Indian Journal of Geo-Marine Sciences Vol. 41(5), October 2012, pp. 418-424

Imposex in spirata (: ) from Pakistan (Northern Arabian Sea)

Nuzhat Afsar1, *Ghazala Siddiqui & Zarrien Ayub Center of Excellence in Marine Biology, University of Karachi, Karachi-75270, Pakistan 1Lesbela University of Agriculture Water and Marine Science (LUAWMS), Uthal, Balochistan, Pakistan *[E-mail: [email protected]]

Received 8 March 2011; resived 18 August 2011

This is the first report of the occurrence of imposex in the neogastropod (Linnaeus, 1758) from Pakistani waters. Studies were carried out at Manora Channel, which is situated in the vicinity of Karachi port and fish harbor, the hub of commercial boating activity in Pakistan. Other two sites Buleji and Sonmiani are free of such activity and were therefore considered as reference sites. Specimens of B. spirata were morphologically and histologically examined. Specimens showing imposex were only found at Manora Channel. Incidence of imposex was 73.7% and the vas deferens sequence (VDS) were ranked to be between 1 to 4+. The average VDSI was 0.42 whereas the average relative penis length index (RPLI) was 14.45 and the relative penis size index (RPSI) was 0.32. Both normal and imposex females generally showed normal gonadal development. However, evidence of an ovo-testis was found in histological sections of normal and imposexed female of B. spirata gonads examined from Manora Channel. At Manora Channel the sex ratio was significantly in favour of females whereas at Buleji and Sonmiani, it was close to unity (1:1).

[Keywords: Ovo-testis, Babylonia spirata, Organotin, Karachi]

Introduction Gastropod belonging to the family Organotin compounds such as tributyltin Babyloniidae are also affected by TBT contamination and triphenyltin (TBT & TPhT) used in the and the reports concerning the imposex have been antifouling paints of ships, boats, fishing gears documented in species of Babylonia, previously and nets instigate imposex (superimposition of placed in the family Buccinidae11 such as B. japonica, male characters) in female snails. This phenomenon B. areolata, B. ambulacrum and B. formosae habei serves as an effective biomonitoring system to from Japan, Thailand, China and Taiwan12-15,9. In determine the presence of organotin contamination neighboring India, imposex have been reported in the in the marine environment1-5. muricid gastropod Cronia konkanensis, the babyloniid TBT has been used extensively in antifouling paints Babylonia. spirata and the cymatiid Gyrineum natator and the incidence of imposex in prosobranch from sites experiencing boating activity16-18. The gastropods has been reported at TBT concentrations phenomenon of imposex has also been observed in as low as 1ngL-1 in sea water1. Due to biocidal many species of muricid gastropods in Pakistan19-20. contamination of TBT in the marine environment, Preliminary investigations for tin measurement from population decline, morphological abnormalities such studied area have been conducted and concentrations as oviduct blockage, penis and vas deferens of organotin compounds:monophenyltin (MPhT) and development, deformed and bifurcate penis, 2-eyed diphenyltin (DPhT) been detected in soft tissues of tentacle, suppression of oogenesis, spermatogenesis three neogastropod species sampled from Manora and seminal vesicle development in females, have Channel20. Here we report the first case of imposex in been reported6-8. The phenomenon of imposex has a buccinoid gastropod, B. spirata from Pakistan. been reported in more than 170 species of gastropods mostly in the members of neogastropod family Materials and Methods Muricidae9, 10. Specimens of Babylonia spirata were collected —————— from Manora Channel, Pakistan (Fig. 1), between *Corresponding author: 1Present address: September and March during 2003-2008. We also AFSAR et al: IMPOSEX IN BABYLONIA FROM PAKISTAN 419

