Hinia reticulata and Nucella lapillus. Comparison of two gastropod tributyltin bioindicators

Hinia reticulata and Nucella lapillus (collected from 1989 to 1991 at Pointe de Pléneuf and Méan Mélen, France, respectively) exhibit imposex in response to tributyltin (TBT) pollution leached from antifouling paints and proved to be good TBT bioindicators. H. reticulata was kept for 18 mo under TBT-free conditions in the laboratory, but no evidence for imposex remission was found. A second series of experiments showed that food chain uptake of TBT from a contaminated diet is an important mode of TBT exposure in H. reticulata. Comparative tank experiments with H. reticulata and N. lapillus demonstrated that the same type of TBT exposure resulted in comparable TBT body burdens, biological concentration factors, and imposex development in both species [measured as increase of VDS (vas deferens sequence index), uncubed RPS (relative penis size index) and average female penis length]. Differences in imposex development of natural and laboratory populations are discussed against the background of different types of TBT contamination of their food. A statistical study, based on an analysis of natural populations of both prosobranch species, makes a comparison of the specific TBT sensitivity of the two bioindicators possible. As a consequence it is proposed that TBT biomonitoring programs in Europe should use both prosobranchs as indicator species.     

Occurrence of tri-n-butyltin-caused imposex in the North Pacific marine snail Nucella lima in Auke Bay,Alaska

Like female dog whelks (Nucella lapillus) in the Atlantic Ocean, females of the Pacific gastropod N. lima respond to low concentrations of tri-n-butyltin (TBT) by growing a penis and vas deferens. This condition, termed imposex, was found to occur in N. lima collected from August 1987 to May 1988 along a TBT pollution gradient associated with a marina in Auke Bay, Alaska. The suite of symptoms characteristic of imposex in N. lima was slightly different than in N. lapillus. Imposex, as measured by relative penis size (RPS) of females to males, increased from 0.0 to 34.27 along this gradient and as measured by vas deferens sequenceindex (VDS) increased similarly from 0.0 to 4.29. Concentrations of TBT in N. lima increased from below detection limits (about 0.010 g Sn g^-1 dry tissue wt) to 0.065 g Sn g^-1 dry tissue wt along the gradient. The gradient was determined by measuring TBT in whole-body tissues of bay mussels (Mytilus edulis). Concentrations of TBT in mussels increased from below detection limits to 0.833 g Sn g^-1 dry tissue wt in mussels from within the marina that was the major local source of TBT. Imposex, tissue TBT burden, and position along a TBT pollution gradient are significantly correlated (P<0.01) in N. lima. TBT was tested as a causative agent for imposex by exposing snails at a distant control site to TBT paint. After 1 mo exposure, 33% of the females grew a penis ranging in size from 0.2 to 0.8 mm. Our results generally corroborate those found for N. lapillus and indicate that imposex in N. lima is caused by environmental TBT exposure. We suggest that the RPS in the genus Nucella may be useful in monitoring TBT in coastal waters worldwide.     

The morphological expression of imposex in Hinia reticulata (Gastropoda: Buccinidae): a potential indicator of tributultin pollution

Specimens from the prosobranch Hinia reticulata collected along the coast of Brittany and Normandy from 1988 to 1991 exhibited imposex (occurrence of male parts in addition to the female genital duct) in response to tributyltin (TBT) pollution. Four stages of imposex development (1 to 4) with two different types in the Stages 1, 3 and 4 could be distinguished and have been documented with scanning electron micrographs for the first time. Furthermore, three additional alterations of the genital tract are shown. Neither TBT-induced sterilization nor sex change occurred. TBT accumulation in whole body and selected tissues is described and sex-related differences are shown. The VDS (vas deferens sequence) index, cubed and uncubed RPS (relative penis size) index and average female penis length of a population were analysed with regard to their quality as indices for TBT biomonitoring. Based on the way imposex develops in this species the VDS is recommended as the most valid index. Only in highly polluted areas should the uncubed RPS be used as a second index.     

