Preliminary report on bottlenose dolphin (Tursiops truncatus) uterine samples for parity analysis

There have been numerous studies on various mammalian species regarding vascular changes in uterine arteries elucidating the effects of parity. In equids, vascular changes of uterine arteries have been demonstrated to occur in uniparous and multiparous mares. The severity of these arteriole changes suggests a link to previous pregnancies. Differences in the number or range of pregnancies can be ascertained through microscopic evaluation of elastin deposition in the arterioles, perivascular fibrosis, and stromal cellularity. There has been little, if any, work performed on parity in the bottlenose dolphin (Tursiops truncatus). The objective of this preliminary study was to determine the feasibility of detecting similar vascular changes in the endometrium of known-aged female bottlenose dolphins to assess parity. Archived formalin fixed samples of uterus were obtained from nine bottlenose dolphins with known age and parity. Four slides were made from each sample and individually stained with four different techniques. From our small sample pool, it appears that uteri from nulliparous animals do not develop perivascular fibrosis. Parous uteri developed perivascular fibrosis and arteriolar elastosis. These changes agree with our expectations that some degeneration (elastosis) and compensation (fibrosis) occurs as a result of uterine expansion of pregnancy. The assessment of this technique for use in bottlenose dolphins would provide an important tool in the determination of the reproductive success of dolphin populations, identify individuals who are sexually mature but nulliparous, which could indicate reproductive dysfunction or increased calving intervals, and increase our knowledge on the role contaminants play in reproductive dysfunction.

Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here.

Microbial community analysis of Acropora palamata mucus swabs, water and sediment samples from Hawksnest Bay, St. John, U.S. Virgin Islands

Colonies of the scleractinian coral Acropora palmata, listed as threatened under the US Endangered Species Act in 2006, have been monitored in Hawksnest Bay, within Virgin Islands National Park, St. John, from 2004 through 2010 by scientists with the US Geological Survey, National Park Service, and the University of the Virgin Islands. The focus has been on documenting the prevalence of disease, including white band, white pox (also called patchy necrosis and white patches), and unidentified diseases (Rogers et al., 2008; Muller et al., 2008). In an effort to learn more about the pathologies that might be involved with the diseases that were observed, samples were collected from apparently healthy and diseased colonies in July 2009 for analysis. Two different microbial assays were performed on Epicentre Biotechnologies DNA swabs containing A. palmata coral mucus, and on water and sediment samples collected in Hawksnest Bay. Both assays are based on polymerase chain reaction (PCR) amplification of portions of the small rRNA gene (16S). The objectives were to determine 1) if known coral bacterial pathogens Serratia marcescens (Acroporid Serratiosis), Vibrio coralliilyticus (temperature-dependent bleaching, White Syndrome), Vibrio shiloi (bleaching, necrosis), and Aurantimonas coralicida (White Plague Type II) were present in any samples, and 2) if there were any differences in microbial community profiles of each healthy, unaffected or diseased coral mucus swab. In addition to coral mucus, water and sediment samples were included to show ambient microbial populations. In the first test, PCR was used to separately amplify the unique and diagnostic region of the 16S rRNA gene for each of the coral pathogens being screened. Each pathogen test was designed so that an amplified DNA fragment could be seen only if the specific pathogen was present in a sample. A positive result was indicated by bands of DNA of the appropriate size on an agarose gel, which separates DNA fragments based on the size of the molecule. DNA from pure cultures of each of the pathogens was used as a positive control for each assay.

Assessing the precision of frequency distributions estimated from trawl-survey samples

In trawl surveys a cluster of fish are caught at each station, and fish caught together tend to have more similar characteristics, such as length, age, stomach contents etc., than those in the entire population. When this is the case, the effective sample size for estimates of the frequency distribution of a population characteristic can, therefore, be much smaller than the number of fish sampled during a survey. As examples, it is shown that the effective sample size for estimates of length-frequency distributions generated by trawl surveys conducted in the Barents Sea, off Namibia, and off South Africa is on average approximately one fish per tow. Thus many more fish than necessary are measured at each station (location). One way to increase the effective sample size for these surveys and, hence, increase the precision of the length-frequency estimates, is to reduce tow duration and use the time saved to collect samples at more stations.

Improving pinniped diet analyses through identification of multiple skeletal structures in fecal samples

Marine mammal diet is typically characterized by identifying fish otoliths and cephalopod beaks retrieved from stomachs and fecal material (scats). The use and applicability of these techniques has been the matter of some debate given inherent biases associated with the method. Recent attempts to identify prey using skeletal remains in addition to beaks and otoliths are an improvement; however, difficulties incorporating these data into quantitative analyses have limited results for descriptive analyses such as frequency of occurrence. We attempted to characterize harbor seal (Phoca vitulina) diet in an area where seals co-occur with several salmon species, some endangered and all managed by state or federal agencies, or both. Although diet was extremely variable within sampling date, season, year, and between years, the frequency and number of individual prey were at least two times greater for most taxa when prey structures in addition to otoliths were identified. Estimating prey mass in addition to frequency and number resulted in an extremely different relative importance of prey in harbor seal diet. These data analyses are a necessary step in generating estimates of the size, total number, and annual biomass of a prey species eaten by pinnipeds for inclusion in fisheries management plans.

