Jittipan Chavadej

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.

Characterization of mucus-associated proteins from abalone (Haliotis) - candidates for chemical signaling

Living in groups is a widespread phenomenon in the animal kingdom. For free‐spawning aquatic animals, such as the abalone (Haliotis), being in the close proximity to potential mating partners enhances reproductive success. In this study, we investigated whether chemical cues could be present in abalone mucus that enable species‐specific aggregation. A comparative MS analysis of mucus obtained from trailing or fixed stationary Haliotis asinina, and from seawater surrounding aggregations, indicated that water‐soluble biomolecules are present and that these can stimulate sensory activity in conspecifics. Purified extracts of trail mucus contain at least three small proteins [termed H. asinina mucus‐associated proteins (Has‐MAPs)‐1–3], which readily diffuse into the surrounding seawater and evoke a robust cephalic tentacle response in conspecifics. Mature Has‐MAP‐1 is approximately 9.9 kDa in size, and has a glycine‐rich N‐terminal region. Has‐MAP‐2 is approximately 6.2 kDa in size, and has similarities to schistosomin, a protein that is known to play a role in mollusc reproduction. The mature Has‐MAP‐3 is approximately 12.5 kDa in size, and could only be identified within trail mucus of animals outside of the reproductive season. All three Has‐MAP genes are expressed at high levels within secretory cells of the juvenile abalone posterior pedal gland, consistent with a role in scent marking. We infer from these results that abalone mucus‐associated proteins are candidate chemical cues that could provide informational cues to conspecifics living in close proximity and, given their apparent stability and hydrophilicity, animals further afield.

Culture and development of the polychaete Perinereis cf. nuntia

Summary The polychaete Perinereis cf. nuntia, a tropical species endemic in Thailand, was cultured in captivity as follows: eggs and sperm from sexually mature (epitokous) P. cf. nuntia, were artificially fertilized, and settled into a sand bed about 30 cm deep at the nectochaete stage. The sand beds were supplied with seawater at 30-ppt salinity and the nectochaetes reared for five months after which time some adults were becoming epitokes. The culture method yielded 3–4 kg polychaetes at an atokous stage per m2 of culture area. Because the polychaetes were to be used to feed shrimp broodstock, samples of the worms were screened for the presence of white-spot syndrome and yellow-head viruses using polymerase chain reaction (PCR) methods prior to introduction to the system as founders and at monthly intervals during their culture. Morphological details of P. cf. nuntia from fertilization to nectochaete stage are described using light microscopy, transmission (TEM) and scanning electron microscopy (SEM). The egg is surrounded by a chorion layer, TEM reveals that the microvillous tip vesicles are putative sites for the binding of the sperm to the egg surface. TEM studies of the cortical reaction show that the cortical alveoli contain a fibrous substance which, after fertilization and membrane fusion, passes into the perivitelline space to form part of the fertilization membrane, as well as being secreted from the egg surface to form a jelly layer surrounding the egg.

Identification of an attractin-like pheromone in the mucus-secreting hypobranchial gland of the abalone Haliotis asinina linnaeus.

ABSTRACT Pheromones are chemicals used to communicate between animals of the same species, and are thought to be used by most marine animals. With limited vision, abalone primarily sense their world chemically, and pheromones may play an important role in settlement, attraction, recognition, alarm, and reproduction. Despite this, there has been no detailed investigation into pheromone substances, both in their precise biochemical nature or pheromonal function. In this study, we investigated the presence of pheromonelike substances from the hypobranchial gland of the abalone Haliotis asinina using bioassays, immunohistochemistry, Western blotting, and reverse-phase high-performance liquid chromatography (RP-HPLC). The hypobranchial gland of many prosobranchial marine molluscs has been classified as a sex auxiliary gland releasing unknown substances during spawning. In our study, cephalic tentacle assays demonstrated that the cell extracts of the hypobranchial gland contain chemical cues that are sensed by conspecifics. An antibody against the sea slug “attractin” pheromone was used as a probe to localize a similar protein in the mucin-secreting cells of the epithelial lining the hypobranchial gland of both male and female abalone. The approximate molecular weight of this abalone attractin-like protein is 30 kDa in both males and females. Fractionation of hypobranchial gland extracts by C5 RP-HPLC could not selectively purify this protein, and no sex-specific differences were observed. We predict that the attractin-like protein could be one of a number of important proteins involved in maturation, aggregation, and/or spawning behavior of abalone. In future research, additional hypobranchial gland components will be tested further for these types of behavior.