Increased sperm numbers in the vas deferens of meadow voles, <Emphasis Type="Italic">Microtus pennsylvanicus,</Emphasis> in response to odors of conspecific males

Sperm competition occurs when the sperm of two or more males compete to fertilize the egg/s of a particular female. Males of some species respond to a high risk of sperm competition by increasing the number of sperm in their ejaculates. Males may accomplish such a response by increasing the intensity or duration of contraction of the cauda epididymidis and vas deferens. During emission (first phase of the ejaculatory process), the vas deferens receives sperm from the cauda epididymidis and propels the sperm to the urethra. In this paper, we tested the hypothesis that males exposed to a high risk of sperm competition mobilize larger numbers of sperm from the cauda epididymidis to the vas deferens before initiation of copulatory behavior. This accumulation of sperm in the vas deferens would result in a larger number of sperm in the ejaculate. To test this hypothesis, we exposed male meadow voles, Microtus pennsylvanicus, to either low or high risks of sperm competition using soiled bedding of conspecific individuals. At three different times after this exposure (15, 30, or 60 min), we removed both vasa deferentia and counted the sperm within them. We found a significant increase in sperm numbers in the vas deferens of males after 30 min of being exposed to a high risk of sperm competition. The lower sperm numbers after 15 and 60 min of exposure suggest that the observed response is relatively slow and that sperm mobilized to the vasa deferentia may return to the cauda epididymides if ejaculation does not occur some time after the observed response. Our results indicate that the physiological response that may result in high sperm numbers in the ejaculate in relation to high risk of sperm competition can occur before initiation of copulatory behavior.     

Origin and nature of spermatophoric mass of the spiny lobster Panulirus homarus

In the spiny lobster Panulirus homarus (Linnaeus) the spermatophoric mass consists of a highly convoluted tube, containing the sperm mass and a gelatinous matrix. The spermatophoric components have their origin in the proximal vas deferens and the completed spermatophoric mass is stored within the distal dilated part of the vas deferens. In the proximal vas deferens the inner glandular epithelial cells give rise to leaf-like typhlosoles which gradually diminish in size and finally disappear in the distal vas deferens. The secretions originating from the typhlosole form the outer gelatinous matrix of the spermatophoric mass. Histochemical observations reveal that the wall of the spermatophore tube consists of neutral mucopolysaccharide, whereas the sperm mass and the gelatinous matrix are rich in acidic mucopolysaccharides. Both polyacrylamide gel electrophoresis and agarose gel electrophoresis were used to separate the acidic mucopolysaccharide (AMPS) complexed with the proteins and the simple proteins. In addition, AMPS have been isolated following the method of Rahemtulla and Lovtrup (1974) and used for electrophoresis. In both the polyacrylamide gel electrophoresis and agarose gel electrophoresis, the extracted acid mucopolysaccharides give only a single fraction which, in comparison with the standard acid mucopolysaccharides, corresponds to chondroitin sulphate. The functional significance of the chondroitin sulphate and neutral mucopolysaccharides in the spermatophoric components of P. homarus is discussed in relation to their functional role in spermatophore hardening and protection of the delicate spermatozoa during their prolonged storage on the sternum of the female.     

Östrogenartige wirkungen von bisphenol a auf vorderkiemenschnecken (Mollusca: Gastropoda: Prosobranchia)

