Isolation of developmental mutants of the ascidian Ciona savignyi

Ascidians have been used extensively as model animals for experimental embryology. We report here the results of a pilot study with the aim of developing genetic methods for the ascidian Ciona savignyi. The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to induce point mutations. F1 animals, produced by using sperm from ENU-treated animals to fertilize untreated eggs, were grown to reproductive age. Sperm and eggs collected from the hermaphrodite F1 adults were used to generate self-fertilized F2 broods, which were then screened for recessive, zygotically acting mutations. Animals carrying potential mutations were outcrossed to wild type to test for the heritability of the phenotypes. We report on a number of mutants isolated using this method, including several with abnormalities in tail and notochord development. Received: 15 March 1999 / Accepted: 6 May 1999     

Heritable Targeted Inactivation of the Rainbow Trout (Oncorhynchus mykiss) Master Sex-Determining Gene Using Zinc-Finger Nucleases

Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.     

Reproductive Strategies of the Insidious Fish Ectoparasite,Neobenedenia sp. (Capsalidae: Monogenea)

Fish monogeneans are lethal parasites in aquaculture. We provide the first experimental evidence that a notorious fish monogenean, Neobenedenia sp., can produce viable eggs in isolation for three consecutive generations. We infected individual, isolated, farmed barramundi, Lates calcarifer (Bloch) with a single oncomiracidium (larva) of the hermaphroditic monogenean Neobenedenia sp. Isolated parasites reached sexual maturity at day 10 post-hatch (24°C, 35‰) and laid ∼3,300 embryonated eggs over 17 days. Egg production rapidly increased following sexually maturity on day 10 (58±15 eggs) and peaked on day 15 (496±68 eggs) before gradually decreasing. Neobenedenia sp. exhibited egg laying and egg hatching rhythms. Parasites laid eggs continuously, but egg production increased in periods of darkness (64.3%), while the majority of oncomiracidia (81%) emerged from eggs in the first three hours of light. Eggs laid by isolated ‘parent’ parasites hatched and individual emerging oncomiracidia were used to infect more individual, isolated fish, with three consecutive, isolated, parasite generations (F1, F2 and F3) raised in the laboratory. Infection success and egg hatching success did not differ between generations. Our data show that one parasite, in the absence of a mate, presents a severe threat to captive fish populations.     

Gamete-associated flavobacteria of the oviparous Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) in lakes Michigan and Huron,North America

Flavobacterial diseases, caused by multiple members of the Family Flavobacteriaceae, elicit serious losses in wild and farmed fish around the world. Flavobacteria are known to be transmitted horizontally; however, vertical transmission has been suspected but proven only for one fish-pathogenic flavobacterial species (e.g., Flavobacterium psychrophilum). Herein, we report on the isolation and molecular identification of multiple Flavobacterium and Chryseobacterium taxa from the ovarian fluid and eggs of feral Great Lakes Chinook salmon (Oncorhynchus tshawytscha). Identified egg- and ovarian fluid-associated flavobacteria were either well-known flavobacterial fish pathogens (e.g., F. psychrophilum and F. columnare), most similar to emerging fish-associated flavobacteria (e.g., F. spartansii, F. tructae, F. piscis, C. piscium, C. scophthalmum), or were distinct from all other described Chryseobacterium and Flavobacterium spp., as determined by phylogenetic analyses using neighbor-joining, Bayesian, and Maximum Likelihood methodologies. The gamete-associated flavobacteria fell into three groups (e.g., those that were recovered from the ovarian fluid but not eggs; those that were recovered from the ovarian fluid and eggs; and those that were recovered from eggs but not ovarian fluid), a portion of which were recovered from eggs that were surface disinfected with iodophor at the commonly used dose and duration for egg disinfection. Some gamete-associated flavobacteria were also found in renal, splenic, and neurological tissues. Systemic polymicrobial infections comprised of F. psychrophilum and F. columnare were also detected at nearly an 11% prevalence. This study highlights the potential role that sexual products of female Great Lakes Chinook salmon may play in the transmission of fish-associated flavobacteria.     

Cultivation in vitro of Schistosomatium douthitti (Trematoda: Schistosomatidae)

Basch P. F. and O'Toole M. L. 1982. Cultivation in vitro of Schistosomatium douthitti (Trematoda: Schistosomatidae). International Journal for Parasitology12: 541–545. We grew S. douthitti in vitro from the cercaria to pairing ovigerous adults, using methods and media previously reported for cultivation of Schistosoma mansoni. Growth of S. douthitti in vitro was much more rapid than that of S. mansoni, but the cultures achieved a similar stage of maximal development. Cultured S. douthitti worms were smaller than those from animals and eggs were inviable. We determined that females of this species, known to produce eggs in unisexual animal infections, will also do so in culture in the absence of males.     

Co-occurrence of Point Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Aedes aegypti Populations in Myanmar

Background

Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar.

Methodology/Principal Findings

We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study.

Conclusions/Significance

Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 μl of L. infantum promastigotes (1 × 10⁶ cells per ml) was injected into the hemocel through the coxa I of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L. infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated.     

Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs

The water flea, Daphnia, has been the subject of study in ecology, evolution, and environmental sciences for decades. Over the last few years, expressed sequence tags and a genome sequence have been determined. In addition, functional approaches of overexpression and gene silencing based on microinjection of RNAs into eggs have been established. However, the transient nature of these approaches prevents us from analyzing gene functions in later stages of development. To overcome this limitation, transgenesis would become a key tool. Here we report establishment of a transgenic line using microinjection of plasmid into Daphnia magna eggs. The green fluorescent protein (GFP) gene fused with the D. magna histone H2B gene under the control of a promoter/enhancer region of the elongation factor 1α-1 (EF1α-1) gene, EF1α-1::H2B-GFP, was used as a reporter providing high resolution visualization of active chromatin. Transgenic lines were obtained from 0.67% of the total fertile adults that survived the injections. One of the transgenic animals, which exhibited fluorescence in the nuclei of cells during embryogenesis and oogenesis, had two copies of EF1α-1::H2B-GFP in a head-to-tail array. This is the first report of a transgenesis technique in Daphnia and, together with emerging genome sequences, will be useful for advancing knowledge of the molecular biology of Daphnia.     

