Eyestalk Research Articles

Characterization and expression analysis of Cyclin-dependent kinase 7 gene and protein in ovaries of the giant tiger shrimp Penaeus monodon

The characterization of genes differentially expressed in ovaries is necessary for understanding the mechanisms involving ovarian developmental processes of the giant tiger shrimp ( Penaeus monodon ). Here, the full-length cDNA of P. monodon Cyclin-dependent kinase 7 ( PmCdk7 ) was characterized. It was 1407 bp in length (ORF of 1062 bp corresponding to 353 amino acids). The expression of PmCdk7 in the ovaries was greater than that in the testes ( P < 0.05). PmCdk7 was significantly up-regulated in stage IV (mature) ovaries in wild intact broodstock ( P < 0.05). In wild eyestalk-ablated broodstock, its expression was earlier increased in stage III (late vitellogenic) ovaries ( P < 0.05). The expression level of PmCdk7 during ovarian development (stages I–IV ovaries) in eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock ( P < 0.05). 5-HT (50 μg/g body weight) injection promoted its expression level in ovaries of domesticated 18-month-old shrimp at 6–48 h post injection (hpi). The rPmCdk7 protein and its polyclonal antibody were successfully produced. The positive immunoreactive band (67 kDa) was not observed in premature ovaries of juveniles. Western blot analysis revealed differential expression of PmCdc7 in stages II–IV ovaries compared with stage I ovaries in intact broodstock but it was comparably expressed among different ovarian developmental stages in eyestalk-ablated broodstock. Immunofluorescence revealed the positive signals of PmCdk7 in the ooplasm of previtellogenic oocytes and its subsequent nucleo-cytoplasmic translocation during oocyte development. Results indicated that PmCdk7 seems to play functional roles in the development and maturation of oocytes/ovaries in P. monodon. • The full-length cDNA of Penaeus monodon Cdk7 (1407 bp) was characterized. • PmCdk7 mRNA and protein were differentially expressed during ovarian development. • The level of PmCdk7 mRNA in stages I–IV ovaries was induced by eyestalk ablation. • 5-HT injection promoted its expression level in domesticated shrimp at 6–48 hpi. • Nucleo-cytoplasmic translocation of PmCdk7 was observed during oocyte development.

Changes in the organics metabolism in the hepatopancreas induced by eyestalk ablation of the Chinese mitten crab Eriocheir sinensis determined via transcriptome and DGE analysis.

BackgroundTo understand the regulation mechanism of eyestalk ablation on the activities of hepatopancreas, Illumina RNA-Seq and digital gene expression (DGE) analyses were performed to investigate the transcriptome of the eyestalk, Y-organ, and hepatopancreas of E. sinensis and to identify the genes associated with the hepatopancreas metabolism that are differentially expressed under eyestalk ablation conditions.ResultsA total of 58,582 unigenes were constructed from 157,168 contigs with SOAPdenovo. A BlastX search against the NCBI Nr database identified 21,678 unigenes with an E-value higher than 10−5. Using the BLAST2Go and BlastAll software programs, 6,883 unigenes (11.75% of the total) were annotated to the Gene Ontology (GO) database, 7,386 (12.6%) unigenes were classified into 25 Clusters of Orthologous Groups of Proteins (COGs), 16,200 (27.7%) unigenes were assigned to 242 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and1,846 unigenes were matched to “metabolism pathways”. The DGE analysis revealed that 1,416 unigenes were significantly differentially expressed in the hepatopancreas, of which 890 unigenes were up-regulated and 526 unigenes were down-regulated. Of the differentially expressed genes, 382 unigenes were annotated and 63 were classified into metabolism pathways. The results of the real-time polymerase chain reaction (PCR) analysis of four unigenes related to carbohydrate metabolism were consistent with those obtained from the DGE analysis, which demonstrates that the sequencing data were satisfactory for further gene expression analyses.ConclusionThis paper reported the transcriptom of the eyestalk, Y-organ, and hepatopancreas from E. sinensis. DGE analysis provided the different expressed genes of the metabolism processes in hepatopancreas that are affected by eyestalk ablation. These findings will facilitate further investigations on the mechanisms of the metabolism of organic substances during development and reproduction in crustaceans.

