0554.p65

M.R. HEUPEL ET AL.
JOURNAL OF EXPERIMENTAL ZOOLOGY 284:586–594 (1999)
Plasma Steroid Hormone Profiles and Reproductive
Biology of the Epaulette Shark, Hemiscyllium
ocellatum

M.R. HEUPEL,* J.M. WHITTIER, AND M.B. BENNETTDepartment of Anatomical Sciences, University of Queensland, St. Lucia,Queensland, Australia 4072 Examination of the reproductive biology of the oviparous epaulette shark, Hemiscyllium ocellatum, was conducted on a wild population. Male sharks were found to reachmaturity at between 55–60 cm total length (TL) and female sharks mature around 55 cm TL.
Blood samples collected from mature male and female sharks were analyzed for sex steroid hor-mones to examine seasonal hormone patterns. Plasma samples were analyzed via radioimmu-noassay techniques with female samples measured for estradiol, progesterone, and androgenconcentrations, and male samples measured for androgen concentrations. Male androgen concen-trations showed a single broad peak from July to October with maximum hormone concentrations(60 ng/ml) occurring in August. Male androgen concentrations were lowest in December–February(<20 ng/ml), and appeared to correlate with reproductive activity and water temperature. Femaleandrogen concentrations were an order of magnitude lower than those for males and showed peaksin June (6 ng/ml) and December (8 ng/ml). Estradiol concentrations in females peaked during themonths of September–November (0.5 ng/ml) coinciding with the egg laying period. Progesteroneconcentrations ranged up to 0.5 ng/ml prior to the mating season. Observations of ova size and eggproduction showed eggs develop in pairs and ova are ovulated at a size of 25–27 mm. Females layeggs from August to January. Males were observed with swollen claspers from July through De-cember, with the highest amount of sperm storage in the epididymis occurring between Augustthrough November. Our observations indicate that epaulette sharks in the waters near HeronIsland mate from July through December. J. Exp. Zool. 284:586–594, 1999. The epaulette shark, Hemiscyllium ocellatum, studied in a range of sharks and rays in order to is a small benthic shark commonly found in shal- characterize the reproductive patterns of these low water on coral reefs in northern Australia and fishes (e.g., Sumpter and Dodd, ’79; Koob et al., New Guinea (Last and Stevens, ’94). Previous re- ’86; Rasmussen and Gruber, ’90, ’93; Callard et search on aquarium-held individuals showed that al., ’91, ’93, ’95; Rasmussen and Murru, ’92; this species bred year-round in a captive environ- Manire et al., ’95; Manire and Rasmussen, ’97).
ment, producing up to 50 eggs per year (West and Callard et al. (’91) described the hormone cycles Carter, ’90), but nothing is known of the repro- for oviparous and viviparous reproductive strate- ductive biology of this species in the wild.
gies as either synchronous or asynchronous. The Defining the breeding cycle of a wild population oviparous strategy was defined as a synchronous is an integral component to understanding the bi- cycle in which estradiol and progesterone concen- ology of a species. Some elasmobranch species, trations peak at the same time. Oviparous spe- such as the lesser spotted dogfish, Scyliorhinus cies were assumed to have a short cycle in which canicula, and the black dogfish, Centroscyllium both hormones increase during the follicular fabricii, appear to have a continuous breeding sea- growth phase and decline during the luteal phase.
son (Sumpter and Dodd, ’79; Yano, ’95), whereas An asynchronous pattern was used to define the many other species including the bonnetheadshark, Sphyrna tiburo, (Manire et al., ’95), the Aus-tralian sharpnose shark, Rhizoprionodon taylori, Grant sponsors: The Australian Coral Reef Society; Great Barrier (Simpfendorfer, ’92) and the blacktip shark, Car- Reef Marine Parks Authority; University of Queensland, AustraliaPostgraduate Research Scholarship.
charhinus limbatus, (Castro, ’96) have distinct sea- Work was conducted under QFMA permit numbers 6435 and PRM000801 and GBRMPA permit numbers G95/454 and G95/595.
*Correspondence to: M.R. Heupel, Mote Marine Lab, 1600 Ken Thompson Parkway, Sarasota, FL 34236-1096.
