Serotonin induces ovarian maturation in giant freshwater prawn broodstock, Macrobrachium rosenbergii de Man

This study investigated the effects of serotonin (5-hydroxytryptamine or 5HT) on ovarian development in Macrobrachium rosenbergii de Man. Adult female prawns at the ovarian stage I (spent) were injected with 5HT at 1, 5, 10, 20 and 50 μg g^− 1 body weight (BW) intramuscularly on days 0, 5 and 10, and sacrificed on day 15. The doses as related to the effect could be categorized into three levels: low (1 and 5 μg g^− 1 BW of 5HT), medium (10 and 20 μg g^− 1 BW of 5HT) and high (50 μg g^− 1 BW of 5HT). The low-dose, especially at 1 μg g^− 1 BW, caused prawns to exhibit a significant increase in ovarian index (ovarian weight/body weight × 100) (5.79 ± 0.09%) as compared to the control (1.49%). The ovaries of most of these prawns could develop to stage IV (mature) and contained synchronously mature oocytes while most of the control ovaries remained at stage I and II (proliferative), and contained only oogonia to previtellogenic (Oc1, Oc2) and early vitellogenic oocytes (Oc3). The medium- and high-dose treated prawns exhibited ovaries that could reach stages III and IV and contained various types of oocytes of different maturity. Pretreatment with 5HT receptor antagonist, cyproheptadine (CYP), at 10 μg g^− 1 BW before 5HT injection significantly suppressed the effect of 5HT. Intramuscular injection of the 5HT-primed thoracic ganglion culture medium into CYP-pretreated prawns resulted in the increase of ovarian index about 5–6 times more than in the control, and in the groups injected with 5HT-primed media from muscle strip, eyestalk and brain. The ovaries of most prawn could develop up to stage IV and contained synchronously developed vitellogenic (Oc4) and mature oocytes (Oc5). These findings suggest that 5HT indirectly induces ovarian development and oocytes maturation in M. rosenbergii, probably via a putative ovarian stimulating factor released from the thoracic ganglia.     

Development of primers and a procedure for specific identification of the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3′-end of 18S rDNA through 5′-end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.     

Enhancement of survival and metamorphosis rates of <Emphasis Type="Italic">Penaeus monodon</Emphasis> larvae by feeding with the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

The purpose of this study was to compare the performance of two species of diatoms, Thalassiosira weissflogii and Chaetoceros gracilis, in the larviculture of the black tiger shrimp Penaeus monodon. Shrimp larvae were fed with either C. gracilis, T. weissflogii, or a combination of the two species of diatoms. The larvae fed solely with T. weissflogii or a combination of the two types of diatom had significantly higher survival rates and faster metamorphosis than those fed solely with C. gracilis. The numbers of diatom cells consumed by larvae during 3-h periods were determined, revealing that larvae consumed significantly higher numbers of C. gracilis than T. weissflogii. However, when the protein, total fatty acid, and polyunsaturated fatty acid content of the two species of diatom are compared, significantly higher amounts of each are found in T. weissflogii. Converting the number of diatom cells consumed into equivalent protein, total fatty acid, eicosopentaenoic acid, and decosahexaenoic acid reveals that larvae fed with T. weissflogii received significantly higher amounts of all the nutrients, compared to those consuming C. gracilis. The results showed an advantage of feeding T. weissflogii to C. gracilis in enhancing survival and metamorphosis in P. monodon larvae.     