Stimulation of Molting and Ovarian Maturation by Methyl Farnesoate in the Pacific White Shrimp Litopenaeus vannamei (Boone, 1931)

Eyestalk ablation (ESA) is commonly used in aquaculture to stimulate ovarian maturation in crustaceans, and methyl farnesoate (MF) affects crustacean molting and reproduction. To investigate the physiological effects of ESA and MF treatments on the shrimp Litopenaeus vannamei, we compared the effects of single eyestalk removal and MF injections. The ESA group had the lowest survival rate (50%), and individuals in the 0.1 µg and 1.0 µg MF-treated groups had survival rates of 80 and 73.3%, respectively. Conversely, molting numbers were highest in the ESA group, and similar to those of the 1.0-µg MF group. To investigate shrimp growth, we measured body weight during the experimental period and found that individuals in the ESA and 1.0 µg MF groups showed significant increases in body weight. Furthermore, to investigate the effects of ESA and MF treatments on gonadal maturation, the gonad somatic index (GSI) was calculated after the experiment. All treated groups (ESA and MF) had higher GSI values than the control group, but the ESA and 1.0 µg MF groups were not significantly different. Using histological ovary analysis, we determined that all treated groups showed indications of the previtellogenic stage, unlike the control group (immature stage). These results suggest that the high-MF-concentration treatment produced effects similar to those of ESA with respect to molting number, growth, and ovarian maturation.

Functional characterization of recombinant gonad-inhibiting hormone (GIH) and implication of antibody neutralization on induction of ovarian maturation in marine shrimp

Ovarian maturation in crustacean is controlled by an eyestalk neuropeptide called gonad-inhibiting hormone (GIH) that is presumed to inhibit vitellogenin synthesis. In this study, a recombinant protein of GIH of Penaeus monodon (rPem-GIH) was expressed in Pichia pastoris expression system and purified to homogeneity by reversed-phase SPE (C18). The purified rPem-GIH significantly reduced vitellogenin mRNA level in primary tissue culture derived from previtellogenic ovary of P. monodon broodstock by 45.7% compared with the untreated group. This effect was similar to that of partially purified optic lobe extract, and demonstrated that the rPem-GIH possessed gonad-inhibiting activity. A monoclonal antibody specific to Pem-GIH (anti-GIH mAb) was able to neutralize the activity of GIH from the partially purified optic lobe extract to about 58% by in vitro assay in primary tissue culture of the ovary. Peptide mapping revealed that amino acids 16 to 20 (MYNKV) of the mature Pem-GIH may serve as an epitope of anti-GIH mAb. A single injection of anti-GIH mAb into wild previtellogenic P. monodon was able to induce ovarian maturation and spawning at a comparable rate to the eyestalk ablation. Our results demonstrate neutralization effect of anti-GIH mAb on vitellogenesis inhibitory action of GIH, and thus provide a potential alternative to induce ovarian maturation in P. monodon broodstock.

Pacific White Shrimp (Litopenaeus vannamei) Vitellogenesis-Inhibiting Hormone (VIH) Is Predominantly Expressed in the Brain and Negatively Regulates Hepatopancreatic Vitellogenin (VTG) Gene Expression1