Seasonal growth of King George whiting (Sillaginodes punctata) estimated from length-at-age samples of the legal-size harvest

Samples of 11,000 King George whiting (Sillaginodes punctata) from the South Australian commercial and recreational catch, supplemented by research samples, were aged from otoliths. Samples were analyzed from three coastal regions and by sex. Most sampling was undertaken at fish processing plants, from which only fish longer than the legal minimum length were obtained. A left-truncated normal distribution of lengths at monthly age was therefore employed as model likelihood. Mean length-at-monthly-age was described by a generalized von Bertalanffy formula with sinusoidal seasonality. Likelihood standard deviation was modeled to vary allometrically with mean length. A range of related formulas (with 6 to 8 parameters) for seasonal mean length at age were compared. In addition to likelihood ratio tests of relative fit, model selection criteria were a minimum occurrence of high uncertainties (>20% SE), of high correlations (>0.9, >0.95, and >0.99) and of parameter estimates at their biological limits, and we sought a model with a minimum number of parameters. A generalized von Bertalanffy formula with t0 fixed at 0 was chosen. The truncated likelihood alleviated the overestimation bias of mean length at age that would otherwise accrue from catch samples being restricted to legal sizes.

Length and age at maturity of female petrale sole (Eopsetta jordani) determined from samples collected prior to spawning aggregation

The problem of bias in female petrale sole age and length-at-maturity relationships caused by sampling from spawning aggregations was investigated. Samples were collected prior to aggregation, and histological methods were used to determine maturity status. Mature and immature fish were classified by inspecting oocytes for the presence of yolk in September, when substantial divergence in yolked and unyolked oocyte diameters had been observed. Comparison of macroscopic and microscopic assessment of maturity showed that maturity status cannot be determined accurately by using macroscopic inspection during the summer. Female petrale sole from the central Oregon coast were 50% mature at 33 cm and 5 years of age. Comparison of data from our study with data used in recent petrale sole stock assessments showed that both sampling bias and the use of samples from sea-sons when status cannot be accurately determined have likely caused errors in fitted maturity relationships.

Lunar, diurnal and tidal periodicity in relation to the prawn abundance and migration in the Godavari estuarine systems

Preliminary investigations to study the lunar, diurnal and tidal periodicity in abundance and migrations of prawns were made in the Bairavapalam distributary of the Goutami branch of the Godavari estuary during the period of November 1961 to July 1962. The study was based on observation of the catches of a stake net (bag net) operated near the mouth of the estuary. Records of the catches were maintained tidewise daily. Comparative estimates of abundance were made on the basis of catch per hour's operation. It was generally observed that the catches were higher during the darker half of the month than in the brighter fortnight. The landings during low tides were generally higher than those during high tides and usually heavier catches were made during nocturnal low tides than during the day low tides. A continuous inward and outward (immigration and emigration) movement of prawns of all size groups was observed in the estuary from November to July. In general, more penaeid prawns were found to be immigrating at dawn than at dusk. Similarly, the number of emigrants was also found to be generally higher during the new moon period than during the full moon. Metapenaeus monoceros showed an almost distinct nocturnal periodicity in migration, while no such periodicity was observed, distinctly, in other species. In the case of Penaeus indicus the movement of migrants was prolonged. In M. brevicornis the migrants were scarce till March and thereafter increased numerically. The migrant forms of Metapenaeus dobsoni continued to be abundant till May with peak periods in January and February. The migratory pattern of Metapenaeus affinis was similar to that of Metapenaeus brevicornis, though the migrants of the former species appeared a month earlier than the latter. Intensive studies over extended areas for longer periods are required to understand clearly the migratory pattern of the various species. The phenomenon of immigration of prawns can be clearly understood only by vital staining or tagging studies. Perhaps the emigrants might be returning with the succeeding changing life. To verify this, laboratory experiments, by vital stains, were conducted. The marked specimens, if released during the low tides on a large scale, may be recaptured during the subsequent high tides and the duration also may be calculated. At least some percentage of the emigrants remains in the sea for maturity and breeding.

Growth of the buccaneer anchovy Encrasicholina punctifer off Mozambique, based on samples collected in research surveys

Growth of Encrasicholina punctifer (Engraulididae) is estimated, based on samples obtained during five surveys on Sofala Bank (central Mozambique). The estimated values of the von Bertalanffy growth function, TL∞=12 cm and K=2.0 yearˉ¹ are compared with results of growth studies elsewhere in the Indo-Pacific region, based mainly on the index Φ=log(sub)10 K+2*log(sub)10L∞ assumed to be constant within species. This comparison is rendered difficult by the uncertain taxonomical status of stolephorid anchovies.

