Origin and occurrence of sexual and mating systems in Crustacea: a progression towards communal living and eusociality.

Crustaceans are known for their unrivalled diversity of sexual systems, as well as peculiar mating associations to achieve maximum mating success and fertilization accomplishment. Although sexes are separate in most species, various types of hermaphroditism characterize these predominantly aquatic arthropods. A low operational sex ratio between female and male, together with temporally limited receptivity of females towards males, imposes restrictions on the structuring of mating systems in crustaceans. The basic mating systems consist of monogamy, polygamy, mate guarding and pure searching. Understandably, ecological influences may also play a determinative role in the evolution of such sexual and mating systems in crustaceans. An important outcome of the crustacean sexual biology is the development of complex social structures in many aquatic species, in much the same way insects have established them in terrestrial conditions. In addition, groups like isopods and certain families of brachyuran crabs have shown terrestrial adaptation, exhibiting peculiar reproductive modes, sometimes reminiscent of their terrestrial counterparts, insects. Many caridean shrimps, living in symbiotic relationship with other marine invertebrates in the coral reef habitats, have reached pinnacle of complexity in sexuality and peculiar mating behaviours, resulting in communal living and establishing advanced social systems, such as eusociality.

Hormonal coordination of molting and female reproduction by ecdysteroids in the mole crab Emerita asiatica (Milne Edwards).

The measurement of hemolymph ecdysteroids using radioimmunoassay (RIA) indicates a biphasic increase during intermolt and premolt stages of the mole crab Emerita asiatica. The gradual rise during intermolt stage corresponds to vitellogenic activities in the ovary; whereas a distinctive premolt peak is characteristic of molting crustaceans. Injection experiments with 20-hydroxyecdysone (20E) during different molt cycle stages revealed the onset of precocious premolt changes, as determined by the epidermal retraction and setal development. Injection of 20E augmented protein synthesis in the ovary, hepatopancreas and integumentary tissues. Quantification of ecdysteroids in different developmental stages of ovary also indicated a progressive increase of ovarian ecdysteroids. Interestingly, the ovarian ecdysteroids after reaching a peak at C3 stage, start declining drastically to reach the lowest level at D(3-4) stage. This decline in the ovarian ecdysteroids is inversely related to rising hemolymph ecdysteroids during these active premolt stages. The hatching of the embryos, attached to the pleopods of the ovigerous females also occurs under a high titer of hemolymph ecdysteroids. In support, 20E injection at C3 stage crabs indicated a significant reduction in time duration of pleopodal embryonic development leading to hatching of zoea larvae. Understandably, the augmented hemolymph ecdysteroid titer helps in the synchronization of embryo hatching and the premolt changes, as occurring under the normal premolt conditions.

Breeding biology of the intertidal sand crab, Emerita (Decapoda: Anomura).

Emerita is a burrowing mole crab or sand crab, adapted to life in wave-washed sandy beaches of temperate and tropical seas. The reproductive biology of this anomuran crab presents several peculiarities, all contributing to its adaptation to this harsh environmental niche. We discuss the following aspects: 1) sex ratio and size at sexual maturity, 2) neoteny and protandric hermaphroditism, 3) mating behaviour and sperm transfer strategy, 4) synchronisation of moulting and reproduction, 5) environmental impact on reproductive cycle and egg production, 6) biochemistry of yolk utilisation and energetics, 7) larval development, dispersal and settlement and 8) the value of Emerita as indicator species. These aspects are discussed in the light of the life history pattern, comprising a sedentary adult and pelagic larval phases. The successful colonisation of the physically challenging habitat of the sandy beach by Emerita is attributable largely to reproductive strategy and the larval developmental and recruitment pattern. Sensitivity to changing environmental conditions, including pollution, make this intertidal crab an indicator species for monitoring anthropogenic impact.

Instability of crab vitellogenin and its immunological relatedness with mammalian atherogenic lipoproteins.

Vitellogenesis is the process of accumulation of vitellogenin (Vg) in rapidly growing oocytes of oviparous animals and its' subsequent transformation into lipovitellin (Lv). Lipovitellin, which forms the major yolk protein, serves as a principal nutrient reserve for the developing embryo. In the present study, Vg and Lv were purified from the hemolymph and ovary, respectively of the crab Scylla serrata by gel filtration followed by preparative gel electrophoresis. It was observed that purified Vg, but not Lv, possessed an intrinsic protease activity with which it underwent autoproteolysis giving rise to several smaller proteins. Furthermore, urea-mediated unfolding studies by UV-spectral analysis revealed clearly that Vg was easily disrupted by urea whereas Lv was resistant. Taken together, these results suggest that although Lv had a stable conformation, its precursor Vg was labile and highly sensitive to degradation. Another aspect that was investigated in the present study was the immunological kinship of crab Vg and Lv to mammalian atherogenic lipoproteins, the low density lipoprotein (LDL), very low density lipoprotein (VLDL), and apolipoprotein B (apoB). By Western blot analysis, it was demonstrated that crab Vg and Lv were immunoreactive to antibodies to human LDL, VLDL, and apoB. These observations suggest the existence of common epitope recognition sites in crab Vg and mammalian lipid transferring proteins. This corroborates well with our earlier study on the recognition of crab Vg receptor by mammalian lipoproteins.

