Sexual Differentiation and Variations Sexual Characteristics Rapana venosa (Valenciennes, 1846)  

Igor P. Bondarev
Benthos Ecology Department, Institute of Biology of the Southern Seas (IBSS), Nakhimov av., 2, Sevastopol, 299011, Russian Federation
Author    Correspondence author
International Journal of Marine Science, 2015, Vol. 5, No. 19   doi: 10.5376/ijms.2015.05.0019
Received: 26 Feb., 2015    Accepted: 15 Mar., 2015    Published: 25 Mar., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Bondarev, 2015, Sexual differentiation and variations sexual characteristics Rapana venosa (Valenciennes, 1846), International Journal of Marine Science, Vol.5, No.19 1-10 (doi: 10.5376/ijms.2015.05.0019)

Abstract

For the first time provides photographic images, and discussed a wide range of normal and abnormal colors of gonads Rapana venosa from the Black Sea. It has been shown that some abnormal variants of gonads color are becoming the norm, at least for some contemporary populations of Crimea. The dependence of the change in color gonads with age of the rapa-whelk is shown. Attention is paid to the color variations of the digestive gland and their relationship to the color of gonads. It is assumed that specified color changes are evidence of environmentally caused of the species’ form-generation in the evolution process. The species intersexuality and absence of imposex females in the R. venosa Crimean populations is discussed.

Keywords
Anomaly; Ecology; Evolution; Gonad color; Sexual differences; Rapana venosa; Imposex females

A large predatory gastropod Rapana venosa was accidentally introduced from the native range (North West Pacific seas) in the Black Sea in the early 1940s (Chuhchin, 1961b, Bondarev, 2014). High ecological plasticity of the species and a favorable complex of environmental factors allowed R. venosa to colonize the Black Sea coastal waters fast enough and continue resettlement through the Mediterranean basin in the various areas of the eastern and western Atlantic (Chuhchin, 1984, Mann et al., 2004, 2006, Lanfrankoni et al, 2009, Bondarev, 2010, Bondarev, 2014). The need for adaptation to different environmental conditions, as well as significant changes in the ecosystem of the Black Sea have occurred since the time of invasion (Bondarev, 2014) led to a diverse morphogenesis (Bondarev, 2010, Bondarev, 2013). First of all morphogenesis appeared in conchological changes, but as shown in the present study, the internal organs of R. venosa also changing. Most importantly these changes relate to the gonads which are color-differentiation of the sexes.
Our present studies found wider spectrum of color gonads of both males and females, than is available in previously published material (Chuhchin, 1961 a, 1984, Mann et al., 2006, Micu et al., 2009, Lanfrankoni et al., 2009, Saglam and Duzgunes, 2014). Moreover it is shown that the diversity of these colors is not (necessarily) associated with intersex status of individuals. Absence or presence of imposex females analyzed against environmental and physiological information. For comparison, we studied samples from several places that differ in their orography and location relative to the port city of Sevastopol (Figure 1) which is a source of seawater pollution. The results are compared with existing data from other R. venosa habitats.


Figure 1 Sampling areas: 1 – Round bay, 2 – Blue bay, 3 – Theothan cap, 4 – Laspi bay

The main purpose of this paper is description and image-fixing of normal and abnormal colors of
R. venosagonads and some other soft parts existing diversity and discuss possible reasons for extensions and change their color spectrum. This baseline information will allow to monitor the further evolution of the species and to identify directions of further research.
1 Results
Rapana venosa is dioecious mollusk with well-defined external sexual characteristics. Its reproductive system adapted to internal fertilization and female laying leatherback cocoons. For the fertilization male has big size penis is usually longer than 2cm. Gonads of males and females differs in color, occupy the upper part of the visceral bag lying on the digestive gland (Figure 2).


