INTRODUCTION
Intersexuality is a rare phenomenon defined as the presence of both male and female reproductive tissue in the same gonad (ovotestis); it is interesting from a biological point of view and gives rise to many questions. The occurrence of intersexuality has been reported in many species of bony fish (Atz, Reference Atz, Armstrong and Marshall1964; Devlin & Nagahama, Reference Devlin and Nagahama2002; Sadovy de Mitcheson & Liu, Reference Sadovy de Mitcheson and Liu2008; Bahamonde et al., Reference Bahamonde, Munkittrick and Martyniuk2013), but it is not typical for marine fish of northern latitudes and has not been confirmed for walleye pollock.
Theragra chalcogramma is quite important for global commercial fishing of the northern hemisphere. Reproduction of walleye pollock has been investigated by many authors (e.g. Hinckley, Reference Hinckley1987; Stahl & Kruse, Reference Stahl, Kruse, Kruse, Drinkwater, Ianelli, Link, Stram, Wespestad and Woodby2008). Most members of the family Gadidae are gonochoristic: with individuals developing into males or females only, and remaining the same sex throughout their life spans (Devlin & Nagahama, Reference Devlin and Nagahama2002; Sadovy de Mitcheson & Liu, Reference Sadovy de Mitcheson and Liu2008).
Rare cases of Gadiformes intersexuality have been previously observed by chance in the field in Merluccius merluccius hubbsi (Ciechomski & Christiansen, Reference Ciechomski and Christiansen1968), Merluccius Merluccius (Belhoucine et al., Reference Belhoucine, Bouderbala, Flower, Francour and Boutiba2012) and Gadus morhua (Hefford, 1908).
In recent years, analyses of large-scale annual commercial catches have detected a constant presence of intersex in walleye pollock. This paper describes the morphology of intersex gonads at the macro- and microscopic level and reports the presence of intersexuality in walleye pollock.
MATERIALS AND METHODS
As part of a fishing expedition, five hermaphrodite specimens were collected from 1987 to 2006, and ten specimens from 2007 to 2014 in the Bering and Okhotsk seas. Each individual was measured for total length, and total and gutted weight. The gonads were harvested and fixed in 10% formalin solution for further investigation in a laboratory. A cross-section from the examined gonad was embedded in paraffin using conventional techniques, sectioned at 5 μm and stained with hematoxylin and Ehrlich's eosin. The Leica DMLC microscope and Leica DC 100 digital camera were used for photography and microscopy. For the examination of walleye pollock gonads, we used a classification scheme modified from Almeida et al. (Reference Almeida, Kristoffersen, Taranger and Schulz2008) and Stahl & Kruse (Reference Stahl, Kruse, Kruse, Drinkwater, Ianelli, Link, Stram, Wespestad and Woodby2008).
RESULTS
The occurrence of intersexuality in walleye pollock based on observations came from annual commercial fishing specimens. The intersex individuals were observed at low but steady rates, in numbers ranging from 3 to 16 per 10,00,000. The length of such individuals (N = 13) varied from 44 to 53.5 cm, the weight range was 610–970 g and the gonadal weights were 23–134 g. The largest recorded specimen was 55 cm long, weighed 1250 g and its gonads weighed 58 g. The smallest was 33 cm long, weighed 220 g and its gonads weighed 1.36 g. The gonadosomatic index of all individuals ranged from 4.6 to 16.9%; the liver somatic index varied from 5.4 to 7.7. The age range was 6–10 y.
The bisexual gonads were delimited (Sadovy & Shapiro, Reference Sadovy and Shapiro1987) and comprised unsymmetrical ovaries, to which several testes were attached via connective tissue ducts. The testicular and ovarian tissues connected in several unique ways. In five cases, the testes were attached to the caudal sides of both ovaries, and in four cases, to the cranial sides. The following gonadal connections were observed only once: two testes connected to the cranial and caudal sides of a single ovary; four testes attached to the cranial and caudal sides of two ovaries; two testes connected to the ventral side of one of the ovaries; two testes attached near the oviduct; one testis connected to the caudal side of the right ovary and two testes with cranial and caudal sides of the left ovary; testes were in the cranial part of both ovaries, at the caudal side of the right ovary and near the oviduct. The number of testes ranged from two to four, but in most cases it was two (Figure 1).
Macroscopic characteristics of Theragra chalcogramma intersex individuals with ovary (O), testis (T), spermatoduct (arrow) and oviduct (arrowhead). Scale bars: 1 cm.
