Vitellogenesis stimulated by thoracic ganglion implants into destalked immature spider crabs, Libinia emarginata.

Since destalked immature female Libinia emarginata fail to molt to maturity (Hinsch, 1972) and implants of thoracic ganglion of adult females into young females of Potamon dehaani caused a doubling of ovarian weight and differentiation of yolk granules (Otsu, 1963), the effects of the thoracic ganglion implantation on sexual maturation in Libinia was investigated. Destalked immature Libinia with a maximum carapace length of 4-5 cm received two implants of adult female thoracic ganglion at 10-day intervals. All of the surviving experimental animals and destalked controls molted to immaturity. Most of the experimental animals failed to complete a successful molt and few survived longer than 3-4 days. The experimental animals that survived for over a week showed signs of yolk deposition not observed in the destalked controls. Eggs in several stages of vitellogenesis were scattered throughout the gonad in the animal showing the most pronounced effect. This was not observed in any of the destalked controls.     

Rodent DNA reassociation kinetics

The kinetics of reassociation of the nuclear DNA of eight rodents has been examined to determine the amount and repetition frequency of various DNA fractions. Each rodent had at least two distinct, repeated DNA fractions, one group repeated about 300 times and the other repeated about 50,000 times. As shown, the actual repetition frequencies and the amount of the various DNA fractions varied from one species to another. Genetic implications of these results are discussed.     

The mandibular organ of the female spider crab,Libinia emarginata,in immature,mature, and ovigerous crabs

The cells of mandibular organs of female Libinia emarginata exhibit changes in substructure during molting and vitellogenesis. The cytoplasm is that of a steroid-hormone producing cell. The cells do not appear to produce ecdysones.     

The vas deferens of the spider crab,Libinia emarginata

Sperm enter the anterior vas deferens individually in the spider crab male. There they become surrounded by secretion products from the cells of the vas deferens, and are compartmentalized into spermatophores of varying size. The anterior vas deferens can be divided into three regions. The epithelium of the anterior vas deferens varies regionally from low to high columnar. The cytoplasm contains vast arrays of rough endoplasmic reticulum and Golgi complexes but few mitochondria. Intercellular spaces contain septate junctions, gap junctions and vesicles. Once the spermatophores have been formed in the anterior vas deferens, they are moved posteriorly to the middle vas deferens where they are stored and surrounded by seminal fluids. The epithelial cells of the middle vas deferens contain large amounts of rough endoplasmic reticulum and Golgi complexes. Numerous micropinocytotic vesicles appear, forming at the cell surface and within the apical cytoplasm. Their suggested function is the resorption of secretion products of the anterior vas deferens which initiated compartmentalization of the spermatozoa into spermatophores. The posterior vas deferens functions primarily as a storage center for spermatophores until they are released at the time of copulation. Seminal fluid surrounding the spermatophores is produced in this region as well as in the middle vas deferens. The cells of this region contain vast arrays of vesicular rough endoplasmic reticulum and Golgi complexes. The cells are multinucleate. Microtubules are numerous throughout the length of the cells and appear to insert on the plasma membrane.     

DNA reassociation kinetics and hybridization in different angiosperms

Reassociation kinetics ofDaucus carota andPetroselinum crispum (Apiaceae), andDatura innoxia (Solanaceae) are presented. Hybridization of³H-labelled DNA of two carrot cultivars indicate strong qualitative homologies of DNA sequences; nevertheless, certain quantitative differences in some Cotregions seem to exist. However, homologous sequences ofDaucus DNA with DNA ofDatura, and, suprisingly, even with DNA ofPetroselinum are very restricted: between 8% in the repeated regions and ca. 7–9% in the unique regions.     

Microspectrophotometry of the visual pigment of the spider crab Libinia emarginata

Summary Isolated dark-adapted rhabdoms from the spider crab Libinia emarginata were examined by microspectrophotometry to determine the visual pigments present and their light-sensitive characteristics. The rhabdoms contain a single pigment with _max=493 nm. Upon one minute irradiation with bright orange light this pigment forms a light-stable photoproduct with nearly the same _max as the parent pigment but with slightly greater absorption to the long wavelength side of the absorption peak. On exposure to orange or yellow light in the presence of 5% glutaraldehyde, however, the pigment of Libinia rhabdoms bleaches slowly.The photosensitive pigment of properly oriented, transversely illuminated rhabdoms shows isotropic and dichroic regions, corresponding to layers of the rhabdom in which the microvilli are respectively parallel and perpendicular to the direction of propagation of the measuring beam. The maximum dichroic ratio is about 2, with most absorption when the plane of polarization is parallel to the microvillar axes.