Fig. 1—Sampling sites for Babylonia spirata in Pakistan. Sonmiani, Buleji & Manora Channel: 1-Sampling site, 2-Karachi Port, 3- Karachi Fishing Harbor. collected specimens from Sonmiani in February 2005 plastic coated millimeter graph paper against the and from Buleji in March, 2008 (Fig. 1). Manora penis/pseudopenis and counting the number of units Channel lies in the vicinity of Karachi Port and occupied between its tip to the junction with the body Karachi Fish Harbor. Karachi port is the largest port wall behind the right tentacle. in the country, about 250-300 shipping vessels from Imposex stages were identified following the all over the world visit the port every month. In general scheme of imposex described by earlier contrast, Sonmiani and Buleji are ‘clean’ sites with workers1,21,22 (Fig. 2). Vas deferens sequence Index little or no maritime activities (Fig. 1). (VDSI) was calculated as the average imposex stage, Specimens of B. spirata were found in the low according to scheme of imposex development in intertidal zone at about 0 m tidal level. The prosobranchs1, 21, 22 (Fig. 2) were handpicked and brought live to the laboratory Relative Penis Length Index (RPLI) and Relative till further examination. Shell length of all specimens Penis Size Index (RPSI) were calculated by the collected was measured to the nearest 0.1 mm using a following formulas1,21,22. vernier caliper. The soft tissue was extracted by breaking the shell and then examined morphologically RPLI= (mean female penis length) /(mean male for the occurrence of imposex by assessing penis length) × 100 anatomical changes and morphological deformities RPSI= (mean female penis length)3 /(mean male such as asymmetry in tentacles or abnormal eye 3 penis length) × 100 growth on tentacles, as well as the presence of pseudopenis and vas deferens in female snails. Male The histological examination of gonads of individuals were identified by the presence of penis 36 individuals collected from Manora Channel was and vas deferens and prostate gland whereas, females carried out. Gonadal tissues were fixed in Davidson’s were distinguished by the presence of a dark sperm fixative23 for 48 hrs, and then preserved in 70% ingesting gland (SIG), posterior to the egg capsule alcohol till further processing. Prior to sectioning gland (ECG), and the presence of an albumen gland the specimens were dehydrated in alcohol, embedded ventral to the SIG. The male penis and female in wax, sectioned at 5-7 µm and stained with pseudopenis were measured by placing a piece of haematoxylin and eosin. 420 INDIAN J. MAR. SCI., VOL. 41, NO. 5, OCTOBER 2012

Fig. 2—General scheme of imposex stages in neogastropods. Bold lines indicate imposex stages observed in the present study. Abbreviations: ac-aborted capsules; cg-capsule gland; gp-genital papilla; obc-open bursa copulatrix; ocg-open capsule gland; ocv-occlusion of the vulva; p-penis; pd-penis duct; pr-prostate; te-tentacle; vdp-vas deferens passage into capsule gland; vds-vas deference sequence (Oehlmann et al. 41). Results of the total number respectively. Whereas incidence Out of 159 individuals of Babylonia spirata of imposex was 73.7%. Incidence of imposex was examined from Manora Channel, 60 were males, calculated as the proportion of imposex females 26 normal females and 73 females were affected by among the total number of females in a sample. imposex (IS), constituting 37.7%, 16.4% and 45.9% Whereas percentage of imposex describes the AFSAR et al: IMPOSEX IN BABYLONIA FROM PAKISTAN 421

frequency of imposex in whole population. The mean blockage or occlusion of vulva by vas deferens shell length of male and female B. spirata was formation was not observed even in specimens with 47.0±6.2 mm and 48.7±8.6 mm respectively. The advanced stages of imposex. Similar conditions were proportion of males was 0.4 and the sex ratio was described in B. japonica from Japan in which no significantly in favour of females (X2 9.66; P≤0.05), blockage of oviduct was observed in imposex therefore, female mortality may not be directly females15,22-25. However, this state differs from the attributable to imposex. In specimens of B. spirata symptoms described in populations of muricid (n=109) studied from Buleji, which is a ‘clean’ site, gastropods including Nucella lapillus, Ocinebrina the sex ratio was close to unity (X2 0.45; P≥0.05) aciculata and Thais clavigera6,7,26,27. In these species and no imposex individuals were found. Similarly oviduct blockage and occlusion of vulva were found in at Sonmiani (n=10) the sex ratio was 1:1 and there specimens showing advanced stages of imposex6,1,26,27. was no incidence of imposex at this site. Imposex indices such as RPLI, RPSI and VDSI The RPLI in this species was 14.5 and RPSI was were determined to assess the effect of imposex in the 0.3. The VDSI was 0.4 in the imposex population. B. spirata population. The VDS stages 1a, 1b, 1c, 2a, The VDS stages 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 2b, 2c, 3a, 3b, 3c, 4 and 4+ were found in B. spirata at 28 4 and 4+ were found in this species (Table 1). Most Manora Channel. Stroben et al. reported VDS stages of the imposex females were in VDS stages 3c (23.3), 1a, 1b, 2a, 3a, 3b, 4 and 4+ in the buccinid Hinia 1c (21.9) and 4+ (21.9), while rest of the VDS stages reticulata whereas, stages 1c, 2b, 2c and 3c were not ranged between 1.4 to 6.9. found in this species. VDS stages up to stage 5 have been described in buccinid gastropod Neptunea Discussion phoenicea29 though stage 4+ has been recommended In the present study of Babylonia spirata examined as the end of imposex development in many from Manora Channel, the incidence of imposex was buccinids28. In imposex gastropods with stage 73.7% and morphological alterations such as 4+ vas deferens bypass the vagina and runs ventrally pseudopenis and VDS development were evident in through the channel of capsule gland, and hence there females affected by imposex. However, oviduct is no restriction of fertility. Whereas in buccinids and muricid gastropods with stage 4 imposex, the penis Table 1—Biometric data of male, female and imposex females of duct and vas deferens are continuous from the penis to Babylonia spirata. SD, standard deviation; VDS, vas deferens sequence. the vulva and in the late stages leads to the blockage of vulva. In extreme conditions the malformation Site VDS No of Shell Penis extends to stage 7 which is the last stage of imposex specimens length±SD length±SD 1 mm mm in muricids . In the nassariid species Hinia reticulata two stages without penis development (1b and 3b) have Manora Male 60 47.02±6.17 6.13±1.31 28 Channel also been described by Stroben et al. . In B. spirata 0 26 48.73±8.56 0.00±0.00 from Manora Channel these two stages were also 1a 1 55.29±7.52 1.29±0.00 present (Table 1). In buccinids a- and c-type of 29 1b 7 50.38±8.15 0.00±0.00 imposex stages are common . The b- and c- types of 1c 16 45.75±6.80 0.00±0.00 imposex stages are characteristic of exposed shores 22 2a 4 48.00±0.00 1.00±0.00 and a-types of sheltered habitats . The occurrence of 2b 1 50.25±8.02 0.00±0.00 a- type imposex stages along with b- and c- types in 2c 4 46.00±0.00 1.00±0.00 B. spirata could be due the fact this species at 3a 1 48.80±10.28 1.40±0.00 Manora Channel is found in semi-exposed condition 3b 5 47.35±6.38 0.00±0.00 close to the mouth of the channel. 3c 17 45.00±0.00 1.00±0.00 Morphological abnormalities, such as abnormal eye 4 1 48.00±0.00 3.00±0.00 spot growth in the tentacles, were observed in three 4+ 16 48.63±6.47 2.69±0.87 specimens collected from Manora Channel. Two eye Female 99 spots were observed in the right tentacle of a male Buleji Male 51 32.75±3.47 4.71±1.32 specimen and in an imposex female with stage Female 58 32.29±4.37 0.00±0.00 4+. One imposex female with stage 4+ was found Sonmiani Male 63 46.80±8.25 5.40±1.140 with three eye spots on its left tentacle. Similar Female 57 46.60±10.43 0.00±0.00 morphological deformities of tentacles (two eyed 422 INDIAN J. MAR. SCI., VOL. 41, NO. 5, OCTOBER 2012