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins.

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.

Occurrence of vertebrate steroids, estradiol 17beta and progesterone in the reproducing females of the mud crab Scylla serrata.

In crustaceans, vitellogenesis is known to be controlled by eyestalk neuropeptides, biogenic amines, ecdysteroids and a juvenile hormone-like compound, methyl farnesoate. In recent years, the occurrence of vertebrate steroid hormones, estradiol 17beta (E2) and progesterone (PG) has also been reported in a few decapods, although their precise role in female reproduction is yet to be determined. The levels of E2 and PG in the ovary, hepatopancreas and the hemolymph of the red mud crab, Scylla serrata were analyzed in different vitellogenic stages in order to establish a correlation between hormone profile and stages of vitellogenesis. It was observed that the levels of both the steroids increased steeply in the tissues at the onset of vitellogenesis (vitellogenic stage I). Maximum levels of estradiol were present in the hepatopancreas whereas the highest concentration of progesterone was seen in the ovary, suggesting dichotomous roles for these hormones in vitellogenesis. Furthermore, levels of these hormones were estimated in different embryonic stages of the eggs of the sand crab Emerita asiatica and mud crab S. serrata. Their levels fluctuated, following a definite pattern in the different stages, suggesting a possible functional role as morphogenetic hormones. This study, in addition, also reports the presence of E2 and PG on lipovitellin purified from ovary and eggs as well as vitellogenin purified from the hemolymph implicating a role for these lipoproteins as steroid carriers.

Carbohydrate components of lipovitellin of the sand crab Emerita asiatica.

Lipovitellin II (Lv II), the major yolk protein of the anomuran crab Emerita asiatica, was purified using heparin-sepharose affinity column chromatography. The purified Lv II was a glycoprotein as it was stainable with periodic acid-Schiff's reagent. Quantitative analysis of sugars showed the presence of fucose, mannose, galactosamine, N-linked oligosaccharides, as well as O-linked oligosaccharides containing N-acetyl hexosamine as the terminal residue. The amount of N-linked oligosaccharides is higher than that of the O-linked oligosaccharides. Biogel P-4 column chromatographic separation of the radiolabeled oligosaccharides of Lv II showed the presence of five different O-linked oligosaccharides and four different N-linked oligosaccharide species. HPTLC separation of the neoglycolipids prepared from the O-linked oligosaccharides also showed the presence of five different O-linked oligosaccharide species. N-linked oligosaccharides contain significant quantities of mannose. Unisil column chromatographic purification in conjunction with HPTLC separation revealed three neutral glycolipid species such as monoglycosylceramide, diglycosylceramide, and triglycosylceramide in the Lv II. The functional significance of these carbohydrate components of the major yolk protein during embryogenesis of the sand crab is discussed.

Development of vitellogenin-ELISA, an in vivo bioassay, and identification of two vitellogenesis-inhibiting hormones of the tiger shrimp Penaeus monodon.

A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of vitellogenin in the hemolymph of Penaeus monodon is reported. Lipovitellin from the mature ovary was purified using hydroxylapatite column chromatography and used as the standard protein, which was serially diluted from 3336 to 6.51 microg and gave a linear plot. Sensitivity results showed ELISA was insensitive to samples that did not contain vitellogenin or lipovitellin. Specificity results showed the degree of the discrimination of the assay between positive samples (having vitellogenin or lipovitellin) and negative samples (devoid of vitellogenin or lipovitellin). Reproducibility studies showed that the intraassay coefficient of variation was 5.1% (n = 11) and the interassay coefficient of variation was 5.15% (n = 13). Separation of X-organ sinus gland peptides using a reversed phase column gave a total of 37 fractions. Two fractions were found to reduce the hemolymph vitellogenin concentration in a time-dependent manner and could be identified as vitellogenesis-inhibiting hormones I and II.

Determination of amino acid sequence and site of mRNA expression of four vitellins in the giant freshwater prawn, Macrobrachium rosenbergii.