Ovarian maturation in crustaceans is temporally orchestrated by two processes: oogenesis and vitellogenesis. The peptide hormone vitellogenesis-inhibiting hormone (VIH), by far the most potent negative regulator of crustacean reproduction known, critically modulates crustacean ovarian maturation by suppressing vitellogenin (VTG) synthesis. In this study, cDNA encoding VIH was cloned from the eyestalk of Pacific white shrimp, Litopenaeus vannamei, a highly significant commercial culture species. Phylogenetic analysis suggests that L. vannamei VIH (lvVIH) can be classified as a member of the type II crustacean hyperglycemic hormone family. Northern blot and RT-PCR results reveal that both the brain and eyestalk were the major sources for lvVIH mRNA expression. In in vitro experiments on primary culture of shrimp hepatopancreatic cells, it was confirmed that some endogenous inhibitory factors existed in L. vannamei hemolymph, brain, and eyestalk that suppressed hepatopancreatic VTG gene expression. Purified recombinant lvVIH protein was effective in inhibiting VTG mRNA expression in both in vitro primary hepatopancreatic cell culture and in vivo injection experiments. Injection of recombinant VIH could also reverse ovarian growth induced by eyestalk ablation. Furthermore, unilateral eyestalk ablation reduced the mRNA level of lvVIH in the brain but not in the remaining contralateral eyestalk. Our study, as a whole, provides new insights on VIH regulation of shrimp reproduction: 1) the brain and eyestalk are both important sites of VIH expression and therefore possible coregulators of hepatopancreatic VTG mRNA expression and 2) eyestalk ablation could increase hepatopancreatic VTG expression by transcriptionally abolishing eyestalk-derived VIH and diminishing brain-derived VIH.

Sex-lethal gene of the Chinese mitten crab Eriocheir sinensis: cDNA cloning, induction by eyestalk ablation, and expression of two splice variants in males and females

Sex-lethal (Sxl) plays an important role in sex determination in insects. In this study, the full-length complementary DNA (cDNA) of Sxl of the Chinese mitten crab Eriocheir sinensis (EsSxl) was cloned. EsSxl is expressed during different developmental stages of embryos, and its expression level in the cleavage stage is lower than that in other stages of embryonic development, such as the original zoea stage when it reaches the highest level. The expression level of EsSxl in eight different tissues was investigated. EsSxl was expressed in seven examined tissues, excluding eyestalk, and the highest expression levels were observed in testis and hepatopancreas. EsSxl expression in testis was 13-fold higher than that in ovary. After induction by eyestalk ablation, changes in EsSxl expression were also tissue-specific, with EsSxl expression increasing by 2.6-fold and 11.5-fold in ovary and muscle, respectively, between 0 and 7 days after eyestalk ablation and decreasing by 2.0-fold in testis between 0 and 3 days after eyestalk ablation and by 265-fold in hepatopancreas between 0 and 7 days after eyestalk ablation. Two splice variants of EsSxl were identified, and the only difference between them was a 77-bp alternatively spliced intron that is transcripted in EsSxl1 and skipped in EsSxl2. Both variants were expressed in males and females, demonstrating the lack of a sex-specific expression pattern for Sxl in Chinese mitten crab.

Calcium–calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting

Calcium–calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium–calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3262bp in length and has an open reading frame (ORF) of 1038bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1day after ESA in hepatopancreas, and 3days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.

Dynamics of Vitellogenin and Vitellogenesis-Inhibiting Hormone Levels in Adult and Subadult Whiteleg Shrimp, Litopenaeus vannamei: Relation to Molting and Eyestalk Ablation1

Levels of vitellogenin (VG) and vitellogenesis-inhibiting hormone (VIH) in the whiteleg shrimp, Litopenaeus vannamei, were measured by time-resolved fluoroimmunoassay in relation to the molting cycle and ovarian maturation induced by eyestalk ablation. During the molt cycle, VG mRNA expression levels and VG concentrations showed similar patterns of fluctuation. VG levels increased significantly at early intermolt (stage C0) in adults, but not in subadults. Unilateral and bilateral eyestalk ablation increased VG levels in adults, whereas only bilateral eyestalk ablation affected subadults. VIH levels showed contrasting patterns between adults and subadults. In adults, levels were high in late postmolt adults (stage B) and then low thereafter, whereas they increased from postmolt (stage A) to intermolt (stage C0) in subadults and remained high. Unilateral eyestalk ablation increased VIH levels 10 days following ablation in adults, after which levels decreased at 20 days. VIH levels decreased from 10 to 20 days after bilateral ablation. Both unilateral and bilateral ablation led to increased VIH levels in subadults. Eyestalk ablation induced ovarian maturation, but did not reduce VIH concentrations in the hemolymph. This phenomenon was perhaps due to other crustacean hyperglycemic hormone peptides having cross-reactivity with VIH antibodies. This is the first report to quantify concentrations of VG and VIH together in L. vannamei hemolymph, and to examine their relative dynamics.

