occurs. A second group (n = 77; 6.2–35.0 mm CL) was collected using bio-

collectors (Wahle et al. 2009) in Alberton Prince Edward Island, Canada (46°50’W:64°00’N), on

24 September 2013, which is just after the pelagic larvae settlement. The CL (to 0.1 mm

accuracy) of each lobster was measured using Vernier calipers. Each lobster was put in an

individual vial, placed on ice packs for an hour, and fixed with Bouin’s solution for at least 2

weeks (Gabe 1968).

In the laboratory, lobsters were washed with 70% ethanol before the stereomicroscopy

observations. The first pair of pleopods was examined to detect the onset of sexual dimorphism,

that is, for males the pleopods change shape, harden, and increase in volume. After the

stereomicroscopy observations, all lobsters were dissected and prepared for light microscopy

(Comeau and Benhalima 2018a, 2018b) to assess the differentiation of internal sexual organs.

Tissues were dehydrated in a graded series of ethanol, cleared in xylene, and embedded in

paraffin in a vacuum chamber. Blocks were sectioned longitudinally at a thickness of 5−6 µm on

a rotary microtome. Slides were stained with Masson’s trichrome (Gabe 1968) and final mounts

were made with cover glass slips using a mounting resin. Microscopic observations were

completed using an Olympus BX51® compound light microscope equipped with a RT color

digital camera (Spot Diagnostic Instrument Inc.) using bright field optics.

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We followed Lawton and Lavalli (1995) to describe the habitat use of juvenile lobsters into three

phases: shelter-restricted, emergent, and vagile. Juvenile lobsters were also grouped into stages

based on growth information (size-at-instar) from Gendron and Sainte-Marie (2006). Most larval

settlement occurs in August in the southern Gulf of St. Lawrence (Hudon et al. 1986);

consequently, young-of-the-year lobsters were only observed from the September sample

collected in Alberton.

RESULTS AND DISCUSSION

The smallest shelter-restricted juvenile lobsters were collected in Alberton and represented the

young-of-the-year in stages V−VIII with a size range of 6.2−11.8 mm CL (Table 1). No stage IX

animals were observed, resulting in a gap in the size range at 11.8−15.7 mm CL. In contrast,

Caraquet samples were collected immediately prior to the annual settlement of stage IV larvae,

and thus the young-of-the-year lobsters from the previous year (i.e., 0+-year old animals from the

2012 settlement) were in the 12.0−20.0 mm CL size range, and represented shelter-restricted

(stages VIII and IX) and emergent (stage X) juvenile lobsters.

The sexual dimorphism of the first pair of pleopods was detected for emergent juveniles at stage

X (for lobsters ≥ 15 mm CL; Fig. 1; Table 1); which means the lobsters were approximately one-

year old. Since the sexual dimorphism lasts throughout the entire lifetime of the lobster, it is the

best external character to use to distinguish between males and females. Interestingly, the first

pair of pleopod was not the first appearance of a secondary sexual characters as female’s

gonopores were observed for the first time for shelter-restricted juvenile at stage IX while the

male’s gonopores were first observed at stage X (Fig. 1b).

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Based on light microscopy, the gonadal differentiation of both emergent male and female

juveniles began at stage XI (for lobsters ≥ 20 mm CL; Table 1), which is the molt following the

pleopod dimorphism. For males, the testes with undifferentiated germinal cells containing

chromatin aggregate in the nucleus (Fig. 2a), which is the first stage of the spermatogenesis

(Comeau and Benhalima 2018a), were observed at stage XI (22 mm CL). However, the

differentiation of the vasa deferentia was only observed two molts later for vagile juveniles at

stage XIII (for males > 30 mm CL), i.e., two-year old males. Histology revealed the presence of

the vasa deferentia, muscle fibers, and connective tissue, but no lumen or activity related to the

formation of spermatophores (Fig. 2b). Simultaneously at stages XIII and XIV (35 mm CL),

follicles filled with spermatocytes connected by the collecting duct were observed in the testes