Normally, developing germ cells were present in both ovarian and testicular tissues. Oocytes at different stages of development were present within the ovigerous lamellae of ovotestis: peri-nucleus, primary, secondary and tertiary yolk oocytes (Figure 2). No differences were identified in oocyte composition and condition of the left and right ovaries. All the testes of one specimen were also equally developed. The testes contained spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa (Figure 2A). The medial areas of the testes were more mature than peripheral regions.
Microscopic characteristics of Theragra chalcogramma intersex gonad: A. Peri-nucleus (PN), tertiary yolk oocytes (TY), ovarian wall (W) and testicular tissues (T); B. Oocyte resorption area near testicular and ovarian tissue junctions showing primary yolk (PY), secondary yolk (SY), peri-nucleus (PN) oocytes and atretic eggs (A). Scale bars: A–B, 100 μm.
In some ovaries, the majority of oocytes were filled with yolk (tertiary yolk stage), concurrently with the testis containing a significant portion of mature sperm. In other cases, a large number of oocytes had reached the secondary yolk stage, whilst the testis contained a small amount of mature sperm. In one specimen, yolk vesicle oocytes predominated along with a small amount of peri-nucleus oocytes; the testicular tissues contained spermatogonia and spermatids. The state of maturity of both male and female intersexual gonads matched normal individuals from the same catch.
In all cases, the spermatoducts developed normally, branching out from the testes, growing into the ovarian wall and then going around it, without opening in the ovarian cavity. There was no evidence of spermatoduct and oviduct connections in any of the examined specimens, apparently these ducts open separately.
Proximal to the testes, oocyte resorption areas were present in ovigerous lamellae. The atretic eggs comprised stratified connective tissue encapsulating a resorbed zona radiata. Among the atretic eggs, a peri-nucleus, and sometimes yolk oocytes, were spread diffusely (Figure 2B). Oocyte resorption areas were localized only near testicular and ovarian tissue junctions.
DISCUSSION
The vast majority of walleye pollock specimens (with normally developing gonads) begin to spawn in the North Pacific regions when they are 4–5 years old and reach a length of 38–42 cm (Shaw & McFarlane, Reference Shaw and McFarlane1986; Fadeyev, Reference Fadeyev1989). Since the investigated intersex specimens were 6–10 years old, if normal gonadal development is assumed, it is likely that they have participated in spawning a number of times. However, we cannot confirm the presence of functional hermaphroditism in walleye pollock unless specimens are caught before spawning or in an obviously spent condition.
The intersex individuals were characterized by normal development of the testes and ovaries, with the exception of ovarian areas proximal to testes, where the process of oocyte maturation was disrupted. Total and partial oocyte atresia also occurs in normally developing walleye pollock females from the Bering and Okhotsk seas (Privalikhin, Reference Privalikhin2003). In this case, we cannot rule out the possibility that the proximity of the testis had a hormonal impact on these oocytes as other and more distant (to the testes) areas of the ovary had no atresia.
Intersex gonads of Theragra chalcogramma had similar morphological features to other Gadiformes: testicular and ovarian partitioning in the same gonad, sex cell maturation and differentiation occurring at a similar rate and stage as in males and females caught that season, the testis displayed normal spermatic development and advanced oocyte degeneration was noted in the ovaries (Ciechomski & Christiansen, Reference Ciechomski and Christiansen1968; Haugen et al., Reference Haugen, Andersson, Norberg and Taranger2011; Belhoucine et al., Reference Belhoucine, Bouderbala, Flower, Francour and Boutiba2012).
Most fish species are labile in their phenotypic sex, whereby exposure to sex steroids during a set development time window can override the genetic sex (Devlin & Nagahama, Reference Devlin and Nagahama2002); with steroid exposure resulting in Gadus morhua hermaphrodites (Haugen et al., Reference Haugen, Andersson, Norberg and Taranger2011).
The occurrence of intersexes may represent a natural level of intersexuality in the walleye pollock population. Contrariwise, the appearance of such individuals in natural populations as well as other gonadal changes (e.g. oocyte resorption) are a response to changing environmental factors and serve as indicators of environmental stress load on the population (Jobling et al., Reference Jobling, Nolan, Tyler, Brighty and Sumpter1998; Blazer, Reference Blazer2002).
Our study extends the understanding of sexual development in walleye pollock, which is an important commercial species in the North Pacific. The results require further, more in-depth study, alongside further measurements in ecosystem changes at the population, as well as at individual, levels.
ACKNOWLEDGEMENTS
We wish to thank the contributions of the staff at the Kamchatka Research Institute of Fisheries and Oceanography (KamchatNIRO), the Pacific Research Fisheries Centre (TINRO-Centre) for their assistance with collecting the material and Dmitry Skarin for style corrections to the paper.
