Brian Tsukimura

In vitro stimulation of oocytes by presumptive mandibular organ secretions in the shrimp, Penaeus vannamei

Abstract Several mandibular organ compounds and their derivatives were assayed in vitro to determine their effects on oocytes from the shrimp, Penaeus vannamei. Pieces of adult immature ovary were incubated for 24 h in modified Medium 199 supplemented with either a steroid or terpenoid hormone. The diameters of late perinucleolus stage oocytes were examined under light microscopy. Of the four steroids assayed, only 17α-hydroxyprogesterone at concentrations of 1 pM–100 nM significantly increased oocyte diameter. Progesterone, 17β-estradiol and 17α,20β-dihydroxyprogesterone had no effect. Terpenoids, methyl farnesoate and juvenile hormone III, also significantly increased oocyte diameter. Combinations of 17α-hydroxyprogesterone and juvenile hormone III showed no additive effect. These data suggest that mandibular organ compounds and their derivatives may be potent in stimulating oocyte growth.

A life history model for the San Francisco Estuary population of the Chinese mitten crab, Eriocheir sinensis (Decapoda: Grapsoidea)

First discovered in San Francisco Bay in 1992, the Chinese mitten crab,Eriocheir sinensis, has become established over hundreds of km2 of the San Francisco Estuary. Ecological and economic impacts of this invasive species motivated our search for a greater understanding of the crab’s life history as an important step in better management and control. Data for this life history model comes from the authors’ research and scientific literature. Juvenile crabs migrate from the Estuary into fresh water where they develop into adults. Environmental signals may stimulate gonad development that is followed by a downstream migration beginning at the end of summer. Mating occurs after the crabs reach saline water. Embryos are carried until hatching, and the larvae undergo five zoeal stages before settlement. Our model projects rates of development at various temperatures and growth increments, supports a minimum of 2 years in low salinity or freshwater habitat, and predicts that most California mitten crabs are at least 3 years old before becoming sexually mature. Environmental factors strongly influence the timing and duration of the crab’s life stages, and are discussed in the context of a gradient of development times for worldwide populations of this important invasive species.

Regulation of methyl farnesoate in the hemolymph and mandibular organ of the lobster, Homarus americanus

Methyl farnesoate (MF) was measured in the hemolymph and mandibular organs (MO) of the lobster. Although a few animals had detectable MF levels in their hemolymph, this compound was undetectable (less than 0.4 ng/ml) in the hemolymph of most animals. One day after bilateral eyestalk ablation, MF was detected in the hemolymph of all animals, reaching variable levels (2.0-31.2 ng/ml) by the fourth day. Unilateral eyestalk ablation caused a smaller increase in hemolymph levels of MF. Similarly, the MF content of the MO, the only known site of MF synthesis, was low in intact lobsters (8.1 ng/gland) and was elevated in unilaterally and bilaterally eyestalk-ablated animals (54.1 and 106.9 ng/gland, respectively). When extracts of the sinus gland (SG), a source of neuropeptides in the eyestalk, were injected into bilaterally ablated lobsters, hemolymph levels of MF dropped to undetectable levels in 2 to 3 hr. The response to SG extract was dose dependent, and MF levels recovered by 12 to 24 hr after treatment. SG extract also lowered the MF content in the MO from 267.6 to 6.6 ng/gland after 4 hr. These results indicate that MF in the hemolymph and MO is negatively regulated by a factor(s) from the SG.

Development of an anti-vitellin ELISA for the assessment of reproduction in the ridgeback shrimp, Sicyonia ingentis

To investigate the reproductive regulation of the ridgeback shrimp, Sicyonia ingentis, vitellin (Vn) synthesis was studied. Using gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Vn was found to have a molecular mass of 322 kDa and to be composed of three subunits of 182, 91 and 85 kDa. Purified Vn was used to prepare anti-Vn antiserum, which was used to develop an enzyme-linked immunosorbent assay (ELISA) with a dynamic range of 0.3-300 ng. The ELISA was used to measure hemolymph levels of yolk proteins. The mean hemolymph concentrations in fresh caught animals ranged from 256 (+/-36.6 S.E.M.) to 1073 (+/-87.6 S.E.M.) mg/ml in stage 2 and 4 animals, respectively. The ELISA was also used to determine the effects of steroid hormone injections in adult non-reproductive female shrimp. One milligram injections of progesterone, 17alpha-hydroxyprogesterone or 17beta-estradiol were administered for three consecutive days to individual females. There were no changes in hemolymph vitellogenin levels during the successive 7-day period following the first injection of any steroid.