1999 WILEY-LISS, INC.
REPRODUCTION IN THE EPAULETTE SHARK
viviparous reproductive strategy with estradiol be- by Maruska et al. (’96): stage 1, primary or germi- ing dominant and peaking in the follicular growth nal zone; stage 2, early spermatocysts; stage 3, phase and progesterone dominant and peaking in spermatocytes; stage 4, spermatids; stage 5, im- the luteal phase which occurs later in the cycle.
mature sperm; stage 6, mature spermatocysts; and Our research into the reproductive biology of the epaulette shark was designed to examine the Blood samples for plasma hormone analysis were reproductive biology of this species and the sea- taken from five mature male and five mature fe- sonality of reproductive activity in the wild on a male sharks for each calendar month. A heparin- tropical reef off Heron Island, Queensland, Aus- ized syringe was used to take a 1 ml blood sample tralia. Components of the study involved anatomi- from the caudal vessel of sharks. Blood was trans- cal observation, histological analysis and sex ferred to an eppendorf tube, stored on ice for up to steroid hormone analysis to determine if there was 2 hr after which samples were centrifuged, the a defined breeding season in this species.
plasma pipetted off into clean eppendorf tubes, andstored frozen at –70°C. Plasma samples from male MATERIALS AND METHODS
sharks were assayed for androgens and samples Hemiscyllium ocellatum were captured during from females were analyzed for estradiol, progest- low tides by hand netting over the reef flat area erone and androgens. Samples were analyzed on Heron Island Reef. This large platform reef sur- using coated-tube radioimmunoassay kits for es- rounds Heron Island, a coral cay situated at tradiol, progesterone, and androgens (ICN Diag- 23°27'S and 151°55'E. Approximately 500 sharks nostics, Costa Mesa, CA) and counted using a were examined during tagging for a mark recap- gamma counter (Beckman, Fullerton, CA). Coated ture study. The reproductive condition (i.e., gravid, tube assay kits were validated by comparing re- carrying egg purses) and any obvious evidence of sults of a pooled sample including five female mating activities of mature females were noted.
samples with results for the same pooled sample The size (inner clasper length) and condition of analyzed by traditional radioimmunoassay meth- claspers of mature males were recorded and cal- ods. Due to practical considerations the assays cification of claspers was examined to estimate were run in two separate batches. Samples from January, May, and August formed the second Mature females from all months except May, batch along with additional samples from other June, July, and December and mature males from months. Due to a change in the assay (by the all months except February, May, June, and July manufacturer) the values for progesterone and es- were collected for reproductive organ examination.
tradiol in the second assay were significantly lower These specimens (32 female and 12 male) were than in the original assay and therefore these data used in morphometric as well as histological ex- amination. Measurements of oviducal glands Hormone concentrations were analyzed statisti- (length, width, thickness) and ova diameter were cally using Sigmastat (Jandel Scientific, San taken and oviducal gland measurements were mul- Raphael, CA). Non-parametric Kruskal-Wallace tiplied to obtain a volume estimate. Lengths and one-way ANOVA on ranks followed by an all widths of testes were measured. These measures pairwise multiple comparison (Dunn’s method) were compared to the size of the animal, time of were conducted using a critical probability value year, and condition of organs (e.g., active, inactive, of 0.05. Mean hormone concentrations were corre- regressing). Samples of testes were immersion fixed lated with mean monthly maximum water tem- in 4% formaldehyde for subsequent examination.
peratures obtained from records held at Heron These samples were processed for histology (Sh- annon Citadel 2000) and embedded in paraffin wax.
Sections (7 µm) were cut and mounted on glass slides before staining with Masson’s trichrome.
Males
Stained sections were examined and photographed Clasper elongation and calcification in male under light microscopy (Zeiss Axiophot, Germany).
sharks generally occurred when sharks were be- Testes were cut in cross section and the number of tween 55–60 cm total length (TL) (Fig. 1) with the spermatocysts in each stage were counted, mea- smallest mature male 54 cm TL and the largest sured, and expressed as a percentage of total immature male 61 cm TL. Inner length measure- spermatocysts. Individual stages of spermatogen- ments of fully calcified claspers were consistently esis were categorized into seven stages as described about 7% of the total body length of the shark.
M.R. HEUPEL ET AL.