The distribution of APGWamide and RFamides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii

Immunohistochemistry was used to identify the distribution of both APGWamide-like and RFamide-like peptides in the central nervous system (CNS) and ovary of the mature female giant freshwater prawn, Macrobrachium rosenbergii. APGWamide-like immunoreactivity (ALP-ir) was found only within the sinus gland (SG) of the eyestalk, in small- and medium-sized neurons of cluster 4, as well as their varicosed axons. RFamide-like immunoreactivity (RF-ir) was detected in neurons of all neuronal clusters of the eyestalk and CNS, except clusters 1 and 5 of the eyestalk, and dorsal clusters of the subesophageal, thoracic, and abdominal ganglia. The RF-ir was also found in all neuropils of the CNS and SG, except the lamina ganglionaris. These immunohistochemical locations of the APGWamide-like and RF-like peptides in the eyestalk indicate that these neuropeptides could modulate the release of the neurohormones in the sinus gland. The presence of RFamide-like peptides in the thoracic and abdominal ganglia suggests that it may act as a neurotransmitter which controls muscular contractions. In the ovary, RF-ir was found predominantly in late previtellogenic and early vitellogenic oocytes, and to a lesser degree in late vitellogenic oocytes. These RFs may be involved with oocyte development, but may also act with other neurohormones and/or neurotransmitters within the oocyte in an autocrine or paracrine manner.

Recovery of Surfactant from an Aqueous Solution Using Continuous Multistage Foam Fractionation: Mixed Surfactant System

A continuous multistage foam fractionation column with bubble caps was used for surfactant recovery from mixed surfactant solutions containing polyethylene glycol tert-octylphenyl (OPEO10) and cetylpyridinium chloride (CPC) and the effects of air flow rate, foam height, and feed flow rate were investigated under a steady state of conditions. For the mixed surfactant system, the effect of synergism in the surfactant adsorption density was found. For separation efficiency, the total residual factor remained unchanged with an increasing feed molar fraction of OPEO10 (α), suggesting that the addition of OPEO10 does not increase the total separation efficiency. The residual factor of CPC increased with an increasing molar fraction of OPEO10 (α), while the residual factor of OPEO10 was lower for the mixed surfactant systems. A competitive removal was found in that the OPEO10 can compete with CPC for the bubble surface. The total separation factors and enrichment ratio of mixed surfactant systems were in-between the two single surfactant systems at a long foam residence time and, in contrast, showed antagonism at short foam residence. This is due to the difference in liquid entrainment in foam at long and short foam residence times.

Proportion of Sperm and Eggs for Maximal in vitro Fertilization in Haliotis asinina and the Chronology of Early Development

ABSTRACT To obtain the highest yield during in vitro fertilization of tropical abalone Haliotis asinina, optimal proportion of the gametes, the timing of sperm-egg interaction, and subsequent development were investigated. The highest yield of fertilization (75%) with fewest abnormal eggs was obtained when incubating eggs and sperm at the ratio of 1:100 in seawater with a salinity of 27.5 ppt, a pH of 7.8, and a temperature range from 27–29°C. After incubation, sperm swim through the eggjelly coat and become bound to the vitelline envelope within 30 sec, followed by an acrosomal reaction at 1 min. The fertilized egg extrudes the first and second polar bodies at 8–10 min, and then the zygote begins cleavage at 15–20 min. This is followed by the second cleavage, and development through the stages of blastula, gastrula, trochophore, veliger, and early creeping larvae, which were completed within 3 days. Noticeably, occurrence of egg jelly condensation after penetration of the first sperm would not allow other sperm bind to the egg jelly and to penetrate through its vitelline envelope. This event is thought to be a weak blocking against polyspermy, because the classic cortical reaction initiated by cortical granule exocytosis could not be observed in this species.