Delay of the egg activation process in the Black Tiger Shrimp Penaeus monodon by manipulation of magnesium levels in spawning water

The aim of this study was to determine whether magnesium (Mg²⁺) in seawater is required for egg activation of the black tiger shrimp Penaeus monodon and whether manipulation of Mg²⁺ levels can be used to delay the process and thereby synchronize egg activation. Female P. monodon broodstock were allowed to spawn in artificial seawater containing Mg²⁺ at varying levels with respect to the normal (100%) level: 100%, 50%, 20% and 0%. Egg activation occurred normally at 100% Mg²⁺, incompletely at 50% and 20% Mg²⁺ levels and did not occur at all with 0% Mg²⁺. The fertilization rate with 100% Mg²⁺ was observed to be 83%, but fertilization failed to take place in all the other groups. The fertilization rate was restored from 0% to 76% following the 20% Mg²⁺ level treatment when Mg²⁺ levels returned to normal (100%) as soon as spawning was completed. This study suggests that the level of Mg²⁺ in seawater plays a vital role in P. monodon egg activation, and that commencement of this process could be delayed by manipulation of the Mg²⁺ level during and immediately after spawning.     

Effect of body size and sub-optimal water quality on some hemato-immunological parameters of spotted babylon snail <Emphasis Type="Italic">Babylonia areolata</Emphasis>

Cellular and humoral effectors are imperative for the innate defense mechanisms of invertebrates, including the spotted babylon snail Babylonia areolata, a marine gastropod belonging to the family Buccinidae. In this study, we have investigated the influence of body size [5–8 g (small), 10–12 g (medium) and 15–17 g (large)], and culture waters of varied quality [natural seawater, low salinity, low alkalinity, high total ammonia nitrogen (TAN) and artificial seawater] on some hemato-immunological parameters [total hemocytic counts (THC), hemolymphatic glucose, total protein concentration, phenoloxidase (PO), agglutinating and lysozyme activity] of the snail. Growth performance measures and survival of test snails were also evaluated after rearing them in culture water of varied quality. Body size did not influence most parameters assessed, except that the lysozyme and PO activities of medium and large-size snails were higher than those of the small-size snails. Sub-optimal culture water quality affected hemato-immunological factors, growth and/or survival of the spotted babylon snail. Seawater with low salinity, low alkalinity and a high TAN level caused decreased THC, hemolymphatic glucose, and PO activity; increased lysozyme activity; and clearly retarded growth of the snail. The snails held in artificial seawater could not survive after 4 weeks, suggesting the lack of certain essential factors necessary for their survival.     

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon (Decapoda,Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)‐labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti‐FITC‐conjugated gold‐lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar‐associated components of the HE. This revealed that both Con A and WGA‐binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold‐labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N‐acetyl‐β‐d ‐glucosamine (sugar affinity to WGA).     

Water quality control in a closed re-circulating system of Pacific white shrimp (Penaeus vannamei) postlarvae co-cultured with immobilized Spirulina mat

Immobilized Spirulina mat was co-cultured with Pacific white shrimp (Penaeus vannamei) postlarvae for water quality control in a closed re-circulating system. During the culture period, the experimental Spirulina mat system showed total ammonia nitrogen and nitrate accumulation rates lower by 32.92 and 32.26 %, respectively, compared with the system without the mat. The water exchange rate in the system with the mat was comparatively reduced by 40 %. Moreover, survival rate, growth rate, and size (length) of the co-cultured shrimp larvae were greater than those of the control (P < 0.05): 72.3 ± 5.2 versus 61.3 ± 4.4 %; 0.55 versus 0.44 mm day^?1; and 9.8 ± 0.5 versus 8.7 ± 0.3 mm, respectively. The most frequently observed microbes by PCR-DGGE, either with or without Spirulina mats, were species related to classes γ-, α-, and β- of Proteobacteria (95–98 % similarity), i.e., Nitrosomonas marina, Pseudomonas sp. ITRI66, Fusobacterium ulcerans, and Exiguobacterium arabatum, and species related to Bacteriodetes, i.e., uncultured Flavobacteriales (with a similarity of 96 %). However, species related to Nitrobacter winogradskyi and Stenotrophomonas maltophilia were present only when the mats were applied, suggesting a mutualistic role in the aquaculture system. These bacterial communities, in cooperation with Spirulina sp., could enhance the shrimp response against disease, as well as growth and survival rates, and also improve the shrimp culturing water quality.     