Within a Federal Environmental Agency research project to develop a biological test for hormone-mimetic compounds using the freshwater snail Marisa cornuarietis, the effects of the suspected xenoestrogenic substance bisphenol A (BPA), not only on freshwater but also on marine prosobranch snails, were investigated. For the laboratory experiments the ramshorn snail Marisa cornuarietis and the ovoviviparous snail Potamopyrgus antipodarum were considered as freshwater species and two marine prosobranchs, the netted whelk Nassarius reticulatus and the dog whelk Nucella lapillus, were additionally employed. N. reticulatus, as a typical sediment-living species, was exposed via artificial sediments, while the three other prosobranchs were exposed via water. The test series with Marisa cornuarietis covered a nominal concentration range between 1 and 100 μg BPA/l in a 5 month experiment with adult snails and a complete life cycle test for 12 months. Additionally, a third test in the nominal range between 0.05 and 1 μg BPA/l (measured: 0.0079–0.404 μg/l) was performed with adults snails for 6 months. In these experiments, BPA induced a complex syndrome of physiological and morphological alterations in female Marisa referred to as the induction of ‘superfemales’. Affected specimens were characterised by the formation of additional female organs, an enlargement of the accessory pallial sex glands, gross malformations of the pallial oviduct section resulting in an increased female mortality, and a massive stimulation of oocyte and spawning mass production. For these tests, an LOEC of 48.3 ng/l, an NOEC of 7.9 ng/l and an EC₁₀ of 13.9 ng/l were calculated. Superfemales occurred also in the BPA exposure experiment with the other snail species, but comparable oviduct malformations as in Marisa were not found, probably due to species differences in the gross anatomical structure of the pallial oviduct. During the 9 week test with Potamopyrgus antipodarum in the nominal concentration range between 1 and 100 μg/l, BPA induced an enhancement of embryo production even in the sexual repose phase of the reproductive cycle. A characteristic inverted U-type concentration response relationship was found. Nassarius reticulatus was exposed via BPA-spiked artificial sediments (nominal concentration range: 10–1000 μg/kg dry wt.) for 3 months. BPA exhibited a significant and concentration dependent uterotrophic effect which could be detected not only by an enlargement but also by a weight increase of the accessory pallial gland complex in the pallial oviduct section. Adult Nucella lapillus were tested for three months in the laboratory in a nominal concentration range between 1 and 100 μg BPA/l. Superfemales in the dog whelk were also characterised by enlarged accessory pallial sex glands and an enhancement of egg production, but the test compound also affected the males in this species. A lower percentage of exposed specimens had ripe sperm stored in their vesicula seminalis and male Nucella exhibited a reduced length of penis and prostate gland when compared to the control. Because statistically significant effects were observed already at the lowest nominal test concentration (1 μg/l), it can be assumed that even lower concentrations may have a negative impact on the snails. The results show that prosobranch snails are affected by BPA at lower concentrations compared to other systematic taxa in the animal kingdom. Consequently, it has to be claimed that the results of these experiment have to be considered for the current EU risk assessment for BPA in order to achieve a sufficient protection of wildlife in aquatic ecosystems.     

Potential histopathological and molecular changes in rat vas deferens inhaled by Boswellia papyrifera and Boswellia carterii

Boswellia papyrifera and Boswellia carterii released from smoke contaminate indoor environment and consequently adversely affect humans as evidenced by respiratory disturbances. The aim of this study was to determine the effects of these plants on pathological and biochemical changes in vas deferens of albino rats. Animals were administered 4 g/kg body weight B. papyrifera and B. carterii daily for 120 days along with controls. Significant changes were observed in epithelial cell types and some cells showed signs of degeneration. The ultrastructural studies revealed marked changes in cytoplasmic organelles. Microvilli were missing and lysosomes were found in the cytoplasm. In addition, all treated groups plasma fructose and other biochemical parameters were decreased indicating reduced energy necessary for motility and contractility of spermatozoa. Many spermatozoa were disorganized and agglomerated. Data suggest that smoke from these plants adversely affects vas deferens.     

Male reproductive biology of the red frog crab,Ranina ranina,off Hachijojima,Izu Islands,Japan