Figure 2 R. venosa soft body with base color of gonad: M – male with dark orange gonad, F – female with yellow gonad; gon – gonad, dg – digestive gland, p – penis

The first detailed description of the rapa-whelk gonads with the sex differences in color indication, gonads’ internal structure and stages of development of germ cells was performed by V.D. Chuhchin on the samplings from the Sevastopol bay of the Black Sea (Chuhchin, 1961a). He also showed that R. venosa (misnamed Rapana bezoar L.) reaches sexual maturity at the age of 2+ years (Chuhchin, 1961a, b). At any time of year the rapa-whelk ovaries contain significant amounts of mature eggs. Changes only the volume of the gonads and the number of young oocytes; gonad never completely released from eggs. In mature females ovary is yellow and presented in the form of a dense compact mass, its thickness reaches 3-6mm (Chuhchin, 1961a, 1970, 1984).
With regard to female gonads color there are no discrepancies between other researchers who have studied the R. venosa in different areas also. In the Uruguayan coastal zone of Rio de la Platafemales was determined by the presence of gonophores and yellow gonads (Lanfrankoni et al., 2009). In the south-eastern Black Sea ofTurkey the color of the females’ gonad specified as yellowish (Saglam and Duzgunes, 2014). In the western part of the Black Sea off the coast of Romania (Agigea littoral) thefemales (with no penis) had yellow gonads (Micu et al., 2009). Rapa whelk female gonads in Chesapeake Bay were completely yellow in color with special indication for “no penis” females (Mann et al., 2006). That qualification “no penis” is because of besides true females in Chesapeake Bay and in the Romanian Black Sea Agigea littoral area as well there were imposex females registered.
R. venosa imposex females in Chesapeake Bay had penis lengths ranging from 1.9mm to 25mm and gonads that were either completely yellow or a mix of yellow and pale orange (Mann et al., 2006). In the Black Sea Agigea littoral area imposex females had penis (1.14mm-6.94mm) and yellow-orange gonads (Micu et al., 2009). Thus the color of the gonads of imposex females individuals approaching the color of the gonads of young males by (Chuhchin, 1961a) and it can be assumed that this is a consequence of masculinization. Number of imposex females (IF) in the western part of the Black Sea is approximately 30% the number of females (F: IF = 3.26:1) after (Micu et al., 2009), whereas in 4 out of 5 Chesapeake Baygeographic regions examined the preponderance of imposex females over ‘normal’ females (penis length = 0) by at least a 2:1 ratio (Mann et al., 2006).
In our samplings the presence of even one female with minimum penis size was not found. And the vast majority of females had gonads in various shades of yellow (Figure 3).


Figure 3 R. venosa female soft bodies with normal gonad color variants 2014 sampling areas (1) and (2) with indication of individual age and the shell length: A-D – Blue bay (2), E-G – Round bay (1), A – 4 years old, 60.0mm, B - 5 years, 58.0mm, C – 7 years, 40.5mm, D – 8 years, 45.5mm, E – 10 years, 42.5mm, F - 10 years, 75.0mm, G – 12 years, 64.1mm

Females with abnormal coloration of gonads in our collections were found sporadically but the variety of colors is large enough (Figure 4) corresponding in some individuals’ gonads normal (Figures 7, 8) or abnormal males (Figure 5).


Figure 4 R. venosa female soft bodies with abnormal gonad color variants of Blue bay 2012 (A, F) and 2014 (B-E, G) sampling with indication of individual age and the shell length: A – gonad cream with light orange staining, 6 years, 63.0mm, B – orange with yellow grains, 5 years, 60.2mm, C – orange with yellow grains, 8 years, 73.1mm, D – beige, 8 years, 68.0mm, E – dark orange, 6 years, 66mm.2, F – brick-red, 8 years, 68,5mm, G – rubicund, 7 years, 60.5mm


Figure 5 Rapana venosa abnormal males with indication of penis degree, gonad color, individual age and the shell length: A - with a strongly reduced penis, beige color of the gonads, 3 years, 48.2mm, B - slightly reduced penis, brick-red gonads, 3 years, 54.1mm, C - normally developed penis, maroon gonads, 8 years, 66.8mm