tentacles) have been observed in Nucella lapillus and In male tissue, four developmental stages were Hinia reticulata from TBT contaminated sites7,28. present that were characterized by the presence To detect abnormal gonadal tissue, histological of spermatogonia, spermatocytes, spermatid and sections of individual B. spirata were studied, spermatozoa (Fig. 3A, C). In normal and imposex which showed different stages of oogenesis and females, different reproductive stages were found spermatogenesis in testicular and ovarian tissues. with oogonia, primary oocytes, pre-vitellogenic

Fig. 3—Histological sections of Babylonia spirata collected from Manora Channel (sampling site 1), Pakistan: A-Male (testis); B-normal female (ovary); C-male seminal vesicles; D-imposex female showing seminal vesicles; E-F- imposex female showing spermatogenesis (ovo-testis). Abbreviations: mo, oocyte; N, nucleus; ovt, ovo-testis; sc, spermatocytes; sg, spermatogonia; st, spermatid; sv, seminal vesicles; sz, spermatozoa; yg, yolk granules. AFSAR et al: IMPOSEX IN BABYLONIA FROM PAKISTAN 423

oocyte, post vitellogenic oocyte and mature oocytes In the present study, specimens showing imposex (Fig. 3B, E-F). However, ovarian spermatogenesis and those with ovarian spermatogenesis were only was observed in two morphologically normal and found in environments with ship-related activities. two imposex females. Imposex females with 3a Imposex is known to be induced by TBT as it (with penis) and 3b (without penis) stages were found has been proved by both laboratory and field with ovo-testis, showing spermatogenesis in ovary experiments6,7,1. In Pakistan there are no aquaculture (Fig. 3E-F). Other than spermatid development, related facilities or marinas present along the coast, seminal vesicles filled with spermatozoa were so the use of toxic biocides is not common. The also observed in the histological sections of a only likely source of TBT contamination here morphologically normal female from Manora Channel are commercial ships which enter the channel to (Fig. 3D). Ovarian dysmaturity was not observed in harbour at the Karachi Port and Karachi Fish Harbor, any specimen from this site. It appears that the TBT which is the hub of commercial shipping activity can also cause masculization in the ovaries without and appears to be the localized area of contamination. showing any sign of morphological alteration such as Despite the fact that the International Maritime penis and vas deferens development in babyloniid Organizations has recommended the ban on the gastropods. In hatchery-reared B. japonica from use of TBT based coatings on vessels and prohibited Tottori Prefectural Sea, Japan, which is a TBT their use from year 2003 onwards, in most South East contaminated site, ovarian spermatogenesis was Asian countries TBT based paints are still being observed in both normal and imposex females with extensively used39, 40. penis and vas deferens formation. 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