Observation on the lunar and tidal influence on gill netting in the Bay of Bengal

The effect of lunar and tidal influence on the landing of pelagic fish with drift gill nets has been studied for the first time from Bay of Bengal along the Orissa coast. The catchability of the gear was highest during the first quarter and lowest in the fourth quarter of the moon. The variation in catch rates during the four different quarters was not statistically significant. Contrary to the earlier belief, landing with gill net was not poor during the moonlit nights. The extensive use of bluish grey nylon twine matching with seawater in place of indigenous hemp or cotton twine, has largely eliminated the visibility of gear during the brighter phase of the moon. The rate of exploitation was found to be significantly higher during the nights of neap tide, compared to those of spring tide. A plausible explanation for this phenomenon has been offered.

Preliminary observations on the lunar and tidal influences on the catches of seer by gill nets

The paper deals with the lunar and tidal influences on the catches of seer by gill nets. The landings during full moon and new moon nights, during low and high tides and during different quarters of the lunar month for three fishing seasons are discussed.

The study of maturational progress in the whitespotted rabbitfish (Siganus sutor) under different photoperiod and light intensity conditions

The rule of light on the timing of maturation and spawning in tropical and subtropical regions is not clear well, because the reproductive cycle in these systems is lunar synchronized. In this study, thus, the effects of different light regimes were investigated on maturational progress of whitespotted rabbitfish, Siganus sutor, the commercial species in Persian Gulf and Oman Sea. During prespawning season, 50 adult fish were randomly divided into ten 300-l tanks (n=5). The fish in control tank received indoor light condition and the fish in each other tanks were exposed to nine different combinations of photoperiod (8L: 16D, 12L: 12D, 16L: 8D) and light intensity (1000, 2000, 3000 lux). After 60 days, GSI and HSI values, serum levels of estradiol (E2), testosterone (T), 17-α hydroxyprogestrone (OHP), calcium (Ca2+) and gonad histology were evaluated for females and males. In females the GSI mean values of exposed fish increased in comparison with control except for fish were kept under 8L, 2000 lux (tank 8). These differences were significant only for fishes in tank 7 (8L, 3000 lux). In the cases of HSI, the results were converse, so that, the most of thanks showed significant decreasing than fishes reared under indoor condition. Morphology and histology study of Ovaries showed three developmental phases including 3, 4 and chiefly 5 that were parallel with GSI values. Fortheremore the serum levels of E2 was recorded between 0.54-15.8 ng/ml in different fish and their mean values were lower than control in all treatmants (P> 0.05). In males, the similar results were obtained. The GSI and HSI mean values in experimental regimes compared with control were upper and lower, respectively, except for fishes were reared in tank 1 (16L, 3000 lux). Testes histology of fishes were reared under different regimes showed signs of stage 5, since no blood vessels observed and thick milt exuded on slight pressure. The mean values of testosterone consentration in fishes were kept in tanks 1 and 6 (12L, 1000 lux) were higher and in other ones were lower than control group. It is also noted that the OHP and Ca2+ had diverse results including increasing and decreasing mean values than control. So, these factors contrary to E2 and T were not suitable to evaluation of maturity in both sexes. On the basis of ovarian structure in stage 5, oocyte development pattern in this species was group synchronous. So, increased mean of GSI versus decreased values of HSI, E2 and perhaps Ca2+ were the signs of improved maturation. But in males, reduced levels of T and similarity of testes morphology in all samples caused that GSI mean value was the only indicator for differentiating among treatments. These findings suggest that alternations were used in light regimes have been the reason of improved maturity in all treatments except fishes reared in tank 8. The ١٠٧ rule of light intensity on induction of maturity was cleared by comparision between fishes in tanks 4, 5, 6 and control group. Because day length was the same whereas fishes in tanks 4, 5 and 6 were exposed to increased light intensity compared with control. This fact verified by results was obtained from fishes in tanks 9 and especially 7, since photoperiod in this group was lower than control. So, higher intensity was considered as the reason of alternations. Contrasting with indoor condition, Induced maturity was also cleared for fishes were kept in tanks 1, 2 and 3, where both of light duration and intensity were increased. But, the rule of photoperiod was individually demonstrated when obtained results were compared with similar light intensities in other treatments. In conclusion, with comparison among different light regimes it is declared that siganids were kept under light condition of tank 2 including 16h light duration combined with 2000 lux intensity showed the best signs of sexual maturation in both sexes. On the basis of this study, setting up the spring light condition during prespawning season induces maturation in withspotted rabbitfish. This improvement not only by influence of photoperiod or light intensity, separately, but obtained through interaction between them. Thus, determination of threshold and resistance to light be recommended before exposure, although using proper regime during suitable time are necessary to achieve purposes considerably.