CDNAs Encoding Chitin Synthase from Shrimp (Pandalopsis Japonica): Molecular Characterization and Expression Analysis

Crustacean growth occurs via molting, the periodic shedding of the exoskeleton. Understanding the genes involved in chitin metabolism associated with the periodic molt cycle is important for various applications to decapod crustacean aquaculture. Chitin synthase is an important enzyme in the chitin biosynthetic pathway that plays a major role in synthesis of new cuticle after molting. In this study, we isolated a full-length cDNA encoding chitin synthase (PajCHS) from Pandalopsis japonica through a combination of PCR (Polymerase chain reaction)- based cloning and bioinformatics analysis. The identified PajCHS encodes a transmembrane protein with 1525 amino acid residues (175 kDa). Comparison with other CHSs from insects revealed that PajCHS contains three domains: N-terminal domain A, catalytic domain B, and C-terminal domain C. Three conserved motifs (EDR, QRRRW, and SWGTR) were also well conserved within and near the catalytic domain B, suggesting that Paj-CHS is functionally active. Variation in the transmembrane helix within the N-terminal and C-terminal domains suggested that the orientation of each CHS may be different. Phylogenetic analysis suggested that PajCHS is an ortholog of CHS1 group members from insect species. However, tissue expression profiles indicated that epidermis, hepatopancreas, intestine, and gill were the major production sites for PajCHS transcript, which is considerably different from insect CHS1. qPCR results showed that eye stalk ablation and 20 hydroxyecdysone (20E) injection increased the expression level of PajCHS mRNA, suggesting that the expression of PajCHS1 may be controlled by endogenous 20E.

NewMiddleCambrian bivalved arthropods from theBurgessShale (BritishColumbia,Canada)

AbstractThe morphology of two new bivalved arthropods,Loricicaris spinocaudatusgen. et sp. nov. andNereocaris briggsisp. nov. from themiddleCambrian (Series 3,Stage 5)BurgessShaleFormation (CollinsQuarry locality onMountStephen,YohoNationalPark,BritishColumbia,Canada), is described. The material was originally assigned to the genusBranchiocaris, but exhibits distinctive character combinations meriting its assignment to other taxa.Loricicaris spinocaudatuspossesses an elongate and spinose abdomen comparable to the contemporaneousPerspicarisandCanadaspis, as well as chelate second head appendages and subtriangular exopods, comparable toBranchiocaris.Nereocaris briggsipossesses a laterally compressed carapace, elongate and delicate appendages and a medial eye located between a pair of lateral eyes on a rhomboidal eye stalk. Although undoubtedly congeneric withNereocaris exilisfrom a slightly younger horizon of theBurgessShaleFormation,N. briggsidiffers in overall proportions and segment number, warranting assignment to a new species. The newly described taxa were coded into an extensive cladistic analysis of 755 characters, and 312 extinct and extant panarthropods, including a variety ofCambrian bivalved arthropods from both theBurgessShale and theChengjiangLagerstätten. Cambrian bivalved arthropods consistently resolved as a paraphyletic assemblage at the base ofArthropoda. Important innovations in arthropod history such as the specialization of the deutocerebral head appendages and a shift from a nekton‐benthic deposit feeding habit to a benthic scavenging/predatory habit, the symplesiomorphic feeding condition ofEuarthropoda (crown‐group arthropods), were found to have occurred among basal bivalved arthropods.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei

BackgroundAbdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp.ResultsA new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3′ and 5′ RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival.ConclusionsOur results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.