TABLE 1. Average monthly water temperatures from Heron stages of spermatogenesis showed the expansion Island Reef recorded by research station staff1 of spermatocysts from stages 1–5 (Fig. 2). Stage 1 cells were generally about 1.0 µm in diameter.
Stage 7 spermatocysts were not measured due to their degenerative state. Sertoli cell size was con- sistent throughout the year and ranged from 0.7– 1.0 µm in diameter. When comparing the size of spermatogenic stages by month it was noted that on average November samples had the largest spermatocysts (i.e., stage 5: 34 mm). Samples from September and December were of similar sizes (stage 5: 30 mm in both), but specimens from Feb- ruary were considerably smaller (stage 5: 16 mm).
A distinct annual cycle in androgen concentra- 1Water temperature was measured daily from the jetty adjacent to tions was observed in male sharks (Fig. 3). Dif- the study site at a depth of 1 m at 8:30 am. Note: the thermometer is ferences in androgen concentrations between attached to a float to maintain a depth of 1 m at all times.
months were statistically significant (Kruskal-Wallace = 48.5, P < 0.01). Hormone concentra- Histological examination of sections of testes tions were significantly lower (P < 0.05) from showed the various stages of sperm production December through February (southern hemi- throughout the year (Table 2). In April sharks had sphere summer) with concentrations of <20 ng/ begun sperm production for the mating season.
ml observed. Concentrations rose gradually and Stages 1–6 were present and 50–75% of sperma- peaked in July–October at about 60 ng/ml before tocytes of individual testes were in stages 3 or 4.
starting to decline in November. There was an In August all stages of sperm production were inverse correlation between androgen concentra- present, with a limited portion of the testis de- tions and water temperature (r2 = 0.93). The high- voted to stages 1 and 7. The epididymis contained est concentrations of androgen coincided with sperm during August–November, with fullness observations of males with red and swollen clasp- appearing to be greatest in November. Testes in ers. Males in this condition were frequently found this condition were observed to be enlarged com- between July to December and were assumed to pared to previous months, and contained about 50% of spermatocysts in stages 5 or 6.
Females
Females were found to mature at approximately 55 cm TL. Females less than 55 cm had thin strap-like ovaries and only small non-yolky ova present.
Females above this size had well developed ova-ries with yolky ova present.
Measurements of 32 oviducal glands from ma- ture females showed a change in size through theyear (Fig. 4). Oviducal glands were smallest inJanuary–April. Subsequently, glands showed anincrease in width during August–November whensharks were reproductively active.
Sizes of vitellogenic ova were variable through- out the year with a range of 3–27 mm (Fig. 5).
Small ova (3–6 mm) were present in all femalesexamined. Females sampled in January had fewlarge ova present, and in February–March femaleshad few ova that appeared to be undergoing re-sorption. By April there were small numbers (10–15 per individual) of yolky ova that were 3–5 mm Percentage of 249 male Hemiscyllium ocellatum in diameter. In August females had at least five with fully calcified claspers as a function of total length.
pairs of large, yolky ova of varying sizes. The larg- REPRODUCTION IN THE EPAULETTE SHARK
TABLE 2. Testicular activity of mature male Hemiscyllium ocellatum throughout the year indicating presence of sperm and stages of spermatogenesis present est observed ova were about 25–27 mm with all 35 mm, green-brown in color with fine hair-like subsequent pairs smaller. The presence of egg pairs clumps of tendrils that covered the entire surface.
at this stage was observed throughout the remain- Androgen concentrations measured in females der of the breeding season (September–November).
were about an order of magnitude lower than No samples from ovaries were obtained in Decem- those in males (2–8 ng/ml) (Fig. 6a). Androgen con- ber, but pairs of egg capsules were collected from centrations were not significantly different be- females during August, October, November, Decem- tween months (Kruskal-Wallace = 11.8, P = 0.38) ber, and January. Examination of females during and there was no correlation between water tem- tagging excursions revealed gravid or pregnant fe- perature and hormone concentrations (r2 < 0.01).
males from August through early January. Females Estradiol and progesterone appeared to have sea- were also noted to have red, irritated tissue around sonal patterns (Fig. 6b, c). Estradiol concentrations the cloaca during the months of July and August.