(Fig. 2c); indicating that the spermatogenesis was progressing but not completed because mature

spermatozoa were not observed (Comeau and Benhalima 2018a). It also explains why no

spermatophores were observed in the vasa deferentia. Hence, the sexual differentiation of

emergent juvenile males is initiated at stage X coinciding with the dimorphism of the first pair of

pleopods, which is three stages later than previously believed (Herrick 1909), and the presence

of gonopores. It is followed at stage XI with the differentiation of the testes, and two molts later

the vasa deferentia, suggesting that the male reproductive system is fully differentiated for vagile

juveniles at stage XIII. Finally, Conan et al. (2001) estimated the size at which 50% of male

lobsters attain maturity at 50 mm CL based on the presence spermatophores in the vasa

deferentia, suggesting that the male reproductive system is fully functional at stage XVI.

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For females, the histology revealed the presence of a germinal zone of oogonia (Fig. 2d)

suggesting that the ovaries were fully differentiated (Comeau and Benhalima 2018b) for

emergent juveniles at stage XI. However, we could not detect the oviducts linking the ovaries to

the gonopores for either emergent or vagile juvenile females in stages XI−XIV (16.0−36.0 mm

CL) indicating that their reproductive system will be fully differentiated at larger sizes, as

described by Comeau and Benhalima (2018b) for females with ovarian Stage 1 at sizes > 50 mm

CL (females in stage XVI). Nonetheless, females will only begin to produce mature oocytes and

spawn at around 60 mm CL in the southern Gulf of St. Lawrence, and the size at which 50% of

females reach the onset of maturity is around 72 mm CL (Comeau and Savoie 2002). This

indicates that the reproductive system of females is fully differentiated and functional at larger

sizes than males.

ACKNOWLEDGEMENTS

The authors wish to thank, S.A. Boudreau, J.M. Hanson, T. Surette, and one anonymous review

for thoughtful suggestions that improved the quality of this manuscript. Thanks to the biologists

and technicians from the Department of Fisheries and Oceans Canada Lobster Section from

Moncton, New Brunswick, that carried out scuba sampling and the bio-collectors sorting. Finally

a special thanks to the biologists, technicians and harvesters from the Prince Edward Island

Fisherman’s Association and the provincial government that carried out the bio-collectors

project. This research was supported through the Fisheries Science Collaborative Program from

the Department of Fisheries and Oceans.

REFERENCES

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Charniaux-Cotton, H. 1954. Découverte chez un crustacé amphipode (Orchestia gammarella)

d’une glande endocrine responsable de la différentiation des caractères sexuels primaires et

secondaires mâles. C. R. Acad. Sci. Paris, 239(13): 780–782.

Charniaux-Cotton, H. 1962. Androgenic gland of crustaceans. Gen. Comp. Endocrinol.

Supplement, 1: 241–247.

Charniaux-Cotton, H., and Payen, G. 1985. Sexual differentiation. In The biology of crustacean.

Vol. 9. Integument, pigments, and hormonal processes. Edited by D.E. Bliss and L.H.

Mantel. Academic Press. Toronto. pp. 217–299.

Comeau, M., and Benhalima, K. 2018a. Functional anatomy of the male reproductive system of

the American lobster (Homarus americanus). J. Morphol. 279(10):1431–1443. doi:

10.1002/jmor.20878.

Comeau, M., and Benhalima, K. 2018b. Functional anatomy of the female reproductive system

of the American lobster (Homarus americanus). J. Morphol. 279(11): 1603−1614. doi:

10.1002/jmor.20889.

Comeau, M., and Savoie, F. 2002. Maturity and reproductive cycle of the female American

lobster, Homarus americanus, in the southern Gulf of St. Lawrence, Canada. J. Crustac.

Biol. 22(4): 762–774.

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Conan, G.Y., Comeau, M., and Moriyasu, M. 2001. Are morphometrical approaches appropriate

to establish size at maturity for male American lobster, Homarus americanus? J. Crustac.

Biol. 21(4): 937-947.

Gabe, M. 1968. Techniques histologiques. Masson et Cie. Paris.

Gendron, L., and Sainte-Marie, B. 2006. Growth of juvenile lobster Homarus americanus off the

Magdalen Islands (Quebec, Canada) and projection of instar and age at commercial size.

Mar. Ecol. Prog. Ser. 326: 221–233.