Alterations of pattern in immune response and vitellogenesis during induced ovarian development by unilateral and bilateral ablation in Litopenaeus vannamei

Eyestalk ablation is often used to induce ovarian development in female shrimp, but it possibly alters the animal’s hormonal balance, and affects its physiology and immune system. Therefore, this study was conducted to investigate alterations to the immune response during induced ovarian development by unilateral and bilateral ablation in Litopenaeus vannamei. Sub-adult female shrimp could be induced to undergo ovarian development over a 20-day period by both unilateral and bilateral eyestalk ablation; however, ovaries did not reach the final mature stage in which cortical rods are observed. In terms of immune response, total hemocyte count decreased significantly at 4 h after unilateral ablation, and at 20 days after bilateral ablation. After 20 days, expression in the hemocytes of the immune-related genes prophenoloxidase and peroxinectin in bilaterally ablated shrimp increased significantly, whereas that for penaeidin, for lipopolysaccharide- and β-glucan-binding protein, and for transglutaminase mRNA expression did not change significantly. The results of this study suggest that, while eyestalk ablation induces ovarian development, it may also bring about a hormonal imbalance that leads to immune-related genes being activated.

Ecdysone receptor gene from the freshwater prawn Macrobrachium nipponense: Identification of different splice variants and sexually dimorphic expression, fluctuation of expression in the molt cycle and effect of eyestalk ablation

The full-length cDNA of an ecdysone receptor gene (MnEcR) from Macrobrachium nipponense was cloned and the expression of the gene was investigated. MnEcR maintained a relatively low expression level in the early stages of embryos, but from nauplius stage, a steady increase in MnEcR expression was detected, it had the highest expression level in zoea stage. MnEcR was highly expressed in the hepatopancreas and gills among ten different tissues examined. MnEcR was rapidly upregulated in the premolt stage and rapidly downregulated in the postmolt stage. The expression of MnEcR was remarkably downregulated after eyestalk ablation in M. nipponense. An 18-amino-acid insertion/deletion and a 49-amino-acid substitution were found in the coding region of MnEcR, resulting in four splice variants: MnEcR-L1, -L2, -S1 and-S2. The expression of four splice variants of MnEcR in gonads was investigated using RT-PCR. Interestingly, the expression patterns of these splice variants differed between males and females. The dominant splice variants in testis were MnEcR-S1 and -S2, while in ovary they were MnEcR-L1 and -S2, indicating specific roles for these splice variants in male and female individuals.

Isolation of cDNA, genomic organization and expression of small androgen receptor-interacting protein 1 (PmSARIP1) in the giant tiger shrimp Penaeus monodon

The full-length cDNA and genomic organization of small androgen receptor-interacting protein 1 (SARIP1; also called RWD-containing protein 1, Rwdd1) in the giant tiger shrimp (Penaeus monodon) was isolated and characterized. PmSARIP1 was 1366bp in length containing an open reading frame (ORF) of 738bp corresponding to a polypeptide of 245 amino acids. Its genomic sequence contained 5 exons, 4 introns and untranslated regions (UTRs) spanning 5937bp in length. Tissue distribution analysis indicated that PmSARIP1 was specifically expressed in gonads (ovaries>testes) but not in other tissues of wild P. monodon adults. The expression level of PmSARIP1 was not differentially expressed during ovarian maturation in intact wild adults (P>0.05). Eyestalk ablation resulted in up-regulation of PmSARIP1 throughout the ovarian maturation of wild adults where the peak level was observed at stage I (previtellogenic) ovaries (P<0.05). PmSARIP1 mRNA was clearly localized in ooplasm of previtellogenic oocytes. Serotonin injection (5-HT, 50μg/g body weight; 18-month-old shrimp) promoted the expression level of ovarian PmSARIP1 at 6–72h post injection (hpi) with the peak level at 12hpi (P<0.05). Exogenous progesterone administration (0.1μg/g BW; 14-month-old shrimp) did not significantly affect the expression level of ovarian PmSARIP1. In contrast, 17β-estradiol treatment (0.01μg/g BW) resulted in an increase of PmSARIP1 in 14-month-old shrimp at 7days post injection (dpi). Results from this study suggested that PmSARIP1 should play an important role during ovarian maturation of P. monodon.