were low during the southern autumn and winter This was probably a result of mating activities. It (March–Aug.) with concentrations of 0.05–0.2 ng/ was presumed that ova were ovulated at a size of ml. Concentrations rose to a peak (0.5 ng/ml) in 25–27 mm since this was the largest size of ova spring and early summer (September–November) before declining again in December–February. Es- Egg capsules were produced in pairs with at tradiol concentrations were significantly different least half of the egg capsule formed before ovula- between months (Kruskal-Wallace = 33.1, P < 0.01) tion. One female collected for dissection in August but sample size was not large enough to distin- had partially developed egg capsules within her guish where differences occurred. There was a uterus. The egg capsules were half formed, but weak inverse correlation between water tempera- no ovum had been ovulated. There were several ture and estradiol concentration (r2 = 0.19). Proges- pairs of large yolky ova present in the ovary sug- terone concentrations showed a different cycle by gesting the female was capable of ovulation. Egg peaking in autumn and winter months (June–July) capsules at deposition were approximately 90 × at concentrations up to 0.5 ng/ml. Concentrationsdecreased slightly in September–October and con- Distribution of average monthly androgen concen- Average diameter of spermatocysts in stages 1–6 trations (ng/ml) with standard errors for male H. ocellatum measured from male H. ocellatum in the month of Novem- sampled on Heron Island Reef. Asterisks indicate months with significantly lower (P < 0.05) androgen values.
M.R. HEUPEL ET AL.
Yearly volume distribution of oviducal glands from 32 mature female H. ocellatum excluding the months of May,June, July, and December.
tinued this trend through the remainder of theyear. Progesterone concentrations were also sig-nificantly different between months (Kruskal-Wallace = 17.6, P = 0.03) but differences could notbe statistically determined due to restricted samplesizes. There was a slightly stronger inverse corre-lation (r2 = 0.37) to water temperature than thatfor estradiol.
DISCUSSION
Information pertaining to the life history of hemiscyllid sharks in Australian waters is lim-ited. Although these species are commonly ob-served and are generally known to be oviparous(Compagno, ’84; Last and Stevens, ’94), their re- Measurements of ova diameter from 32 mature productive timing and periodicity are unknown.
female H. ocellatum throughout the year. Graphs depict the The limited research available on H. ocellatum in- progression of ovum size, number, and development throughthe year.
cludes one study on sharks maintained in a con-trolled aquarium environment (West and Carter,’90). There are no data available concerning the bonnethead shark, S. tiburo (Manire and Ras- reproductive activities of H. ocellatum in its natu- mussen, ’97). The claspers of mature R. terrae- novae were found to be about 7–8% of total body Male and female H. ocellatum were determined length (Parsons, ’83), similar to the measures to be reproductively mature at similar sizes. This found for H. ocellatum in this study.
was based on male clasper calcification and ex- Histological examination of testes showed that amination of female reproductive tracts. Using cal- sperm production had a seasonal cycle. Testes cification of claspers to determine sexual maturity were found to be inactive during the months of in male sharks has been used in many studies on January–March, a period when androgen concen- elasmobranch species, including the Atlantic trations were at their lowest. Sperm production sharpnose shark, Rhizoprionodon terraenovae began in April and continued to increase through (Parsons, ’83), the blue shark, Prionace glauca the months of August–November with all stages (Pratt, ’79), the chain dogfish, Scyliorhinus retifer of sperm production present. Sperm production (Castro et al., ’88) the sandbar shark, Carchar- increased as androgen concentrations also began hinus plumbeus (Joung and Chen, ’95) and the to increase. The epididymis contained the larg- REPRODUCTION IN THE EPAULETTE SHARK
rose prior to the breeding season and remainedhigh throughout the remainder of the egg layingseason. Testosterone concentrations in the lemonshark, Negaprion brevirostris, and other carchar-hinids were high during the breeding season, butconcentrations vary widely among species. Ne-gaprion brevirostris had a range of 75–110 ng/mltestosterone, while a study of several species ofcarcharhinid sharks reported a range of 0.85–358ng/ml. Our results from male H. ocellatum fallslightly below concentrations for N. brevirostris,but were within the range of those found for othercarcharhinid species. Based on these results, wepropose that testosterone may be important insexual behaviors, reproductive functions, or mayserve as a precursor for other unidentified steroids(Rasmussen and Gruber, ’90, ’93). Reproductive ac-tivity in male sharks may also be related to watertemperature based on the inverse correlation be-tween water temperature and androgen concentra-tions. The end of the mating season and decreasein androgens coincide with water temperature in-creases during summer months. Whether watertemperature plays any role as a reproductive cuefor male H. ocellatum is unknown, but should beinvestigated further.