Gilgan, M.W., and Idler, P.R. 1967. The conversion of androstenedione to testosterone by some

lobster (Homarus americanus Milner-Edwards) tissues. Gen. Comp. Endocrinol. 9(3):

319−324. doi: 10.1016/0016-6480(67)90025-1

Hasegawa, Y., Hirose, E., and Katakura, Y. 1993. Hormonal control of sexual differentiation and

reproduction in crustacea. Am. Zool. 33(3): 403–411.

Herrick, F.H. 1909. Natural history of the American lobster. Bull. U.S. Bur. Fish. Vol. 29: 149–

408.

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Hudon, C., Fradette, P., and Legendre, P. 1986. La répartition horizontale et verticale des larves

de homard (Homarus americanus) autour des Îles de la Madeleine, golfe du Saint-Laurent.

Can. J. Fish. Aquat. Sci. 43(11): 2164–2176.

Lawton, P., and Lavalli, K.L. 1995. Postlarval, juvenile, adolescent, and adult ecology. In Biology

of the lobster Homarus americanus. Edited by J.R. Factor. Academic Press. Toronto. pp.

47–88.

Talbot, P., and Helluy, S. 1995. Reproduction and embryonic development. In Biology of the

lobster Homarus americanus. Edited by J.R. Factor. Academic Press. Toronto. pp. 177–

216.

Wahle, R.A., Wilson, C., Parkhurst, M., and Bergeron, C.E. 2009. A vessel-deployed passive

postlarval collector to assess settlement of the American lobster Homarus americanus. N.

Z. J. Mar. Freshw. Res. 43(1): 465–474.

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Table 1. Number of juvenile American lobsters (Homarus americanus (H. Milne Edwards,

1837)) collected in Alberton (Prince Edward Island) and Caraquet (New Brunswick), Canada,

and the size range in carapace length (mm) for stages V to XIV observed by stereomicroscopy

and light microscopy (histology) to establish the pleopod dimorphism and the gonadal

differentiation, respectively.

Alberton Caraquet

Phase Stage Size Range n Stereo Histology n Stereo Histology

Shelter-restricted

juvenileV

6.2–6.75 No No 0 – –

VI 7.6–8.2 6 No No 0 – –

VII 8.9 2 No No 0 – –

VIII 9.4–12.0 16 No No 2 No No

IX 13.0–14.0 0 – – 7 No* No

Emergent juvenile X 15.7–20.0 21 Yes No 9 Yes No

XI 20.8–24.0 12 Yes Yes 6 Yes Yes

Vagile juvenile XII 24.9–29.2 6 Yes Yes 7 Yes Yes

XIII 30.5–34.0 8 Yes Yes† 8 Yes Yes†

XIV 35♂ and 36♀ 1 Yes Yes† 1 Yes Yes

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Note: The juvenile phase are described in Lawton and Lavalli (1995) and the stages are

based on the size-at-instar proposed by Gendron and Sainte-Marie (2006). * first appearance of

the gonopore for females; † differentiation of the vasa deferentia for males.

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Fig. 1. Stereo micrographs of (a) the ventral view of a juvenile American lobster (Homarus americanus (H. Milne Edwards, 1837)) of 10.2 mm carapace length (CL) with undifferentiated pleopods (they have been highlighted with lines), and (b) differentiated pleopods and a gonopore of a 16.0 mm CL male lobster.

Abbreviations: Go, gonopore; Pl, pleopod

203x254mm (300 x 300 DPI)

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Fig. 2. Light micrographs (Masson’s trichrome staining) of a cross section from: (a) a differentiated testis of a 20.8 mm CL male American lobster (Homarus americanus (H. Milne Edwards, 1837)); (b) a vas deferens and (c) testis with follicles of a 32.7 mm CL male lobster; and (d) a 21.2 mm carapace length (CL) female lobster with a differentiated ovary showing primary oocytes and the hepatopancreas. Abbreviations: Cd,

collecting duct; Ct, connective tissue; E, epithelium; F, follicle; H, hepatopancreas; M, muscle; N, nucleus, O, ovary; Po, primary oocyte; Sy, spermatocyte; T, testis; UGC, undifferentiated germinal cell

203x254mm (300 x 300 DPI)

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