Biochemical characterization and physiological role of cortical rods in black tiger shrimp, Penaeus monodon

Cortical rods (CRs), precursors of egg jelly investment in many penaeoid shrimp, are composed of different proportions of proteins and carbohydrates, the physiological role of which still requires extensive investigation. In this study, we demonstrated the biochemical properties of the CRs and their role in the induction of the acrosome reaction (AR). Profiles of the isolated CRs revealed a number of major protein bands ranging from 35 to 230 kDa. These CR proteins were extensively glycosylated and sulfated. Lectin-based carbohydrate analysis further revealed the highest reactivity of concanavalin A (Con A) among other lectins used. In addition, the selective interference of Con A binding with mannose but not glucose indicated that CR glycoproteins were of high-mannose type. Using immunoblotting with anti-CR antibody, we further demonstrated that part of egg water (EW, a natural AR inducer) was derived from miscible components of the CRs. Physiological tests of water-soluble CR (wsCR) revealed its high AR inducing competency comparable to that of EW, which was far superior to that of acid-urea treated CR (auCR). Furthermore, the wsCR-induced AR was selectively inhibited by Con A, suggesting the significance of the exposing mannose residues in regulating P. monodon sperm AR response.     

Sperm capacitation of the shrimp Litopenaeus vannamei

Litopenaeus vannamei is one of the most important species of farmed shrimp. The females have an ‘open’ thelycum. Mating is accomplished by attaching the male spermatophore onto the surface of the thelycum 4–6 h before spawning. During this period, sperm may have to undergo morphological changes associated with a capacitation process that has been described for other shrimp species. The objective of this research was to extend research on sperm capacitation in L. vannamei by ultrastructural and biochemical means. The sperm of L. vannamei were divided into those freshly prepared from the spermatophore (S‐sperm), extracted from the male gonopores, and those extracted from the female thelycum (T‐sperm). Under transmission electron microscopy, ultrastructural differences were detected between the S‐ and the T‐sperm in the nuclear material, the filamentous meshwork and the cytoplasmic particles. Under scanning electron microscopy, the difference was observed in the cap and spike regions. Immunofluorescence using confocal microscopy to detect tyrosine phosphorylated proteins revealed different distribution patterns between S‐ and T‐sperm. The location of phosphorylation activity changed from the spike in S‐sperm, to the filamentous meshwork in the T‐sperm. These morphological and biochemical changes confirm that capacitation of L. vannamei sperm takes place following mating.     

Vertical transmission and concurrent infection of multiple bacterial pathogens in naturally infected red tilapia (Oreochromis spp.)

Horizontal transmission of various bacterial pathogens in tilapia is well described, but there is scant information regarding their vertical transmission. This study aimed to determine the possibility of vertical transmission of two commonly reported bacterial pathogens (Francisella noatunensis subsp. orientalis and Shewanella putrefaciens) in natural stocks of red tilapia (Oreochromis spp.). Vertical transmission of these pathogens via gametes was evaluated using in vitro fertilization from 10 different families and analysing for the presence of bacteria in milt, unfertilized eggs, fertilized eggs and offspring at various ages (1‐day‐old larvae, 10‐day‐old fry and 30‐day‐old fingerlings), as well as water samples using colorimetric LAMP assay. The study revealed that both F. n. orientalis (6/10) and S. putrefaciens (4/10) was transmitted vertically to the fertilized eggs. Analysis of the water samples from different water sources (brood stock tanks, hatching chamber and larval rearing tanks) showed that both the pathogens were present in water samples with highest prevalence for F. n. orientalis followed by S. putrefaciens. Analyses for the presence of two pathogens in various organs (gonads, gill, liver, spleen, kidney and brain) of the healthy tilapia broodstock without any clinical symptoms of disease demonstrated they were carriers of S. putrefaciens and F. n. orientalis. This is the first documented evidence that vertical transmission via the broodstock of tilapia may also play an important role in transmitting these problematic pathogens to their progeny and underlines the necessity to modify the current disease management strategies in tilapia aquaculture.