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Zusammenfassung Die isolierten, dunkeladaptierten Rhabdome von Libinia emarginata wurden mikrospektrophotometrisch untersucht, um die Sehfarbstoffe und ihre lichtempfindlichen Eigenschaften zu entdecken. Die Rhabdome enthalten ein einziges Pigment (_max 493 nm). Nach einer Bestrahlungszeit von 1 min mit hellem orangem Licht entsteht ein lichtfestes Photoprodukt mit beinahe dem gleichen _max wie das ursprüngliche Pigment, aber mit etwas größerer Absorption gegen die langwellige Seite des Absorptionsmaximums hin. Das Pigment von Libinia-Rhabdomen bleicht langsam aus, wenn man es orangem oder gelbem Licht in Gegenwart von 5% Glutaraldehyd aussetzt.Werden richtig orientierte Rhabdome von der Seite belichtet, so zeigt das lichtempfindliche Pigment teils Isotropismus teils Dichroismus. Im ersten Fall sind die Mikrovilli parallel zur Richtung des Meßstrahles orientiert, im zweiten Fall stehen sie senkrecht dazu. Das größte Dichroismus-Verhältnis liegt bei 2. Man erhält die größte Absorption, wenn die Polarisationsrichtung parallel zur Achse der Mikrovilli steht.

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This work was supported by U.S.P.H.S. grant NB-03333. Some of the experiments were done at The Marine Biological Laboratory, Woods Hole, Mass.     

Methyl farnesoate induced ovarian maturation in the spider crab,Libinia emarginata

Summary

To overcome the problem of getting crustaceans to reproduce in captivity, eyestalk ablation or X-organ sinus gland removal is commonly utilized in commercially important species such as shrimp. We have investigated the effect of unilateral and bilateral eyestalk ablation on methyl farnesoate (MF) production by mandibular organs (MOs) and on ovarian maturation in female spider crabs Libinia emarginata, a useful model since these animals are in a terminal molt and are devoid of a functional Y-organ. Non-reproductive, over-wintering female L. emarginata were induced to be reproductive by feeding and increasing the holding temperature to stimulate the endocrine system. In addition, we removed X-organ sinus glands by eyestalk ablation either unilaterally (UEA) or bilaterally (BEA) to further stimulate MF synthesis by MOs. Endogenous MF in the hemolymph was extracted and quantified by means of HPLC and in some cases by GC/MS. Oocyte growth and egg quality were studied simultaneously to determine how they were related to MF levels found during vitellogenesis. The initial MF concentration in unablated controls was low, 0.31 ng/ml of hemolymph, and this increased (p<0.05) to about 1 ng/ml by 2 weeks, remaining at about that level for the remainder of the experiment. Eyestalk ablation significantly stimulated MF concentrations by week 1 to nearly 2 and 3.5ng/ml in the UEA (p <0.01) and BEA (p <0.001) animals, respectively. Oocytes appeared to respond to increased MF levels, as ovarian maturation was initiated from the point at which MF increased (p <0.05). Thereafter, the rate of oocyte growth was directly correlated with the extent of elevation of MF. The gonado-somatic index [(GSI) = gonad weight/body weight × 100] of controls at the start was about 1.5 and increased to 6.5 by week 4. Mature oocytes were reached at a GSI around 7. Oocyte maturation was accomplished at week 2 in BEA, week 3 in UEA, and later than week 4 in controls. After maturation, oocytes started to degrade in some ablated animals, particularly in the bilaterally ablated ones where the highest MF concentrations were observed. These data indicate that MF elevations are required for stimulating ovarian maturation in Crustacea. MF appears to accelerate gonad development during the vitellogenic process, but may be deleterious at high concentrations. These results have a significant and important application and implications for aquaculture.     

DNA reassociation kinetics of closely related species

The kinetics of reassociation of the DNA of three groups of closely related organisms were examined. The laboratory mouse was compared to an Asiatic mouse, whose chromosome number is the same but whose chromosome organization is different. Chinese hamster (2N=22) was compared to Syrian hamster (2N=44), and Haplopappus gracilis (2N=4) was compared to H. ravenit (2N=8). It was found that the most highly repeated DNA fractions of the three comparative sets of organisms differ in their reaction rates. However, these fractions of the related hamsters, haplopappi, and probably the mice, do not differ in the amount of DNA composing the fractions. The intermediately fast reassociating DNA and the unique DNA do not differ between members of related pairs of organisms. The implication of these results is that a short sequence of DNA may be highly copied in one organism, while in a related organism a longer DNA sequence is repeated a fewer number of times, and the total amount of repeated DNA may be the same in both related organisms.     

Fluorometric measurement of DNA reassociation kinetics.

A new radioimmunoassay (RIA) method is proposed, utilizing cellulose disks coated with a ${}^{125}\text{I-}\text{Ag}\text{Ab}$ complex as a single reagent. The labeled antigen is released into the incubation medium proportionally to the concentration of the corresponding antigen in the sample. The method is comparable to the classical RIA with regard to sensitivity, precision, and specificity, but it has the advantage of a wide range of nondependence on the sample volume and greater practicability (the analysis is performed using a single-step procedure). The assay of human chorionic somatomammotropin is adopted as a model.     

Cytoplasmic DNA: Differentiation by reassociation kinetics

Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.03}$ while 65% of the particulate (microsomal) DNA reassociated with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.14}$ Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences.     