Attraction of eyestalk ablated Lymnaea acuminata towards the different photo and chemo stimulants

In the eye of Lymnaea acuminata cornea, lens, microvillar layer, pigmented layer, cell layer and photoreceptors works in well organized manner and respond to photic stimulation. In the present study attraction of eye stalk ablated snail Lymnaea acuminata towards the different visible monochromatic light was studied. The photosensitivity of snails to different monochromatic light was wavelength as well as time dependent. In left or right eye stalk ablated snails response to monochromatic light was noted after 3 and 7 days of ablation. However, when both eyes were ablated the response to photic stimulation was observed after 9 days. This functional recovery indicates that type and number of photoreceptors as well as their regenerative capacity are not same in left and right eye of uninfected and infected snails. Sixty minute exposure of red light+ serine as chemo stimulant in bait, caused maximum attraction of snails (62%) than single exposure of chemo (36%) or red (26%) light stimulus.

Effects of Water Temperature, Photoperiod, Eyestalk Ablation, and Non‐Hormonal Treatments on Spawning of Ovary‐Mature Red Swamp Crayfish

AbstractThis study evaluated the temperature and photoperiod requirements of spawning in ovary‐mature red swamp crayfish Procambarus clarkii and examined the feasibility of inducing spawning in them by inexpensive and practical, nonhormonal treatments. In a spawning induction experiment lasting 20 d, four water temperature gradients (12–14°C, 16–18°C, 20–22°C, 24–26°C) and four photoperiods (completely light [CL], 16 h light: 8 h dark [16L:8D], 12 h light: 12 h dark [12L:12D], and completely dark [CD]) were evaluated. Spawning in ovary‐mature red swamp crayfish could be observed in a relatively broad temperature range, from 16°C to 22°C. A period (5–14 d) of low temperature (16–18°C) significantly induced spawning in ovary‐mature females (p &lt; 0.01), but hibernation began to occur and spawning could not be observed when the temperature was below 14°C. Spawning occurred in both CD and CL circumstances, but long‐period light‐groups showed a higher spawning rate—68.9 ± 10.2% (mean ± SD) for CL and 69.6 ± 6.4% for 16L:8D—than that in short‐period light‐groups—53.3 ± 5.7% for 12L:12D and 33.3 ± 8.3% for CD (p &lt; 0.05). In another spawning induction experiment lasting 3 d, eyestalk interventional stimulating with a high concentration of Na+ combined with a low concentration of Ca2+ induced spawning with a high survival rate (SR = 95.8 ± 7.3% [mean ± SD]) in ovary‐mature females. A high concentration of K+ combined with a high concentration of Ca2+ caused no spawning and a high mortality rate (SR = 20.8 ± 19.0% [mean ± SD]). In this study we also observed that water removal or removing from water can be a stimulating factor of inducing fast and synchronous spawning in ovary‐mature crayfish, inducing 46 (16.0%) of 288 ovary‐mature red swamp crayfish spawned in 10–185 s.

The effect of Ibuprofen on female and male reproduction of the open thelyca marine shrimp,Litopenaeus

A research was designed to explore the effect of ibuprofen (IBU) as an intervening agent for prostaglandin biosynthesis on male and female reproduction of three species of Litopenaeus. Ovarian maturation was studied in L. stylirostris and L. vannamei injected periodically with IBU at 0.01 and 0.1 μg g−1 body weight (b.w.) and treated for unilateral eyestalk ablation. IBU at 0.1 μg induced a statistically (P < 0.05) higher rate of L. vannamei females with developing ovaries (45 ± 19%) as compared with controls (12 ± 8%) during 5 weeks; L. stylirostris did not activate vitellogenesis under this experimental protocol. IBU was also evaluated for male sexuality in L. occidentalis, L. stylirostris and L. vannamei. Periodic injections of IBU had no effect on sperm counts and spermatophore weights; however, sperm abnormalities in IBU 0.1 μg treated L. occidentalis and L. stylirostris were very low. In L. stylirostris, the renovation of normal spermatophores was remarkably improved in IBU treated males as compared with controls, but in L. vannamei spermatophore deterioration was not affected. The findings of this research support the hypothesis that prostaglandins are involved in penaeid reproduction playing a negative physiological role; however, altered steroidogenic pathways could also be involved.