Quantification of methyl farnesoate levels in hemolymph by high-performance liquid chromatography

Methyl farnesoate (MF) is an acyclic sesquiterpenoid that has been detected in hemolymph and other tissues of crustaceans and insects. This paper describes a rapid and sensitive method for measuring MF in crustacean hemolymph. Extracts of hemolymph samples were separated by normal-phase high-performance liquid chromatography (5-microns silica, 250 x 4.6 mm I.D., 1.3% diethyl ether in hexane) and detected by UV (220 nm). The limit of detection with this method was less than 250 pg/ml. This method should be useful for studying the physiological functions of MF in crustaceans and other arthropods.

Cloning and expression study of the lobster (Homarus americanus) vitellogenin: Conservation in gene structure among decapods

This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.

Regional Differences in Methyl Farnesoate Production by the Lobster Mandibular Organ

Visual examination of the mandibular organ (MO) from the lobster, Homarus americanus, disclosed two distinct morphological regions: a fan-folded region along one edge of the gland, and a smooth, unfolded region comprising the rest of the gland. Because MOs produce methyl farnesoate (MF), the MF content of both regions was measured. In freshly dissected glands, more than 95% of the MF was found in the fan-folded region of the gland. In MO sections incubated with [3H-methyl]methionine (a radiolabeled precursor of MF), more than 90% of MF synthesis was found in the fan-folded region. Eyestalk ablation, a procedure that increases MO activity, caused the MF content of MOs to increase more than 130-fold, but had little effect on the regional distribution of MF. Histological observations indicated that these two regions had different cellular compositions. The fan-folded region contained two cell types (A and B). The A cells were mitotically active and appeared to be undifferentiated. The B cells contained a large number of small vacuoles. The unfolded region was largely composed of a third cell type (C). The C cells were large and morphologically complex, containing many mitochondria and large vacuolar-like structures. They contained relatively few small vacuoles. On the basis of appearance and location, B cells appear to be the likely site of MF synthesis. The physiological importance of C cells is unknown.

Yolk protein synthesis in the riceland tadpole shrimp, Triops longicaudatus, measured by in vitro incorporation of 3H-leucine.

Investigation of reproductive control within the tadpole shrimp, Triops longicaudatus, required the isolation and characterization of the yolk protein (vitellin, Vn). To this end, tadpole shrimp were cultured in environmental chambers (29 degrees C-22 degrees C, with 14:10 light:dark cycle). Desiccated cysts hatched in 2-3 days after inundation. The tadpole shrimp began egg deposition 7 to 8 days after hatching and exhibited a mean growth rate of 1.85 +/- 0.24 mm/day. It was observed that 4-day-old shrimp had visible eggs in their ovaries. In addition, Vn was isolated and characterized from reproductive animals, resolving as one protein on native PAGE, and possessing a molecular weight (MW) of 376,000 +/- 2,900 as determined by FPLC. Examination by SDS-PAGE revealed that Vn is composed of a single molecule with a MW of 214,000 +/- 2,000. Methyl farnesoate (MF), a crustacean compound whose role in reproduction is still being elucidated and is structurally similar to juvenile hormone III (JH III) was incubated with ovarian explants. These explants were incubated for 24 h at room temperature in EAGLEs medium adjusted to Van Harrevelds solution in six concentrations (1 pM to 100 nM) of MF and JH III. Methyl farnesoate and JH III had no direct in vitro effect on yolk protein synthesis (P < or = 0.545 and P < or = 0.815, respectively).

A KEY TO THE IDENTIFICATION OF BRACHYURAN ZOEAE OF THE SAN FRANCISCO BAY ESTUARY

Abstract San Francisco Bay and its estuary is a heavily invaded region due to organisms arriving in ballast water and on fouled ships, as well as with the intentional release of non-native species. Recent crab invasions rendered previous means to identify crab larvae unusable. We provide an illustrated dichotomous key with descriptions of stage I zoeae, and to facilitate identification use external characters that remain definitive through all zoeal stages. Crab zoeae included in this key are as follows: Cancer antennarius, Cancer gracilis, Cancer magister, Cancer productus, Carcinus maenas, Eriocheir sinensis, Hemigrapsus nudus, Hemigrapsus oregonensis, Lophopanopeus bellus, Rhithropanopeus harrisii, Pachygrapsus crassipes, Pyromaia tuberculata, and the family Pinnotheridae. This work aims to provide a tool to aid in the understanding of brachyuran zoeae of the San Francisco Bay and estuary.

Workshop on the Implementation of the Grand Challenges

This is a pre-copyedited, author-produced PDF of an article accepted for publication in Integrative and Comparative Biology following peer review. The version of record [Tsukimura, B., H.V. Carey and D.K. Padilla. 2010. Workshop on the Implementation of the Grand Challenges. Intetgrative Comparative Biology 50: 945-947] is available online at: http://icb.oxfordjournals.org/content/50/6/945.full.