Estradiol concentrations vary among species as well as throughout the reproductive cycle. Stud-ies on various carcharhinid sharks report estra- Distribution of average monthly (a) androgen, (b)
diol concentrations ranging from 0.4–4.5 ng/ml estradiol, and (c) progesterone concentrations (ng/ml) with
and 0.6–2.0 ng/ml (Rasmussen and Gruber, ’90; standard errors for mature female H. ocellatum sampled on Rasmussen and Murru, ’92). Raja erinacea had estradiol concentrations between 0.2–2.0 ng/mldepending on the reproductive status of the fe- est amount of sperm during November and in De- male (Koob et al., ’86). Estradiol concentrations cember sperm production dropped off as the of the bonnethead shark, S. tiburo, were analyzed mating season ended. This suggests that male ep- throughout the reproductive cycle. Concentrations aulette sharks are producing sperm for mating were lowest during early pregnancy (mean = 0.20 during the second half of the year. Males were ng/ml) but increased at mating (mean = 8.98 ng/ observed to have red, swollen claspers from July– ml) and peaked prior to ovulation (mean = 25.03 November. These observations support the hor- ng/ml) (Manire et al., ’95). Although estradiol con- monal data where androgen concentrations were centrations measured in H. ocellatum appeared highest from June to October and suggest that low with a peak of 0.5 ng/ml, these results are males generally mate between July and Novem- similar to other oviparous species (e.g., Koob et ber. Parsons and Grier (’92) defined a seven-stage al., ’86; Callard et al., ’91). Estradiol concentra- process of spermatogenesis for S. tiburo and tions in H. ocellatum increased to their peak dur- stated that not all spermatogenic stages were ing the period of egg laying while decreasing and present throughout the year. Based on their study remaining low during the period of regression and Parsons and Grier (’92) concluded that many shark species may undergo an annual testicular Increases in estradiol concentrations during the cycle of regression and recrudescence, while fewer follicular growth phase are common and have been species may have spermatogenic stages present linked to follicle size in the skate, R. erinacea, with estradiol concentrations increasing in parallel In H. ocellatum, male androgen concentrations with follicle size (Koob et al., ’86). Further re- M.R. HEUPEL ET AL.
search on R. erinacea and Squalus acanthias sup- tion season. Manire et al. (’95) reported progest- ported these data and showed that increases in erone increased during preovulation (mean = 8.9 both estradiol and testosterone characterized the ng/ml) and ovulation (mean = 16.6 ng/ml) prior to follicular phase (Callard et al., ’93). Research on a peak after ovulation (mean = 26.6 ng/ml) in S. carcharhinid sharks also showed an increase in tiburo. This result is similar to that described for estradiol just prior to mating as oocytes were ma- the dogfish S. acanthias (Callard et al., ’93). How- turing (Rasmussen and Gruber, ’90, ’93; Ras- ever, both S. tiburo and S. acanthias are vivipa- mussen and Murru, ’92). This increase is thought rous species and show a different pattern from to set ovulatory events in motion or may regulate the one described for the oviparous skate R. the reproductive cycle (Rasmussen and Murru, ’92; erinacea. Koob et al. (’86) reported an elevation Rasmussen and Gruber, ’93). Estradiol concentra- in progesterone for a restricted two day period be- tions in female H. ocellatum were highest in the fore encapsulation with a sharp drop on the day second half of the year and would coincide with of encapsulation and low concentrations through- maximum ova sizes, ovulation and egg laying.
