F. Norrbin. Ultra-structural changes in the reproductive system of overwintering females of Acartia longiremis. Received: 30 August 2000 / Accepted: 4 May 2001 ...
Marine Biology (2001) 139: 697±704 DOI 10.1007/s002270100627
F. Norrbin
Ultra-structural changes in the reproductive system of overwintering females of Acartia longiremis
Received: 30 August 2000 / Accepted: 4 May 2001 / Published online: 7 July 2001 Ó Springer-Verlag 2001
Abstract As the only member of its genus, and along with only a few other marine copepod species, Acartia longiremis Lilljeborg overwinters as adult females in ``active diapause'' in the plankton. This study describes the changes in ultra-structure of the resting reproductive system from August to January, and evidence for fertilization in overwintering North Norwegian A. longiremis. Gonads in overwintering individuals remain in an undierentiated state from September to January, i.e. without oocyte development or oocyte migration into the oviduct and its diverticulae. Lipid stores in close association with the reproductive system appear to be utilized for the development of primary oocytes after the overwintering period.
Introduction Dealing with the winter season is one of the great challenges in the life history of high-latitude marine copepods. The problem is solved in several ways, the most common of which is dormancy 1of eggs and copepodites. In only a few species is the population maintained by adult, fertilized females during winter (Miller and Clemons 1988; Nñss and Nilssen 1991), and
Communicated by L. Hagerman, Helsingùr F. Norrbin Department of Marine and Freshwater Biology, The Norwegian College of Fishery Science, University of Tromsù, Breivika, 9037 Tromsù, Norway Tel.: +47-77644747 Fax: +47-77646020 1 Dormancy is a general term to describe conditions of resting. In diapause, an endogenous mechanism keeps the organism dormant, and, in quiescence, a hypometabolic state is imposed by environmental conditions (Grice and Marcus 1981; Drinkwater and Crowe 1987).
Acartia longiremis Lilljeborg is one of these (Davis 1976; Norrbin 1994). However, its internal morphology has never been described, including possible dormant ovaries and stored sperm. Acartia is a successful neritic genus of highly productive omnivores. Several species frequently coexist in an area, where the species usually appear in succession during the season. The production and hatching of dormant eggs by many Acartia species is probably responsible for appearance when the environment changes (Uye 1985). Activation can then be triggered by environmental stimuli. A. longiremis is the only species of its genus to reach subarctic areas, and is found in the fjords of Spitsbergen at 77°N. Dormant eggs have never been described for A. longiremis, but it has a cold-season (i.e. early spring) abundance peak in temperate regions, where it coexists with A. clausi (Eriksson 1973; Norrbin and BaÊmstedt, unpublished data). One interesting question arises: how does this species get such an early start without resting eggs? My hypothesis is that selection has caused dormancy to occur early in oogenesis instead of after the eggs have been deposited.. An adult female carrying a dormant ovary may be more viable, and have higher ®tness, than an exposed dormant egg in the subarctic fjords where A. longiremis is common. This may be because benthic conditions are unsuitable for diapause eggs (Lindley 1990; Norrbin 1992), or because environmental stimuli for diapause induction and/or termination, e.g. light, salinity or temperature changes (Uye and Fleminger 1976), are less eective or well-timed for dormant eggs than for the adult female. In Tromsù, A. longiremis females emerge from diapause in late January, upon the return of light after the dark season, but do not begin to lay eggs until March (Norrbin 1992, 1994). This study was made in order: (1) to ®nd out if overwintering A. longiremis have been fertilized, i.e. carry stores of sperm; and (2) to study ultrastructural changes in the reproductive system, gut or lipid stores during dormancy.
698 Table 1 Acartia longiremis. List of analyzed females, with an indication of the presence of oil and primary oocytes in the sections analyzed (NA not analyzed)
Month
Ind. no.
Analysis
Oil in 4th segment
Oil in 5th segment
Primary oocytes
Aug Sep Sep Nov Nov Jan Jan Jan Jan Jan Jan Jan Jan Jan Jan
3 13 2 11 13 2 3 4 7 8 10 11 12 14 18
Light/TEM Light Light/TEM Light/TEM Light, 1 lm section Light Light, 2 lm section Light Light Light Light Light, 1.5 lm section Light/TEM Light Light
Little + + + + + + NA + + + + + ± +
± + + NA + + + + + ± + + ± + +
+ + + ± ± ± ± ± ± ± + + + + ±
Materials and methods Zooplankton samples were taken in HaÊkùybotn, a semi-enclosed bay near Tromsù (69°40¢N; 18°45¢E), on 11 August, 13 September and 1 November 1993, and on 12 January 1994. Females of Acartia longiremis Lilljeborg were ®xed in 2.5% glutardialdehyde and 2.5% paraformaldehyde in 0.05 M cacodylate-buer, adjusted to pH 7.2. Post-®xation was made using 2% OsO4 in distilled water, rinsing before and after with Sorensen's phosphate buer. The copepods were dehydrated in ethanol, and transferred to propylene oxide before being ®nally embedded in Epon. A few nicely positioned and undamaged specimens from each sampling were chosen for sectioning (Table 1). Transverse sections were made using a Reichert± Jung Ultracut E ultramicrotome. Sections were made in the genital segment of the urosome, and in the third to ®fth thoracic segments (Fig. 1). Sections (1±2 lm) for light microscopy were used to map the position of signi®cant organs, and series of thin sections (80 nm) were taken in speci®c areas for use in transmission electron microscopy (TEM). These were stained in uranyl acetate and lead citrate before TEM. A JEOL JEM-1010 transmission electron microscope at the Institute of Medicine, University of Tromsù, was used. Sections for light microscopy of females from the January sampling (Table 1) were used to check the degree of oocyte maturation and oil content. These were photographed or drawn using a light microscope with a camera lucida. Prosome area, oil area and oocyte area were determined by cutting out the relevant areas printed on paper and weighing the pieces. Identi®cation of tissues and organelles was mainly done with reference to Hilton (1931) and Blades-Eckelbarger and Youngbluth (1984). Relative oil content in the fourth and ®fth cephalothorax segments and relative oocyte content were compared for resting females (stage A) and early maturing females (stage B) in January, using a one-factor ANOVA analysis (Zar 1984).