Male red frog crabs, Ranina ranina, were collected year round in 1990 and 1991 off Hachijojima for histological study of the reproductive system and cycle. The testis containing the lobules and seminiferous ducts is surrounded by connective tissue. The seminiferous duct connects to the anterior end of the vas deferens, which can be histologically divided into three portions similar to one another in appearance. It was surrounded by fibrous connective tissue, muscle fibrils and columnar epithelium. Muscle fibrils were absent in the anterior portion. Multiple sperm masses were not formed in the vas deferens and ejaculatory duct, but the sperm mass was covered with a capsule composed of two layers. The outer layer of the capsule was periodic acid Schiff (PAS)-positive, but the inner layer was negative. Both layers were Alcian Blue negative, except the vacuoles in the outer layer that were stained blue. The small round androgenic gland was attached to the posterior end of the vas deferens of the coxa of the eighth thoracic appendage. The ejaculatory duct was distinguishable from the vas deferens by the absence of columnar epithelium and the presence of thick longitudinal muscle fibers. Spermatogenesis was histochemically examined. The acrosomal vesicle appeared to be derived from PAS-positive vesicles in the cytoplasm of the spermatid at the early stage of spermiogenesis. The arms were positive to the Feulgen reaction and the subacrosomal region was negative to PAS. Seasonal changes in reproductive cycle were inconspicuous histologically and microscopically. Sperm were always present in the testis and vas deferens throughout the year and occupied 5.1 to 19.6% of testis observed in cross sections. The minimum size of maturity is less than 39 mm carapace length, but the minimum size capable of successful mating was estimated to be ca. 55 mm.     

Imposex in Hexaplex trunculus (Gastropoda: Muricidae): first results from biomonitoring of tributyltin contamination in the Mediterranean

Imposex (the occurrence of penis and vas deferens in females) in Hexaplex trunculus collected from Malta (Central Mediterranean) in 1992 is reported. This phenomenon is related to the levels of tributyltin (TBT) in the organisms as well as in sediments. Imposex indices (relative penis size and vas deferens sequence) are used to biomonitor coastal contamination by TBT for the first time in the Mediterranean. In its response to TBT, H. trunculus is ranked as one of the most sensitive neogastropod species studied so far. Accumulation of TBT and its metabolites in the digestive glands, gonads and the rest of the body are described, and sex-related differences are shown. No preferential female mortalities are recorded in populations exposed to high levels of TBT. However, a reported shift in the size frequency distribution of H. trunculus in contaminated sites, towards bigger snails, may suggest reduced reproductive potential.     

Physiological adaptations of the invasive cordgrass<Emphasis Type="Italic"> Spartina anglica</Emphasis> to reducing sediments: rhizome metabolic gas fluxes and enhanced O<Subscript>2</Subscript> and H<Subscript>2</Subscript>S transport

Cordgrasses of the genus Spartina form dense monospecific stands worldwide, profoundly influencing the ecology of estuaries. One species, Spartina anglica, originated by allopolyploidy in the 1800s and has been particularly prolific as an invasive species worldwide. S. anglica tolerates low-lying estuarine mudflats that its progenitor species and other coastal halophytes cannot. However, very little is known of the physiology of S. anglica. In the present study, an automated flow-through respirometry system was used to quantify metabolic gas fluxes (O₂, H₂S, CO₂, and NH₃) of S. anglica rhizomes. Enhanced physiological mechanisms to transport O₂ and H₂S in both directions between the rhizosphere and the atmosphere were exhibited by S. anglica, but not by the native North American species S. alterniflora. These results suggest that tolerance of anoxia and H₂S may assist S. anglica in colonizing extensively flooded environments. Enhanced sediment oxygenation by S. anglica may be potentially useful for phytoremediation of contaminated sediments, since microbial degradation of organic pollutants is often limited by O₂ availability.     

Phylogeography of surfclams,<Emphasis Type="Italic"> Spisula solidissima</Emphasis>, in the western North Atlantic based on mitochondrial and nuclear DNA sequences