Four major yolk proteins, designated as vitellins (Vns) Macr-VnA, B, C, and D, were extracted from mature ovaries of Macrobrachium rosenbergii. These were purified to homogeneity by reversed-phase high performance liquid chromatography (HPLC) employing a unique separation system based on the hydrophobic properties of the Vn molecule. Using standard techniques of protein sequencing, more than 33 N-terminal and 57 internal amino acid residues were determined for each of the four Vns. The cDNA fragments encoding the four Vns were amplified by PCR using degenerate oligonucleotide primers derived from the N-terminal and internal amino acid sequences. These cDNA fragments were cloned, sequenced, and used as probes to examine the transcription of mRNAs encoding the four Vns. Significant accumulations of these mRNAs were observed in female hepatopancreas only, while mRNA expression was not detected in male hepatopancreas or any other female tissue including ovary, subepidermal adipose tissue, gill, and muscle. This is the first occasion in Crustacea in which multiple Vns were demonstrated to be synthesized simultaneously in a single tissue.

Relative tolerance of cirripede larval stages to acute thermal shock: A laboratory study.

(1) Meroplankters drawn into once-through cooling circuits of coastal power plants are subjected to transient thermal stress. The effect of such acute thermal shock on the development of barnacle larvae was studied in the laboratory.(2) The response of the barnacle larvae (naupliar and cyprid stages) to elevated temperature was dependent on exposure time and their stage of development.(3) Among the stages tested, N-6 larvae showed maximum tolerance. Exposure to 37 degrees C did not affect larval survival, but delayed development of N-2 larva to cypris by one day.(4) Exposure at 40 degrees C delayed, hastened or did not affect the development time of N-2 and N-4 larvae through cypris, depending on exposure time.(5) Ten mins exposure at 43 degrees C proved lethal to all larval stages with mortality ranging from 20 to 86%.(6) Development success of the surviving larvae, measured in terms of cypris yield, showed no significant difference from controls, at temperatures below 40 degrees C.(7) Settlement activity was significantly affected in only those cyprid larvae which were exposed to 43 degrees C for 10 min.(8) Results of the present study indicate that thermal stress experienced in the once-through cooling system does not have significant impact on survival and development of the barnacle larvae at temperatures of 37-40 degrees C.

Crustacean ecdysteriods in reproduction and embryogenesis.

Ecdysteroids are the molting hormones in Crustacea, as in other arthropods. They also subserve functions in the control of reproduction and embryogenesis. The available evidence indicate that the ecdysteroids are sequestered into the ovary by binding to yolk precursor proteins. Steroidogenic ability of the ovary is yet to be demonstrated in Crustacea. Despite several investigations, the role of ecdysteroids in oocyte maturation is not fully known. However, the embryonic ecdysteroids undergo significant fluctuation, correlated to specific developmental stages, including the secretion of embryonic envelopes and cuticle. Ecdysteroid metabolism in the eggs seems to be active throughout embryogenesis inasmuch as the free ecdysteroids are rapidly converted into conjugates, and vice versa; in addition to their inactivation into excretory ecdysteroidic acids. Eyestalk neuropeptides such as molt inhibiting hormones have a dominant role on the ecdysteroid synthesis by Y-organ, although recent evidence suggests a stimulatory role for yet another endocrine gland, the mandibular organ on Y-organ synthesis.

Purification and characterization of vitellogenin and lipovitellins of the sand crab Emerita asiatica: molecular aspects of crab yolk proteins.

In the mole crab Emerita asiatica, the main yolk proteins consist of two slow moving lipovitellins (Lv I and Lv II) of glycolipoprotein nature. Lv I cleaves into subunits (MW: 109,000 and 105,000) and Lv II gives rise to six subunits (MW: 65,000, 54,000, 50,000, 47,000, 44,000, and 42,000) in SDS-PAGE (with beta-mercaptoethanol). In order to observe the stability of Lv II as well as to achieve better resolution of the proteins, two different buffer systems (Phosphate buffered saline and tris-buffered saline), 40% sucrose, and glass distilled water were used as homogenizing media. Among them, better resolution was achieved with tris-buffered saline and 40% sucrose, and tris-buffered saline seems to be the ideal medium for elution of Lv II. The analysis of biochemical constituents of the major Lv II reveals a percentage composition of 69.325, 27.927, and 2.753 respectively for protein, lipid, and bound sugars. In the I stage embryo, protein comprises about 67.276%, lipid 29.65%, and bound sugars 3.015%. Vitellogenin (Vg) electrophoretically corresponding to the Lv I and Lv II was present in the female haemolymph during the entire period of embryogenesis. The number of subunits (8) of Vg in all stages remained unaltered and their approximate molecular weights were Vg1, 91,000; Vg2, 87,000; Vg3, 83,000; Vg4, 61,000; Vg5, 58,000; Vg6, 45,000; Vg7, 42,000; and Vg8, 38,000. Different proteins present in the embryos (I and IV stage) and the serum obtained from the animal carrying the I stage embryo were separated by gel-filtration in high performance liquid chromatography (HPLC). Sephadex (G-200) gel filtration chromatography was used to purify the Lv II in large quantity. Total lipid extracted from Lv II as well as the embryos belonging to different stages of development were separated into their constituent neutral, glycolipids, and phospholipids, using silicic acid column chromatography. Thin layer chromatography (TLC) was used to isolate the different phospholipids purified from various stages of embryos and Lv II. As many as seven different phospholipids were separated from Lv II and I and IX stage embryos; and whereas thin layer chromatogram of V and VI stage embryos showed six different phospholipids, embryos of VII and VIII stage contained four phospholipid species. Cholesterol, glycolipids, and individual phospholipids isolated from the Lv II and I stage embryo were quantified spectrophotometrically and the results were discussed.

Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female.

Biochemical studies on the male reproductive tissues and seminal secretions have been made with reference to sperm metabolism and different stages of maturity in the crab Scylla serrata. The results reveal that the seminal plasma and spermatophores are rich in protein, carbohydrate, and lipid. In general, organic components of spermatophores are considerably higher than those of seminal plasma. Enzyme studies show that the succinate dehydrogenase (SDH) activity is very low, whereas fumarate reductase (FR) and lactate dehydrogenase (LDH) exhibit high activity. Electrophoretic studies on LDH show that, in addition to the occurrence of a sperm-specific fraction, LDHx, the M-type subunits are predominant in the mature spermatophores. These results from enzyme studies suggest that sperm metabolism is mainly anaerobic, utilizing the carbohydrates as substrates. The results for maturational changes reveal that the male reproductive tissues and their secretions contain lesser quantity of organic components in the immature crabs; as the maturity proceeds, there is not only concentration of organic substances but also an increase in the size of spermatophores. The concentration of biochemical constituents is highest in the proximal vas deferens (PVD), suggesting that the granular seminal plasma as well as the sperm-agglutinating substance and spermatophoric wall are secreted in this region. The spermatheca of the unmated female crabs are poor in organic constituents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. During sperm storage in the spermatheca, only the carbohydrates decline steeply. A low activity of SDH, but a moderate level of LDH and a high level of FR activity, is recorded in the spermathecal content of mated crabs, providing further evidence for anaerobic metabolism of sperm during storage in female. A sharp fall in the stored carbohydrates constitutes further evidence in this regard.

Biochemical composition of seminal secretions with special reference to LDH activity in the reproductive tissues of the field crab, Paratelphusa hydrodromous (Herbst).

Biochemical composition of seminal secretions as well as the male and female reproductive tissues, with special reference to lactate dehydrogenase (LDH) activity, were determined in the field crab, Paratelphusa hydrodromous. The seminal secretions comprising spermatophores and spermatophore-carrying seminal plasma (hereafter referred to as seminal plasma) are rich in protein, free carbohydrates and lipids. The seminal plasma contains large quantities of free carbohydrates. LDH activity, as measured by UV spectrophotometric method, is very high within the spermatophores. Electrophoretic separation of LDH isozymes reveals the occurrence of 6 fractions in spermatophores, of which, the most conspicuous fraction resolves in between LDH3 and LDH4. The use of L-lactate as substrate and consideration of its relative mobility show that this fraction is homologous to the mammalian sperm-specific LDHx fraction. Among the 6 fractions, the majority of them, including LDHx, fall under M-type suggesting that they are mainly involved in the anaerobic metabolism of spermatozoa. When compared to anterior vas deferens and mid vas deferens, LDH activity is maximum in the posterior vas deferens which contains seminal secretions prior to their ejaculation. Though the LDH activity is found to be moderate in the spermathecal contents of freshly mated females, the carbohydrate reserves are very minimal. Interestingly, spermathecal contents are rich in lipid substances, thereby indicating that the spermatozoa, when stored in the spermatheca, may utilise fatty substances for oxidative metabolism.

RNA synthesis in the ovarian follicle cells of Periplaneta americana during vitellogenesis.

The RNA and DNA pattern in the follicle cells of cockroach ovary during vitellogenesis has been studied. Evidences indicate that there is an active synthesis of these two nucleic acids in the nucleus. It has been suggested from the observations presented that RNA is synthesized in the chromosomes and are then transported to nucleolus before being sent to the cytoplasm. It is also shown that the follicle cells do not contribute any RNA material to the vitellogenic oocyte.