In individuals with abnormal coloration of gonads there were no conchological differences. All females with abnormal coloration of gonads were aged at least 5 years which corresponds the observation made for imposex females were not among young aged individuals (Mann et al., 2006). At the same time abnormal coloration of gonads among males in our collections marked the age of 3 years. And given the fact that in our samplings for the study of the gonads individuals aged 2 years or less among both sexes were less than 5%, then the question of the age of the initial formation of abnormal coloration remains open.
Several individuals with reduced penis and abnormal coloration of gonads of our collections (Figures 5 A, B) were males. Studying under the microscope showed that the gonads of these specimens a significant difference in color contain only male sex cells.
At any time of the year the male vas deferens is filled with matured sperm and in testis germ cells are found in all stages of development. In the two-year male gonads are yellow or yellow-orange and testis of mature males have light or dark orange color (Chuhchin, 1961a, 1970, 1984). In the late 1990s - early 2000s the color of the R. venosa males gonads in the Black Sea match this description (Figure 2) according to our unpublished observations.
Rapana venosa male gonads in Chesapeake Bay were bright orange-red (Mann et al., 2006), reddish-brown in the Uruguayan coastal zone of Rio de la Plata (Lanfrankoni et al., 2009), and red-brown gonads from Romanian Agigea coast (Micu et al., 2009) that is also close to basic description of specimen from Sevastopol Bay (Chuhchin, 1961a, 1970, 1984). Quite different is brownish for males’ color of the gonad of Rapana venosa from the south-eastern Black Sea ofTurkey (Saglam and Duzgunes, 2014).
In 2012 R. venosa male with gonad dark brown almost black in color (Figure 6 L) was found. Further investigation showed that the dark brown “abnormal” color actually was normal for males older than 6 years of the studied populations in 2012-2014 bays (Figure 6 F-L, 8K, L, M) and does not occur in younger individuals. Very dark brown to black-brown gonads are characteristic specimens older than 8-10 years (Figures 6 K, L; 8Q, R, S). Young adult males age 2+ - 3-4 years gonads much lighter, they are painted in cream and beige or light brown tone (Figures 6 A, B; 8F, G).


Figure 6 R. venosa males’ soft bodies of Round (A-C, L) and Laspi (D-K) bays 2014 sampling with indication of individual age and the shell length: A – 3 years, 43.1mm, B – 4, years 58.2mm, C – 4 years, 57.5mm, D – 5 years, 61.5mm, E – 6 years, 46.5mm, F – 6 years, 59.2mm, G – years 6, 66.0mm, H – 6 years, 46.5mm, I – 7 years, 53.5mm, J – 8 years, 70.1mm, K – 8 years, 50.4mm, L – 10 years, 52.1mm

Enhancing the intensity of brown color gonads with advancing age is a trend for the majority of the Crimean R. venosa populations at the present stage of the species development (Figures 6, 8). Moreover, it was found that the previously typical males’ gonads orange color began seldom met after 2012 for the local populations inhabiting on the sandy bottom where the main nutrition object is bivalve Chamelea gallina (L., 1758).
The population, which still prevail R. venosa males with red and orange colors and shades of gonads (Figure 7) inhabits the rocky surface Cape Theophane where Rapana feeds mainly on bivalves Mytilus galloprovincialis Lam., 1819 and Mytilaster lineatus (Gmelin, 1791).


Figure 7 R. venosa males’ soft bodies of cap Theothan 2014 sampling with indication of individual age and the shell length: A – 3 year, 48.2mm, B – 3 years, 54.0mm, C – 8 years, 66.8mm

Orange-red part of the spectrum is present in the color of the Rapana gonads from sandy bays as an impurity to brown, creating a sufficiently large variety of shades (Figure 8). It is possible to trace the two color lines in the color of the gonads, characterized by the presence and amount of impurities to the basic brown of red and orange hues (Figure 8). It is possible that such a pattern characteristic of Blue Bay population as a result of interaction with the located nearby Cape Theophane rocks’ biocoenosis. The presence of numerous transitional variants in the color of the male gonads Rapana does not allow estimating the proportion of individuals of each base color concretely in number. You can only talk about a distinct change in the original orange to brown and the predominance of the last color on the current stage of development of individual populations. Color palette shown in Figure 8 reflects the current ratio for 2014 in Blue bay.


Figure 8 R. venosa males’ soft bodies of Blue bay 2012-14 sampling with indication of individual age and the shell length: A – 3 years, 51.5mm, B – 3, years 51.0mm, C – 3 years, 51.1mm, D – 3 years, 47.0mm, E – 4 years, 55.1mm, F – 4 years, 53.5mm, G – 4 years, 43.2mm, H – years 5, 51.2mm, I – 5 years, 52.2mm, J – 5 years, 58.8mm, K – 5 years, 52.0mm, L – 6 years, 56.2mm, M – 6 years, 39.6mm, N – 7 years, 43.0mm, O – 6 years, 55.0mm, P – 7 years, 71.1mm, Q – 8 years, 64.1mm, R – 8 years, 45.5mm, S – 9 years, 49.9mm