DNA reassociation kinetics in diploid and phylogenetically tetraploid cyprinidae.

Four diploid and three phylogenetically tetraploid Cyprinidae (Ostariophysi) have been characterized as for nuclear DNA content, modal chromosome number and DNA reassociation kinetics (hydroxyapatite chromatography). Among the diploid species nuclear DNA content (10(-12) g DNA/2C) was 1.62 for Tinca tinca, 1.87 for Scardinius erythrophthalmus, 2.53 for Leuciscus cephalus and 2.75 for Alburnus alburnus, while the phylogenetically tetraploid species Carassius auratus, Barbus barbus and Cyprinus carpio attained 3.40, 3.66 and 3.80 respectively. Modal chromosome number was 2n = 48-50 for diploid individuals and 2n = 100-104 for phylogenetically tetraploid ones. In all the species 5--8% of the genome is represented by highly repetitive and foldback DNA. In DNA reassociation kinetics of phylogenetically tetraploid Cyprinidae a distinct plateau separates an intermediate reassociating sequence fraction (about 22% of the genome; with average repetition frequencies between 1,000 and 1,400) from a slow reassociating one (unique DNA; about 72% of the genome). These two genome fractions are not clearly distinguishable from each other in Cot curves of the diploid Cyprinidae, where a similar plateau is not evident. Since simple ploidy changes are not expected to affect DNA reassociation kinetics we suggest a different evolution in the genome organization of the two ploidy groups. Some possible hypotheses are discussed.     

Correlation between animal radiosensitivity and kinetics of DNA reassociation

A study was made of DNA of different animal species with various radiosensitivity: guinea pig, man, rat and rabbit. It was shown that the radioresistance of the organism increases with increasing relative content of a rapidly reassociating fraction in DNA. As the kinetic complexity of moderately repetitive sequences increases and the total percentage of meaddle and unique DNA fractions grows, the radioresistance of the organism decreases.     

Vitellogenocytes in the Hepatopancreas of Carcinus maenas and Libinia emarginata (Decapoda brachyura)

Summary

In addition to the ovary, the hepatopanocreas of female decapod crustaceans, Carcinus maenas, and Libinia emarginata is a source of yolk protein. The specific cells in the hepatopancreas that localize vitellogenins on tissue sections are revealed with lipovitellin-specific antiserum. These cells, designated vitellogenocytes, are believed to be responsible for vitellogenin synthesis in the hepatopanocreas. This conclusion is based upon immunolocalization which demonstrates a temporal relationship with vitellogenin synthesis in the hepatopanocreas. Specifically, when the oocytes are most active in vitellogenin uptake, the hepatopanocreas is producing vitellogenins most abundantly. Vitellogenocytes are relatively large and polymorphic, similar to the reserve-inclusion cells that were described by others. Yolk protein was not detected in other cells of the hepatopancreas, male reserve-inclusion cells, or pre-vitellogenic oocytes by the same method of staining. Vitellogenocytes resemble cyanocytes, the source of hemocyanin. Whether the vitellogenocytes and their precursors are related to other populations of hepatocytes, such as cyanocytes, is not known and has not yet been studied.     

Application of DNA sonication in the study of reassociation kinetics]

Single-stranded breaks arising is DNA under sonication divide the molecule in short double-stranded regions of lengths depending on sonication conditions. The reassociation rate of DNA with single-stranded breaks is considerably lowered as compared with intact DNA of the same length. For sonicated DNA the slowing of reassociation and the deflection to higher orders of C(o)t of the reaction is observed at the late stages. Further process of sonication occurs likely on single-stranded breaks and DNA fragments obtained as a result of infinite sonication are practically intact. Reassociation of DNA, degraded to a "saturation", proceeds as a second order reaction in precise accordance with the obtained fragment length.     

Seasonal differences in methyl farnesoate esterase activity in tissues of the spider crab Libinia emarginata

Summary

Methyl farnesoate (MF), an unepoxidated form of insect JH III, is present in Crustacea. MF is synthesized by the mandibular organs and is degraded to fomesoic acid (FA) by peripheral tissues. In this study we investigated MF degradation by esterases in hepatopancreas, ovary, testes and hemolymph of the spider crab Libinia emarginata collected at different times of the year to determine seasonal differences. The conversion of MF to FA varied among the tissues. In the summer, the hepatopancreas showed the greatest esterase activity (52.8% conversion in females and 59.16% in males), and it was twice as high (28.86%) in ovaries than in the testes (12.16%), but was low in the hemolymph of both sexes (10.84% in males, and 6.97% in females). In the fall, the conversion of MF to FA was significantly reduced in all tissues (ovary 8.55%, testes 6.21%, hepatopancreas 10.22%, hemolymph 3.96%). Eyestalk ablation of animals in the fall restored MF esterase activity to summer levels. When tissues from these animals were incubated with OTFP, a specific inhibitor of JH esterase, MF metabolism was significantly reduced. These results suggest that MF esterase activity depends on direct induction by MF, and its degradation is by a specific esterase(s).