The Atlantic surfclam, Spisula solidissima (Dillwyn), is broadly distributed in sandy sediments of the western North Atlantic between the Gulf of St. Lawrence and the Gulf of Mexico. In the United States, a substantial commercial fishery between Long Island and Cape Hatteras harvests offshore populations of one subspecies, S. s. solidissima. A smaller coastal form, S. s. similis Say (also known as S. s. raveneli Conrad), has a partially sympatric geographic distribution, but differs in several life-history characteristics. DNA sequence variation in mitochondrial cytochrome oxidase I (COI) and in introns at two nuclear calmodulin loci was examined to measure genetic divergence between the two subspecies and to test for population structure among populations of S. s. solidissima. Surfclams were collected from seven localities between 1994 and 2001. Based on both mitochondrial and nuclear DNA variation, the two subspecies of S. solidissima are reciprocally monophyletic, with a net COI divergence of 13.9%, indicating long-term reproductive isolation. The only significant differentiation among populations of S. s. solidissima (based on an AMOVA analysis of COI sequences) was between the Gulf of St. Lawrence and more southerly populations. A long internal branch in the S. s. solidissima genealogy coupled with low haplotype diversity in the northern-most population suggests that populations north and south of Nova Scotia have been isolated from each other in the past, with gene exchange more recently. Populations of S. s. similis from Atlantic and Gulf of Mexico coasts had a net COI divergence of 9.2%. Thus, diversification of Spisula spp. clams in the western North Atlantic involved an early adaptive divergence between coastal and offshore forms, with later barriers to dispersal emerging in the offshore form from north to south and in the coastal form between Atlantic and Gulf of Mexico populations.     

The androgenic gland of the shrimp Palaemon dayanus

The androgenic gland of Palaemon dayanus Henderson differs in morphology and anatomy from that of all other known natantian decapods. The gland is attached to the distal extremity of the was deferens as a thin, plate-like structure made up of numerous parallel strands in close juxtaposition. The gland is clearly separated by intervening thick muscle bands from the epithelium of the vas deferens. The structure of the gland shows distinct zones of activity such as growth and degenerating zones. The presence of degenerating zones together with zones of activity in the same gland at the same time is attributed to a holocrine mode of secretion in the androgenic gland.     

Predation on egg capsules of <Emphasis Type="Italic">Zidona</Emphasis><Emphasis Type="Italic">dufresnei</Emphasis> (Volutidae): ecological implications

Among the diverse patterns of energy allocation to the offspring of gastropods, the presence of egg capsules to protect embryos is common. Females of the edible snail Zidona dufresnei attach egg capsules to hard substrates in shallow Argentine Patagonian waters (40°45′S, 64°56′W) during spring-summer. Embryonic development takes about 30 days at 22°C. In this study, three likely capsule predator species and the marks left by each on egg capsule walls were identified in laboratory experiments in February 2010. Abundances of predators and egg capsules with evidence of predation were assessed in the field in the summers of 2010 and 2011. Under laboratory conditions (N = 10 replicates per treatment and control), the predation rate by the chiton Chaetopleura isabellei was the highest (up to 90%), followed by the gastropod Tegula patagonica and the crab Neohelice granulata (~20% each). Nearly 60% of 41 capsules found in the field showed signs of predation. According to the marks identified in the laboratory, C. isabellei was responsible for 79% of this predation, and T. patagonica for the rest. Predation appears to be important during the encapsulated early life and could be an agent for selecting for resistant capsule walls and a relatively shorter development time.     

Age,growth and population structure of <Emphasis Type="Italic">Modiolus barbatus</Emphasis> from the Adriatic