An obvious connection color gonad of individuals with shell color is not established. Although previously known differences in the prevalence of light-colored individuals inhabiting sandy bottom in comparison with on rock living ones (Savini et al, 2004, Bondarev, 2010) can be linked to a predominance of brown gonads just at R. venosa of sandy bays. The weakening of the surface shell color up to albinism is often associated with age also correlated with age-connected increasing the intensity of brown color gonads noted above. It is possible that the lack of melanin in the shell inversely related to its large number of soft tissue.
Marked by a clear link between R. venosa male gonads color intensity and color of the digestive gland. This connection is direct - the darker the gonads, the more intense the color of the digestive gland. And at gerontic individuals of “brown color line” the digestive gland can almost merge with the color black-brown gonads (Figures 6 K, L). In females between the color of the gonad and digestive gland so explicit dependence is not established, but noted that more young individuals are usually lighter in color of the digestive gland.
2 Discussion
Pay attention to the description of the male gonad color for the Black Sea by various authors in chronological order. Light- or dark- orange color (Chuhchin, 1961a, 1970, 1984), red-brown (Micu et al., 2009), brownish (Saglam and Duzgunes, 2014) and from beige to dark brown almost black in color according to our study. Definitely we deal with a trend change color spectrum gonads though the original and transitional color options also take place in contemporary populations.
Shown above dependence of the amplification in the color intensity of the gonad and digestive gland of males from the individuals’ age in common with data of age-related changes color vas deferens: "Seminal duct is a whitish winding tube ... The old Rapana vas deferens becomes dark brown" (Chuhchin, 1961a). Probably is talk about the accumulation of pigments in the generative tissues with increasing age. The same can be said of the digestive gland.
Stained tissues depend on the composition and amount of pigments. In 2009 studies were conducted on the number and composition of carotenoids in the tissues of R. venosa. Thin layer chromatography (TLC) chromatograms fractions were labeled with the following pigments and their mobilities (Rf): 1- yellow (0.88), 2- pink and yellow (0.53 - 0.48), 3- pink (0.35), 4 - yellow double (0.33), 5 - red (0.31). Analysis of the average distribution of carotenoids in the tissues of R. venosa showed that most of them fell on the gonads of both sexes mollusks (61-65%). Rf indicators suggest that they are present: 1 - β-carotene, 3 - pectenolon, 4 - a mixture diatoxanthin and alloxanthin, 5 - mitiloxsanthin. In generative tissue R. venosa alloxanthin clearly dominates in the amount with their esters it is more than 90% of carotenoids (Shulman et al, 2014).
Analysis of the average distribution of carotenoids in the tissues of R. venosa showed that the main proportion (61-65%) accounted for the gonads of both sexes molluscs. During spawning, the carotenoid content in the gonads of males maximum and almost 10 times their values in females. The main part of the carotenoids in the remaining tissues in the digestive gland concentrated where their contents are almost 2 times lower than in the gonads. The level of carotenoids in the foot clam was negligible (3-9% of the amount of carotenoids) (Shulman et al, 2014). These data are consistent with the visual evaluation of the ratio of pigments in various tissues of different sexes at the time of research. Obviously, the formerly it is a pigment carotenoids, determines the color of tissue R. venosa and the color of the gonads most of all. Pink and even red color corresponding to specific carotenoids on TLC chromatograms, probably determine the color of the gonads of females anomaly (Figure 4). Various shades of brown and somewhat red in color gonad and digestive gland are likely determined by melanin. One of the probable reasons for changing the composition of pigments in R. venosa is a change in the spectrum of power that occurred in the majority of the population of Crimea in the late 1990s - early 2000s (Bondarev, 2010, 2014).