Maturation of ovary and oocytes The ovary was situated dorsally in the thorax, and was seen as a shallow ``U'' with the tips pointing anteriorly (Fig. 1). Developing oocytes were found in the oviduct ventrally and laterally of the ovary (Fig. 4). Early vitellogenic cells were in the peripheral layer of the oviduct, and oocytes in a later stage were in the inner layer (Fig. 5). Mitochondria, endoplasmic reticulum, vesicles for yolk synthesis, type 1 and type 2 yolk spheres, and lipid droplets were present in vitellogenic oocytes (Figs. 5, 6, 7), as also described for Labidocera aestiva (Blades-Eckelbarger and Youngbluth 1984). According to Blades-Eckelbarger and Youngbluth (1984), type 1 yolk is found in other Crustacea as well, while type 2 yolk has not been described for other Crustacea, and is formed at a later stage of vitellogenesis. Synthesis of type 1 yolk from electron-dense
Results Fertilization All analyzed specimens had stored sperm in the spermathecal sacs of the genital segment (Figs. 2, 3). The remains of cement from attached spermatophores could be observed in the gonopore (Fig. 2).
Fig. 1 Acartia longiremis female, dorsal view. Drawing shows gut (G), ovary (OV), and approximate positions of sections for TEM microscopy in this study. Transition between third and fourth cephalothorax segments; location of germinal zone of ovary (I). Transition between fourth and ®fth cephalothorax segments; maturing oocytes (II). Genital segment; spermathaeca (III)
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Fig. 2 Acartia longiremis. Genital segment of reproductive female, August. Figure shows gonopore and spermathecal sacs ®lled with sperm (sp). Sacs are multilobed chambers, walled with chitin (CH). The perimeter of one chamber is indicated with small arrows. Gonopore openings are plugged closed with cement left from spermatophore attachment (arrow). Inset shows detail of spermatheca, with individual sperm cells
were observed (Figs. 9, 10, 11). The boundaries between adjacent oocytes were not clear, as in a syncytium. Numerous multilamellar structures (R. Myklebust,
Fig. 3 Acartia longiremis. Spermathecal sac of overwintering female, January. Detail, showing sperm (sp) packed between the chitinous walls (CH) of the members
granules could be observed in cisternae of the endoplasmic reticulum (Fig. 6), as described by BladesEckelbarger and Youngbluth (1984). Fully reproductive females were found in August and September. These had maturing oocytes in the oviduct (Figs. 4, 5), and a small lipid body between the vitellogenic oocytes and the ovary (Fig. 4). During the passage of the oocytes, the gut was sometimes partially closed. Laid eggs are ca. 85 lm in diameter (personal observations), i.e. relatively large compared to female size (Fig. 4). Oogonia of the ovary had numerous mitochondria in the cytoplasm (Fig. 5). November and January females had no oocytes in the oviducts, but large oil sacs adjacent to the ovary (Figs. 8, 9). The ovary seemed less compact than in the August specimen, and fewer mitochondria were seen in the cytoplasm (Fig. 8). A section from the fourth thoracic segment in one January female (no. 12) shows a large lipid body, and a group of primary oocytes, in the oviducts (Fig. 9). Sevxzeral rounded, empty spaces between the oocytes
Fig. 4 Acartia longiremis. Fifth thoracic segment of reproductive female, August. Transverse section, overview. Most of the body cavity is ®lled with two ripe oocytes (oc). Also visible are dorsal and ventral longitudinal muscles, and lateral transversal muscles (M), gut (G), and ventral nerve chord (VNC)
700
701 b
Fig. 5 Acartia longiremis. Fourth thoracic segment of reproductive female, August, showing dorsal ovary (OV), vitellogenic oocytes in dierent stages of development, and follicle cell (fc). Oocyte with large nucleus (N) has numerous mitochondria (m) and empty vesicles that are associated with type 2 yolk formation (unlabelled arrows). Oocytes in the lower lefthand corner are more advanced, with pale type 2 (2) yolk bodies, smaller and darker type 1 yolk bodies (1), and dark lipid droplets Fig. 6 Acartia longiremis. Detail of oocyte, September, showing type 1 yolk formation from granules in cisternae of the endoplasmic reticulum (er; arrows) Fig. 7 Acartia longiremis. Detail of oocyte, August. Type 1 and type 2 yolk bodies, and lipid droplets (L)
personal communication) could be observed in the cytoplasm (Fig. 11). Lipid storage and utilization In the reproductive female of August, there was only a small body of fat, next to the ovary (Fig. 5). Lipid bodies in November and January specimens were large and situated dorsally of the gut, and below the dorsal muscle groups and ovary (Fig. 8). Fig. 8 Acartia longiremis. Germinal zone in ovary of resting female, November. Ovary (OV) is situated dorsally of large fat body (L) and below longitudinal muscles (M). Note nucleus (N) of fat cell to the left. Inset shows detail of ovary, with oogonia
In January females, a visual examination of relative oil content showed a dierence between immature specimens without primary oocytes (stage A), and maturing specimens with oocytes (stage B). A one-factor ANOVA analysis revealed a signi®cant dierence in oil content between immature and maturing females in the fourth segment, but not in the ®fth (n=9, df=7, F=7.701; P