Effects of ornamentation and phylogeny on the evolution of wing shape in stalk‐eyed flies (Diopsidae)

Exaggerated male ornaments are predicted to be costly to their bearers, but these negative effects may be offset by the correlated evolution of compensatory traits. However, when locomotor systems, such as wings in flying species, evolve to decrease such costs, it remains unclear whether functional changes across related species are achieved via the same morphological route or via alternate changes that have similar function. We conducted a comparative analysis of wing shape in relation to eye-stalk elongation across 24 species of stalk-eyed flies, using geometric morphometrics to determine how species with increased eye span, a sexually selected trait, have modified wing morphology as a compensatory mechanism. Using traditional and phylogenetically informed multivariate analyses of shape in combination with phenotypic trajectory analysis, we found a strong phylogenetic signal in wing shape. However, dimorphic species possessed shifted wing veins with the result of lengthening and narrowing wings compared to monomorphic species. Dimorphic species also had changes that seem unrelated to wing size, but instead may govern wing flexion. Nevertheless, the lack of a uniform, compensatory pattern suggests that stalk-eyed flies used alternative modifications in wing structure to increase wing area and aspect ratio, thus taking divergent morphological routes to compensate for exaggerated eye stalks.

Molecular Characterisation of Colour Formation in the Prawn Fenneropenaeus merguiensis

IntroductionBody colouration in animals can have a range of functions, with predator protection an important aspect of colour in crustaceans. Colour determination is associated with the carotenoid astaxanthin, which is taken up through the diet and stabilised in the tissues by the protein crustacyanin. As a variety of genes are found to play a role in colour formation in other systems, a holistic approach was employed in this study to determine the factors involved in Fenneropenaeus merguiensis colouration.ResultsFull length F. merguiensis crustacyanin subunit A and C sequences were isolated. Crustacyanin subunit A and C were found in the F. merguiensis transcriptomes of the muscle/cuticle tissue, hepatopancreas, eye stalk and nervous system, using 454 next generation sequencing technology. Custom microarray analysis of albino, light and dark F. merguiensis cuticle tissue showed genes encoding actin, sarcoplasmic calcium-binding protein and arginine kinase to be 4-fold or greater differentially expressed (p<0.05) and down-regulated in albinos when compared to light and dark samples. QPCR expression analysis of crustacyanin and total astaxanthin pigment extraction revealed significantly (p<0.05) lower crustacyanin subunit A and C gene transcript copy numbers and total astaxanthin levels in albinos than in the light and dark samples. Additionally, crustacyanin subunit A and C expression levels correlated positively with each other.ConclusionsThis study identified gene products putatively involved in crustacean colouration, such as crustacyanin, sarcoplasmic calcium-binding protein and forms of actin, and investigated differences in gene expression and astaxanthin levels between albino, light and dark coloured prawns. These genes open a path to enhance our understanding of the biology and regulation of colour formation.

Participation of calmodulin in ovarian maturation induced by eyestalk ablation in red swamp crayfishProcambarus clarkii

Eyestalk ablation is involved in the ovarian development and maturation of crustaceans held in captivity. However, the molecular mechanism of eyestalk ablation induction of ovarian maturation remains unclear. Reports show that calmodulin (CaM) transduces Ca2+ signals into downstream effects, which is a potent critical factor in the induction course of Penaeus monodon. The effects of unilateral eyestalk ablation on the ovarian growth and maturation of domesticated female Procambarus clarkii were investigated in this study. The expression patterns of CaM gene during the maturation course induced by eyestalk ablation were analysed using reverse-transcriptase quantitative PCR. RNA interference technique was used to elucidate the role of CaM in the crayfish eyestalk ablation mechanism to induce ovarian maturation. The results demonstrated that the expression level of CaM was significantly induced to 8.7-fold 1 day after the eyestalk ablation (P < 0.05), and then gradually reduced on the following days. The CaM knockdown which is lethal to crayfish inhibited the ovarian maturation induced by eyestalk ablation. Thus, the CaM calcium signalling pathway is important in the induction of ovarian maturation by eyestalk ablation in crustaceans.