Trophic system, "mollusc-filter feeder → mollusc predator" is considered as an example of carotenoids bivalves and R. venosa. Basic Rapana carotenoids coincide with those of the mussels, in this connection it may be assumed that their presence is due normal accumulation rather than metabolic transformation. Prevailing in the Rapana gonads alloxanthin is typical algal pigment and get it predatory R. venosa can only when feeding bivalves. The initial R. venosa main food - M. galloprovincialis identified 20 carotenoids (about 99% of composition), in R. venosa - only 6 carotenoids (about 85% of the composition). A significant part of the mussel carotenoids underwent metabolic transformation R. venosa (Shulman et al, 2014).
The population which is dominated by the colors and shades of orange-red "carotenoid line» gonads (Figure 7) inhabits the rocky surface Cape Theophane where R. venosa feeds mainly bivalves genera Mytilus and Mytilaster. It is obvious that this explains the predominance of males with orange-red-brownish gonads in the area even in 2014. The presence of single males with dark brown gonads in this local population can be explained that these individuals previously lived on sand bottom and feed mainly on Ch. gallina. In favor of this version is not typical of rock populations light color shells these single individuals but is characteristic of populations living on sandy bottom. In turn, the presence in the Blue Bay population of individuals “carotene line” (Figure 8, left) is a consequence of interaction with adjacent local populations and ecological communities living on the rocks.
In the investigated bays in R. venosa food range prevail bivalves Veneridae and Cardiidae, average size of about 1.5 cm that essentially limits the growth of the predator, and also provide the specific composition of the pigments. Data on quantitative and qualitative composition of pigments are absent but it is possible that they are differ significantly from pigments M. galloprovincialis and this is the cause of the gonads color change and the formation of brown "melanin line" (Figure 6, 8, right side). Biochemical differences in ecologically different groups of bivalves allow us to assume because in Anadara kagoshimensis (Tokunaga, 1906) (misnamed A. inaequivalvis) there are only 6 of carotenoids (more than 90% of the composition) against 20 in M. galloprovincialis (Shulman et al, 2014). A. kagoshimensis – invader that takes in the Black Sea an increasingly significant role is now one of the main R. venosa food objects in some areas.
However, R. venosa internally switched to nutrition bivalves inhabitants sandy bottom about 15 years ago when en masse Rapana with basic brown gonads are marked only after 2012. Fact that changes in the color of the gonads occurred with sufficiently long delay of approximately 10-15 years, possibly indicative of their threshold manner. This threshold obviously associated with a qualitative leap in the R. venosa biochemical processes providing quantitative predominance of melanin (?) over the carotenoids. To confirm or refute this version is necessary to conduct studies with brown pigmented gonads Rapana and pray bivalves Veneridae and Cardiidae also.
Probably males "melanin" line appeared singly before but have not been marked since in various types of studies to determine the sex is not necessary to extract from the shell of the soft body entirely. The presence of a large penis allows for sexual differentiation without gonads, which are located in the upper part of the shell spire and frequent breakages remain inside. The fact that Turkish researchers (Saglam and Duzgunes, 2014) identified the male gonad color as “brownish” on 2004 samplings says that at least in the shade of the gonads at the time was already marked brown.
It is difficult to say how much change in the spectrum color gonads is irreversible but it’s possible to say that this phenomenon is fairly widespread and is not a casual anomaly. Perhaps we are dealing with a steady R. venosa evolutionary change having an ecological basis.
One explanation for the presence of abnormal colors females mainly in the Blue bay is greater number of examined specimens (260) than in the other our regions combined (115 specimens). Variations in the color of the gonads of females associated with their imposex status (Mann et al., 2006, Micu et al., 2009). In our Crimean samplings imposex females were not found is not the result of inadequate sampling because sex determination by the presence of the penis without an accent on the gonads were held in more than 1,500 specimens of R. venosa. The lack of mention of imposex females by Chuhchin V.D. who described in detail the Rapana differences in the gonads and studied of intersex phenomenon in other species of the Black Sea gastropods (Chuhchin, 1961a, b, 1984) means that the Rapana intersex individuals were absent before of Sevastopol bay.