out the rest of the year. Callard et al. (’93) re- Fluctuations in estradiol concentrations were cor- ported elevated concentrations of progesterone related with changes in water temperature, but pre- and peri-ovulation in R. erinacea. Serial because the relationship was weak it is unlikely samples examined from one captive female H. that water temperature plays a role in the timing ocellatum showed a peak in progesterone the morning the eggs had been laid (Heupel, unpub- Androgen concentrations in females of other spe- lished data). Concentrations previous to and af- cies are generally found to parallel estradiol con- ter this point were essentially undetectable, centrations. Several species have been analyzed suggesting that progesterone is most active at ovi- and showed an increase in testosterone concen- position in H. ocellatum. Progesterone is thought trations (along with estradiol) prior to and dur- to regulate events associated with ovulation, en- ing mating (Callard et al., ’91; Rasmussen and capsulation, and egg retention in oviparous spe- Murru, ’92; Rasmussen and Gruber, ’93; Manire cies and may have specific triggering roles in et al., ’95). Koob et al. (’86) found testosterone fluc- viviparous species. Progesterone may also inhibit tuated with estradiol, but was present in higher activities such as vitellogenesis (Koob et al., ’86; concentrations. Testosterone concentrations de- Rasmussen and Murru, ’92; Callard et al., ’93; crease after mating and remain low throughout the rest of the reproductive cycle of many elas- It has been suggested that hormone concentra- mobranch species (Rasmussen and Murru, ’92; tions in females of oviparous species peak more Rasmussen and Gruber, ’93). Testosterone may be than once during a season (Koob et al., ’86; Callard important in initiating some sequential ovulatory et al., ’91, ’93, ’95). However, this was not the case events and may have a role in courtship. Due to in our studies of H. ocellatum, and was not seen its lower concentrations throughout the rest of the in several other studies on oviparous species. Re- cycle, it does not appear to be important during search by Sumpter and Dodd (’79) examined the pregnancy in viviparous species (Rasmussen and hormone cycles of the lesser spotted dogfish, S. Murru, ’92; Rasmussen and Gruber, ’93). Andro- canicula. This species is oviparous and has an ex- gen concentrations in female H. ocellatum did not tended, if not continuous, breeding season. Despite vary significantly throughout the reproductive sea- the extended reproductive cycle of this species, es- son. Although it is possible that androgens are tradiol and testosterone concentrations displayed important in initiating changes in the reproduc- a distinct annual cycle. Both hormones fluctuated tive tract, no supporting evidence was found from together, rising as the ovary recrudesced and fall- androgen levels in H. ocellatum.
ing as the rate of egg laying decreased. Although Progesterone concentrations in female H. ocell- this study did not include progesterone analysis atum were usually low except for a peak from it clearly defined one estradiol peak rather than April–July prior to the egg laying period. As with several throughout the season. This pattern of one estradiol, there was a weak correlation between single peak in hormone concentrations per year water temperature and hormone concentrations, is similar to that observed for H. ocellatum but water temperature probably does not play a major role in progesterone activity. Peaks in Most oviparous elasmobranchs produce eggs in progesterone concentrations are thought to help pairs (Luer and Gilbert, ’85; Castro et al., ’88; Ellis prepare the reproductive tract for the egg produc- and Shackley, ’95; Yano, ’95). Epaulette sharks also REPRODUCTION IN THE EPAULETTE SHARK
produce eggs in pairs and appear to ovulate ova egg pairs is variable among oviparous elasmo- into egg capsules after they are at least half branch species. The thornback ray, Raja clavata, formed. Studies of at least two other oviparous can produce a pair of eggs from 0–2 days after elasmobranchs, the dogfish S. canicula, and the the previous pair (Ellis and Shackley, ’95). The clearnose skate, R. eglanteria, have shown simi- clearnose skate, R. eglanteria, takes slightly lar patterns of ovulation. Ova were not present longer with 4.5 ± 2.2 days between egg pairs (Luer in egg capsules less than three-fourths formed in and Gilbert, ’85) and the chain dogfish, S. retifer, S. canicula (Metten, ’39) and R. eglanteria formed requires 14–16 days between laying egg pairs two-thirds of the egg capsule prior to ovulation (Castro et al., ’88). The period between egg pair and fertilization (Luer and Gilbert, ’85). Metten production for H. ocellatum was not determined (’39) also described one pair of egg capsules that in the present study. Observation of captive (wild were fully formed but smaller than normal and caught) females at Heron Island Reef showed that without ova. No explanation was given for eggs none produced more than one pair of eggs (Heupel, in this condition, and no explanation is obvious unpublished data). This may have resulted from for the same condition observed in H. ocellatum females being kept in isolation when found to be gravid. One female kept isolated from male sharks The egg laying behavior of dogfishes has been produced a pair of empty egg capsules. Whether well documented with detailed descriptions of at- this was the result of not having a male present tachment of the long tendrils of the egg capsule in the tank, or was due to some other influence, to a vertical structure and the use of this struc- is unknown. However, the presence of red, swol- ture to pull the egg capsule from the oviduct len claspers and sperm production from July to (Castro et al., ’88). However, due to the difference December suggests that males are capable of mat- in tendrils found on egg capsules of H. ocellatum, ing throughout the egg laying season.