Age, growth and population structure of Modiolus barbatus from Mali Ston Bay, Croatia were determined using modal size (age) classes in length frequency distributions, annual pallial line scars on the inner shell surface, internal annual growth lines in shell sections of the middle nacreous layer and Calcein marked and transplanted mussels. The length frequency distributions indicated that M. barbatus attain a length of ∼40 mm in 5–6 years indicating that a large proportion of the population in Mali Ston Bay is <5 years old. Some mussels of ∼60 mm were predicted to be 14 years old using the Von Bertalanffy growth (VBG) equation. Up to the first 6 pallial line scars were visible in young (<6 years) mussels but in older shells the first scars became obscured by nacre deposition as the mussel increased in length and age. The age of the older shells (>6 years) was determined from the middle nacreous lines in shell section, which formed annually in winter between February and March; the wider dark increments forming during summer (June to September). The oldest mussel, determined from the middle nacreous lines, was >12 years, with the majority of mussels aged between 3 and 6 years of age. The ages of mussels ascertained using the growth lines were not dissimilar to the ages predicted from the length frequency distributions. Age at length curves produced using modal size class data were not different from the data obtained using the pallial scar rings and internal growth lines. Taken together these data suggest that M. barbatus attains a length of 40 and 50 mm within 5 and 8 years, respectively. Eighty one percent of individual M. barbatus injected with a Calcein seawater solution (300 mg Calcein l⁻¹), into their mantle cavity successfully deposited a fluorescent line, which was visible in suitably prepared shell sections under ultra violet light. Incorporation of Calcein into the mussel shells was seasonally variable with the lowest frequency of incorporation in mussels marked in February and recovered in May. Seasonal shell growth was observed with significantly higher growth rates in mussels marked in May and removed in August (ANCOVA, F _3,149 = 23.11, P < 0.001). Mussels (∼18 to 22 mm) marked in May and recovered in August displayed maximal growth rates of >2.5 mm month⁻¹ compared with a mean mussel growth rate of 1.2 ± 0.6 mm month⁻¹. At other times of the year mussel shell growth ranged from immeasurable to 1.48 mm month⁻¹.     

Measurement of calcium carbonate deposition in molluscs by controlled etching of radioactively labeled shells

Rates of calcium carbonate removal from shell pieces of Argopecten irradians (Say) and Mercenaria mercenaria (L.) were dependent on the type of etching fluid used and not shell origin. Etching was uniform over the entire shell surface, but surface morphology differed with etching fluids. Peak radioactivity was in early eluant fractions of shells etched immediately after radioactive labeling, and in later fractions when individuals were placed in isotope-free seawater after labeling. The etching technique can measure growth during the labeling period and subsequently by estimating the amount of calcium in fractions prior to the radioactive peak. Geometry of shell layers influenced the pattern of radioactivity seen in fractions. Peak location varied inside and outside the pallial line of individual M. mercenaria. A significant portion of the inorganic carbon used in shell formation by M. mercenaria was derived from metabolic CO₂.     

Survival of Nucella lapillus in a tributyltin-polluted area in west Brittany: a further example of a male genital defect (Dumpton syndrome) favouring survival

A male genital defect was reported in a Nucella lapillus (L.) population at Dumpton Gap (England). This defect was termed Dumpton syndrome (DS) and appears to be a genetic feature. Its main characteristic is the absence of penis (aphally) in males and in females. In 1992, such a phenomenon was discovered in populations in the vicinity of Brest (Brittany, France). DS-affected females exhibit fewer tributyltin-induced imposex characteristics than expected in normal individuals. The percentage of female sterilization is thus lower, favouring population survival. In consequence, the DS is considered to be a pollution-resistance feature. Comparison with the Dumpton population revealed similarities and differences in the DS characteristics. It is thus hypothesized that the DS observed at the two locations is due to two different biological mechanisms. Indeed, aphallic males with a split prostate were observed but no underdevelopment of their vas deferens and testis was noted in the present study. In this gonochoristic gastropod species, the most DS-affected males in Brest possess an ovotestis and it is thus hypothesized that feminity is remnant in N. lapillus. Incidence of abnormality is ten times higher in females than in males. This suggested that a sex-difference operates in the Brest phenomenon.     

Stereoisomeric composition of the sex pheromone of the pine sawfly <Emphasis Type="Italic">Microdiprion pallipes</Emphasis> (Hym., Diprionidae)