Considered to that the appearance of intersex individuals is due to unfavorable environmental conditions and, above all, the presence of various types of pollutants. Observations of small-sized gastropods genus Rissoa the free-water coast and in the clear-water bays females with a penis are rare whereas in polluted bays (Sevastopol bay) almost all females Rissoa splendida Eichwald, 1830 have underdeveloped penis. Strengthening masculinization populations in polluted bays is happened when the deterioration of living conditions in the Black Sea Rissoa parva (da Costa, 1778) and R. membranacea (J. Adams, 1800) also (Chuhchin, 1984).
Since the development of imposex in gastropods is related to cumulative exposure to tributyltin (TBT) and related chemical compounds (e.g. Smith 1971, Gibbs et al. 1987, 1988, Bryan and Gibbs 1991, Mensink et al. 1996, Oberdorster & McClellan-Green 2002, Stickle and Zhang 2003, all after Mann et al., 2006), it is possible that female whelks are younger than imposex female whelks and have thus been exposed to environmental sources of TBT for less time. TBT is a documented environmental pollutant in the Chesapeake Bay (Mann et al., 2006).
The age range of the female population we investigated was 2-11 years which almost corresponds to the limits of life of more than 98% R. venosa. Thus the age of the individuals is not a reason for the lack imposex females in our samplings.
A special study on the presence and concentration of TBT which is a main ingredient of marine antifouling paints in the waters of the Crimea was not conducted. However Sevastopol is a large navy, civilian cargo and fishing port with a large number of small boats. Thus the number of TBT-painted boats must be significant and it can be assumed that the concentration of TBT in the adjoining areas should be high.
The level of pollution lindane, DDT and DDD in mollusks from the Romanian region Mamaia (Badiu et al, 2008) corresponded to the average values of the range of concentrations of the sum of α-, β- and γ-hexachlorocyclohexane isomers, as well as pesticide DDT in the Round bay. And the maximum values of the above pollutants in mollusks of the Round bay 1.5- 2 times or more above (Malakhova et al, 2014).
Sampling locations for our research have sufficient ecological differences to evaluate in general terms the dependence of potential probability of occurrence R. venosa intersex from pollution. The purest is Laspi Bay (area 4) when the Blue Bay (2) and Cape Theophane (3) areas have intermediate indicators of pollution and the most exposed of household water pollution Bay Round (area 1). Sevastopol bay in which sampled Chuhchin (1961a, b) is even more disadvantaged in terms of environmentally contaminated areas than the Round bay. Regardless of the degree of chemical contamination in any of our areas of research were not found imposex females. It does not speak in favor of dependence the presence imposex females in R. venosa from contamination.
Judging by the fact that the Turkish researchers (Saglam and Duzgunes, 2014) divided R. venosa for males and females without mentioning imposex females one can assume that this category was missing from collections in the area. No reported observations of imposex in rapa whelks from native populations in Korea (Chung et al. 1993) also could be evidence of their absence.
It is believed that the lack of imposex signs in females could be interpreted as a symptom of a healthy population
or a signal that this population had not been exposed enough time to TBT to develop signs of imposex Uruguayan Rio de la Plata coastal zone (Lanfrankoni et al, 2009). It is possible that Korean, Turkish and Uruguayan waters are cleaner than the waters of Chesapeake Bay (USA) and Romania coast but it hardly applies to the waters adjacent to Sevastopol. The health of the population can be judged by the physiological condition of the individual that indicators are the weight and size of individuals. Table 1 shows the weight and size of individuals from different areas. It is easy to notice that the size-weight characteristics of individuals from areas where registered imposex females exceed the corresponding parameters in most areas where such individuals were not found. And in the Chesapeake Bay area which the preponderance of imposex females were over ‘normal’ females by at least a 2:1 ratio (Mann et al, 2006) size-weight indicators are higher significantly (Table 1).