it is unlikely that they use this type of strategy.
Wourms (’77) defined three types of reproductive The long hair-like tendrils would appear to be cycle in elasmobranchs: (1) breeding throughout the more suited for egg laying similar to that de- year; (2) partially defined annual cycle with one or scribed for the clearnose skate, R. eglanteria. The two peaks during the year; and (3) a well defined egg laying behavior of R. eglanteria described by annual or biennial cycle. Although epaulette sharks Luer and Gilbert (’85) included the female set- held in a captive aquarium environment fell into tling quietly on the sediment before contracting the first category of Wourms’ description (West and the pelvic fins ventrally, shaking the pelvis from Carter, ’90), animals sampled in the natural envi- side to side, and swimming away leaving a single ronment fell into the last category. The differences egg capsule on the sediment. This activity was in results between aquarium-held sharks and wild- violent enough to leave the egg capsule covered caught sharks may be due to a lack of seasonal tem- in sediment from the bottom of the tank. This type perature variation in the aquarium environment.
of egg laying behavior would appear to be effec- As shown by correlation of water temperature and tive for depositing eggs under and among coral, male testosterone concentrations, seasonal tempera- and because the egg capsules of H. ocellatum lack ture changes may be a cue for commencement and long tendrils, it is likely that this type of method conclusion of the mating period. Removal of this would be used to attach egg capsules to coral. One cue may result in continuous mating activities. Fur- female H. ocellatum was held in a large aquarium ther examination of the effects of water tempera- with a number of different types of shelter and coral including one small piece of Acropora coral.
Although there were several other types of coral ACKNOWLEDGMENTS
present, the shark placed both egg capsules on We thank the staff at Heron Island Research the one piece of Acropora. We were unable to re- Station for their help throughout this research; move the egg from the coral by gently pulling the A. Chan for technical assistance, K. Townsend, two apart. Although no egg capsules have been T. Turner, and S. Bennett for field assistance.
discovered on the reef flat at Heron Island Reef, We also thank Dr. C. Manire and two anonymous the habits of this species suggest eggs are depos- reviewers for their advice and comments. This ited under coral heads. The presence of very small work was undertaken while the primary author juvenile sharks in Acropora beds suggest they may was in receipt of an Overseas Postgraduate Re- search Scholarship at the University of Queens- The length of time between laying of successive M.R. HEUPEL ET AL.
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Source: http://www.sharkmans-world.eu/research/epaulette_reproduction.pdf

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LAPORAN HASIL PENELITIAN GAMBARAN BAKTERI PADA INFEKSI SALURAN KEMIH PASIEN DENGAN BATU GINJAL DI RSUP SARDJITO dr. EKA YUDHA RAHMAN, M.Kes, SpU THE BACTERIAL PATTERN IN URINARY TRACT INFECTION AT THE KIDNEY STONE PATIENTS IN Dr. SARDJITO HOSPITAL EKA YUDHA RAHMAN1 , Aries Alpendri 2 , R HR Danarto3 1) Departement of Microbiology, Division of Urology, Departemen

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Physician Preference for Antiepileptic Drug Concentration Testing Robert J. Baumann, MD*†, Melody Ryan, PharmD*‡, and Aaron Yelowitz, PhD§ A four-item questionnaire asked active U.S. members Monitoring antiepileptic concentrations is also useful of the Child Neurology Society to value painless anti- in evaluating patient adherence to the treatment plan epileptic drug concen

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