Summary. The basic chemical structure of the sex pheromone of the pine sawfly Microdiprion pallipes (Fallén) has earlier been identified as the propionate ester of (2S,3S,7R/S,11R/S)/(2R,3R,7R/S,11R/S)-3,7,11- trimethyl-2-tridecanol. We now report the results from further investigations on the male response to individual stereoisomers and to blends of stereoisomers, both in electroantennographic (EAG) recordings and in field trapping experiments. We also present our attempts to determine the stereochemistry of the compounds present in females of M. pallipes. By comparing gas chromatograms and mass spectra obtained from natural extracts with those from synthetic compounds it was found that the females contain one or more of the four (2S,3S,7R/S,11R/S)-3,7,11-trimethyl-2- tridecanol isomers (SS++-1). The active pheromone component is the corresponding propionate ester 2. In EAG experiments, males responded most strongly to five propionate ester samples, namely two four-isomer blends: SS++-2 and SR++-2, and three individual stereoisomers: SSSR-, SRRR- and SRSR-2. In a series of field trapping experiments it was found that males were attracted to the SR++-2 four-isomer blend and to the individual isomer SSSR-2. Based on the EAG-recordings and field responses of males and the stereoisomers found in the females, we suggest that the propionate ester of (2S,3S,7S,11R)-3,7,11-trimethyl-2- tridecanol (SSSR-2) is used as a main component of the sex pheromone in M. pallipes. Apparently the males react to other stereoisomers in addition to that or those produced by the females.     

Reproductive cycle and seasonal feeding activity of the neogastropod Buccinum undatum

The whelk Buccinum undatum L. in the northern Gulf of St. Lawrence has a distinct annual reproductive cycle. The ovary and pallial oviduct of the female, and the seminal vesicle of the male, show parallel development, whereas the testis has an inverse cycle. Thus, at the end of winter when the ovary, pallial oviduct and seminal vesicle reach maximal size, the testis is small and undeveloped, whereas the reverse situation occurs in summer. This is the first report of a totally inverse relationship between the ovary and testis for a marine invertebrate. This unusual pattern is due to the long-term storage of sperm in the seminal vesicle of the male prior to copulation. There is no resting period at the end of oogenesis, but rather renewed ovarian development starts shortly after gamete release. By contrast, in the testis after transfer of sperm to the seminal vesicle at the end of spermatogenesis, there is a distinct period of phagocytosis. Mating begins in mid-May, reaches a peak during June and terminates prior to July. Egg laying begins in late May, reaches a peak in mid-June to mid-July and can continue until late August. Examination of the incidence of individuals with food in their stomachs showed that feeding activity is maximal in late autumn through to early spring and decreases sharply coincident with the onset of breeding in late May.     

Specific effects of thiosulphate and L-lactate on hemocyanin-O2 affinity in a brachyuran hydrothermal vent crab

The hydrothermal vent crab Bythograea thermydron Williams (Brachyura: Bythograeidae) is exposed to high environmental concentrations of hydrogen sulphide. Hydrogen sulphide has previously been shown to be oxidized to a non-toxic form, thiosulphate (S₂O₃ ^2-), that accumulates in the hemolymph (to concentrations>1 mmoll^-1). Hemocyanin-oxygen (Hc-O₂) affinity was determined in dialysed, fresh or frozen hemolymph samples from B. thermydron. Although freezing is known to alter the affinity or cooperativity of some decapod crustacean hemocyanins, neither Hc-O₂ affinity nor cooperativity was significantly altered in B. thermydron hemolymph samples that had been frozen, consistent with previous findings. Oxygen affinity of B. thermydron hemocyanin was significantly increased by the presence of L-lactate. Likewise, Hc-O₂ affinity was significantly increased by the presence of 1.5 mmol S₂O₃ ^2- I^-1. The magnitude of this effect was the same as for similar concentrations of L-lactate. This specific effect of S₂O₃ ^2- does not appear to be a general property of crustacean hemocyanins, as there was no significant effect of S₂O₃ ^2- on Hc-O₂ affinity or cooperativity in dialysed hemolymph samples from the brachyuran crabs Cancer anthonyi Rathbun and C. antennarius Stimpson, or the thalassinid ghost shrimp Callianassa californiensis Dana. In the context of high environmental sulphide concentrations coupled with low P_{O 2}, and the subsequent accumulation of S₂O₃ ^2- in crab hemolymph, the increase in Hc-O₂ affinity due to thiosulphate appears to be an adaptive response in B. thermydron.