Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.     

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum)

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.     

Temporal changes in the cyst densities of Pyrodinium bahamense var. compressum and other dinoflagellates in Manila Bay, Philippines

Temporal variation in the type and abundance of dinoflagellate cysts in Manila Bay, Philippines, is established using ²¹⁰Pb-dated sediment cores. At least 17 dinoflagellate cyst species, including those of the toxic species, Pyrodinium bahamense var. compressum, were identified. P. bahamense may have been present in the area since at least the 1920s. Total cyst density has increased beginning about 1988 to 1998 coinciding with records of P. bahamense blooms in the area. Heterotrophs have always dominated the cysts assemblage. These changes in the dinoflagellate record and the P. bahamense blooms in recent years may have been induced by the interplay of warmer temperatures, high rainfall leading to higher river discharge and less turbulent waters due to passage of few tropical cyclones.     

Somatic embryogenesis and plantlet regeneration in mango (Mangifera indica L.)

Summary The ‘Carabao’ or ‘Manila Super’ mango (Mangifera indica L.), a virtually neglected fruit before the advent of KNO₃ flower induction in the early 1970s, is now the third leading Philippine export fruit after banana and pineapple. To apply biotechnology for improvement, a reliable embryogenesis and regeneration protocol is required. We have developed a protocol for somatic embryogenesis and plantlet regeneration in mango: eight strains of ‘Carabao’ and two unidentified varieties, PHL 12384 and PHL 12378. Over 40 batches of nucellar explants from immature fruis (0.75–5.0 cm long) were cultured in vitro from April 1999 to April 2000. Two media were used, MMSE. Mango Medium for Somatic Embryo Induction, Proliferation and Germination and MMPR, Mango Medium for Plantlet Regeneration. These are now routinely used. The protocol is reproducible in 14 other varieties of mango. Shifting the base medium from Gamborg's B5 medium to our own formulation. BP medium (Barba and Pate?a's formulation) effectively controlled browning. Browning has limited the successful in vitro culture of many woody species including the mango. Crop Science Society of the Philippines (CSSP) 2001 Best Paper Award, Asian Agriculture Congress, Westin Philippine Plaza, Manila, Philippines, April 24–27, 2001 and Philippine Fruit Association 2000 Best Poster Award, 8th National Symposium. PCARRD, Los Ba?os, Laguna, Philippines, November 14–16, 2000.     

At the tipping point: Differential influences of warming and deoxygenation on the survival,emergence, and respiration of cosmopolitan clams

Although warming and low dissolved oxygen (DO) levels are co‐occurring significant climatic stressors in the ocean, the combined effects of these stressors on marine benthic animals have not been well established. Here, we tested the effects of elevated temperatures and low dissolved oxygen levels on the survival, emerging behavior from sediment, and the respiration of juvenile cosmopolitan Manila clams (Venerupis philippinarum) by exposing them to two temperatures (20 and 23.5°C) and DO levels (3.5 and 6–7 mg/L). Although within previously described tolerable ranges of temperature and DO, this 3.5°C increase in temperature combined with a 50% decrease in DO had a devastating effect on the survival of clams (85% mortality after 8 days). The mortality of clams under normoxia at 23.5°C appeared to be higher than under the low DO condition at 20°C. On the other hand, more clams emerged from sediment under the low DO condition at 20°C than under any other conditions. Oxygen consumption rates were not significantly affected by different conditions. Our results suggest temperature elevation combined with low oxygen additively increases stress on Manila clams and that warming is at least as stressful as low DO in terms of mortality. However, low DO poses another threat as it may induce emergence from sediment, and, thus increase predation risk. This is the first evidence that a combination of warming and deoxygenation stressors should reduce population survival of clams much more so than changes in a single stressor.     

Effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum

The effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum at the temperature of 19.6–21.6 °C, the salinity of 34‰ and pH of 8.0 were investigated from May 18 to July 18, 2006. In this study, the early, middle and late umbo-veliger larvae with the shell lengths of 100, 140, and 190 μm were subject to temporary food deprivation for up to 4.5, 20, and 25d at 0.5, 4, 5d intervals, followed by refeeding for the remaining of a 24, 20, 25d period, respectively. The results suggested that the larvae should have shown considerable tolerance to starvation due to their endogenous and exterior nutrition material, for larvae and time to the point-of-no-return (PNR: the threshold point during starvation after which larvae could no longer metamorphose even if food is provided) were calculated to be 4.25, 17.54, and 22.17d. As the starvation period prolonged, the mean shell length of larvae starved got close to constants at 1.5, 4, and 15d after starvation, which were different for larvae at different stages when starvation began, survival of larvae decreased, and was lower in treatments starved earlier in development than those starved later, for the early, middle and late umbo-veliger larvae, after 4.5, 20 and 25d of starvation period, few larvaes were alive. After starvation period, the alive larvaes were able to metamorphose and had a capability of compensatory growth when refeeding was given. Starvation not only affected metamorphosis rate, but also caused the delay in the time to metamorphosis and the decrease in the metamorphosed sizes. For example, for the continuously-fed larvae, duration to metamorphosis was 20.7d, for larvae with a size of 100-μm starved for up to 4d, larvae with a size of 140-μm starved for up to 16d, larvae with a size of 190-μm starved for up to 20d, duration to metamorphosis were 29.7, 31.7, and 37.7d, the delay in duration to metamorphosis were 9, 11, and 17d, respectively. Furthermore, importance of nutrition material for maintaining larval survival during starvation and the compensatory growth on larvae at the same feeding time were discussed.     

Enhanced chilling tolerance in Zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine

Following leaf application of salicylic acid (SA), calcium chloride, hydrogen peroxide and 6-benzylaminopurine (BA), Manila grass (Zoysia matrella) plants were exposed to day/night temperature of 7/2 °C for 120 h in a growth chamber. The lower content of malondialdehyde (MDA) and H₂O₂ and higher activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) during exposure to low temperature in pre-treated plants in comparison with control plants demonstrated that these compounds improved the chilling tolerance of Manila grass.