Table 1 Some comparative characteristics for R. venosa mature specimens of the investigated areas (according Figure 1) and other regions after the following authors: Black Sea, Turkey [Saglam and Duzgunes, 2014], Agigea coast, Romania [Micu et al., 2009], Uruguayan Rio de la Platacoastal zone [Lanfranconi et al, 2009], Chesapeake Bay, USA [Mann et al., 2006]. SL – shell length (mm), W – whelk total wet weight (g), TW - whelk wet tissue weight (g), Imposex, (% of total specimens), ND – no data

The fact that shell lengths and wet tissue weights (g) of both imposex females and males were approximately the same when the true female rapa whelks had lower wet tissue weights than either imposex females or males (Mann et al., 2006) not talking about the suppression of physiological functions but the appearance of excess reserves.
At the same time analysis of data on Muricidae Hexaplex trunculus (L., 1758) from two fishing harbours showed that in the first site, the concentration of TBT was quite low, but imposex indices were high. On the contrary, TBT concentrations in the second site were high, but imposex indices were low. Besides this, an initial development of imposex in H. trunculus specimens from museum collections collected in the period between 1845 and 1930, i.e. 30–115 years before the use of TBT have found. These observations suggest that factors other than TBT could be implicated on the development of imposex (Lahbib et al, 2009).
The correlation of various indicators is not always the result of a cause-effect relationship. As regards pollutants, among them may be present simultaneously as inhibitors or stimulators of organisms. Eutrophication of waters caused by human activities can sometimes stimulate the development of individual species. Still S.A. Zernov was noted that the most abundant M. galloprovincialis settlement developed on the rocks in close proximity to Sevastopol (Zernov, 1913) which domestic waste water are man-made eutrophicators. As filter feeders like mussels are the main food of Rapana and many other muricides, the size and number of the predators in areas of anthropogenic eutrophication may even be higher than originally clear waters. In the investigated bays the lowest average size and weight of R. venosa individuals correspond to the purest of them - the Laspi bay. And the greatest individual maximum weight of Rapana and the number of the oldest (12 years of age) specimens were marked for the most polluted Round bay.
Based on the above the change in color of R. venosa females gonads not necessarily related to imposex status and pollutants. Change in color of gonads R. venosa females without a penis on the colors typical of males can also be attributed to the manifestation of intersexuality. Supposedly this is the result of changes in physiological processes caused by external factors. But it remains unclear exactly which environmental factors are responsible for the occurrence of such physiological abnormalities.
Changes in the color spectrum gonads of male R. venosa in some populations probably due to a change of the food spectrum and reflects some evolutionary change of the species. Further study of the changes in the generative and other soft tissues in conjunction with the ecological and biochemical studies will help open a bit the “door to the kitchen” of Evolution.
3 Material and Methods
Collection of samples for this study was carried out in the vicinity of the city of Sevastopol and the southern coast of Crimea (Laspi bay). Sampling conducted at depths of 3 - 6 meters in ecologically different habitats: 1- open shallow bay in the city (Round Bay), 2 open bay on the border of the city limits (Blue bay), 3- rocky cape Theophane, 4 - open bay in the recreational area of Crimea (Laspi bay) (Figure 1).
In the Round and Blue bays samplings were made in June-September 2012 - 2014, at Cape Theophane - in August and September 2014, in the Bay of Laspi - in July and August 2014.
The number of individuals examined by area and year was as follows: Round bay: 2013 - 35 individuals, 2014 - 40 individuals; Blue bay: 2012 -30 individuals, 2013 - 50 individuals, 2014 - 180 individuals; cape Theophane: 2014 - 25 individuals; Laspi bay: 2014 - 15 individuals. In addition to specialized collections considered unpublished authors' data for the study of R. venosa 1990-2010 periods.
After the sampling R. venosa individuals subjected to freezing followed defrost for testing and photographing in the laboratory. Empirically it has been found that the color of the mollusks soft parts after thawing remained unchanged. After freezing and defrosting the percentage of extracted from the shell without significant damages soft organs is increased compared with freshly caught from 10-30% to 60-90% in different populations. Soft body was extracted by a metal hook that allows getting inside the shell and tearing columellar muscle at the place of attachment. This method allows keeping intact the shell which is necessary to determine the age of mollusk. Individual age R. venosa determined based on annual spawning marks on the shell in accordance with the method proposed Chuhchin V.D. (1961a,b) as represented (Bondarev, 2010). To more accurately determine the age the specimen shell must be cleaned of various types of surface growths. The outgrowth from the surface of the shell removed using a chlorine-containing alkali (the first stage) then mechanically and hydrochloric acid (the final stage) to remove any of remains calcareous encrustations. Age of the investigated specimens ranged from 2 to 12 years. specimens
Total shell length (SL) was measured using a caliper (0.1mm) total animal wet weight (W) and whelk wet tissue weight (TW) were recorded using a digital scale balance (0.1g). Since soft body mollusk difficult to measure accurately the Figures in the article are accompanied by data on total shell length (SL).
Each whelk was classified as male or female by penis presence/absence respectively and gonad color. Sex determination in males with reduced penis was performed under an optical microscope at 40x magnification.
Photographing samples using digital cameras carried out selectively directly after removing the soft body of the shell. Abnormal specimens photographed everything and typical ones in the number necessary to cover age and color range.
The results are compared with published data from other areas of the Black Sea and the Atlantic coast of North and South America.
Acknowledgements
The author is grateful to IBSS staff member Kovaleva M.A. for microscopic diagnosis of R. venosa two specimens’ gonad.
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