A clonal analysis of the roles of somatic cells and germ line during oogenesis in Drosophila

In order to test whether particular female sterile mutations block functions which normally occur in somatic or germ line derivatives, clones homozygous for each mutation were X-ray induced in heterozygous females. Using the germ line-dependent egg marker, fs(1)K10, it was possible to identify the eggs derived from clones which had been induced in the germ line. Mutations were classified as germ line dependent when these eggs also showed the phenotype associated with the female sterile mutation. Two mutations which caused early abnormalities in oogenesis (fs(1)116, fs(1)1304) were shown to affect germ cells, whereas two mutations which caused egg retention (fs(1)462, fs(1)1001) were somatically dependent. A mutation altering egg dimensions without affecting egg volume (short egg) was also shown to depend on somatic cells in the ovary. With one exception (fs(1)K4), mutations which caused production of fragile, collapsed eggs (fs(1)180, fs(1)473, fs(1)384, and fs(1)1163) were somatically dependent. Patches of mutant fs(1)384 morphology were found in the chorions of the eggs not derived from germ line clones. These patches are interpreted as being caused by homozygous clones in the somatically derived follicle cell epithelium and suggest that fs(1)384 affects processes occurring in these cells during the synthesis of the egg coverings.     

Generation of Ci-Brachyury-GFP stable transgenic lines in the ascidian Ciona savignyi

We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F(0) adults tested, were found to transmit the transgene to their offspring (F(1)s) at frequencies of 42% and 23%. The transgene was further inherited by the F(2) in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F(3) and F(4) generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos.     

Fresh and frozen-thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765 mg/kg C57BL6/J or 600 mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P < 0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P < 0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P < 0.05).     

Sequestration and Transfer of Cry Entomotoxin to the Eggs of a Predaceous Ladybird Beetle

In the past 10 years, sequestration of Cry toxins and transfer to offspring has been indicated in three insect species in laboratory studies. This work directly demonstrates the sequestration and intergenerational transfer of Cry1F by the parents of the aphidophagous coccinellid predator, Harmonia axyridis, to its offspring. Recently emerged adults (10 individual couples/cage/treatment) were exposed during 20 days to aphids (100 Myzus persicae each day) that fed on a holidic diet containing 20 μg/mL Cry1F (and a control-group). Egg batches and neonate larvae were monitored daily, and counted and weighed for immunodetection of Cry1F by ELISA. At the end of the bioassay, the parents were weighed and analyzed by ELISA. Cry1F was detected in the offspring, both eggs and neonate larvae, of exposed H. axyridis adults. On average the neonate larvae had 60% of the Cry1F concentration of the eggs from the same egg batch. The Cry1F concentration in the adults was positively correlated with the concentration in their eggs. These three results provided independent evidence of transfer to offspring. No detrimental effects of Cry1F were observed on the age of first reproduction, total number of eggs laid per female, age-specific fecundity, egg development time, hatching rate, or fertility rate. The occurrence and generality of intergenerational transfer of Cry toxins should be investigated in the field to determine its potential ecological implications.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.     

A C. elegans mutant screen based on antibody or histochemical staining

A method has been developed for isolating mutations in Caenorhabditis elegans that alter antibody or histochemical staining patterns. The basis for this method is a new procedure for making C. elegans permeable that does not kill the eggs contained within the uterus of gravid adult hermaphrodites. A mutagenized population of gravid hermaphrodites is made permeable and then stained with either an antibody or a histochemical stain. Animals that stain aberrantly are picked to individual petri plates and the eggs within the uterus of the stained mother hatch and establish a new genetic line. Antibody and histochemical stains are especially useful phenotypes because the staining pattern will usually directly reflect the gene expression pattern of the gene that codes for the antigen or enzyme. This method was used to isolate mutants that alter the expression of a mec-7lacZ fusion gene. Transgenic animals that contained the mec-7lacZ gene integrated into chromosome I were treated with the mutagen ethylmethanesulfonate, allowed to self-fertilize for two generations and then stained with X-gal or antibodies against β-galactosidase. Gravid animals that stained abnormally were picked to fresh petri plates and offspring were used to establish new mutant lines.     

Generation of albino Xenopus tropicalis using zinc‐finger nucleases

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc‐finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN‐induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.     

Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER

Three dominant female-sterile mutations were isolated following ethyl methanesulfonate (EMS) mutagenesis. Females heterozygous for two of these mutations show atrophy of the ovaries and produce no eggs (ovo ^D1) or few eggs (ovo^D2); females heterozygous for the third mutation, ovo^D3, lay flaccid eggs. All three mutations are germ line-dependent and map to the cytological region 4D-E on the X chromosome; they represent a single allelic series. Two doses of the wild-type allele restore fertility to females carrying ovo^D3 and ovo^D2, but females carrying ovo^D1 and three doses of the wild-type allele remain sterile. The three mutations are stable in males but are capable of reversion in females; reversion of the dominant mutations is accompanied by the appearance, in the same region, of a recessive mutation causing female sterility. We discuss the utility of these mutations as markers of clones induced in the female germ line by mitotic recombination as well as the nature of the mutations.     

NT5E Mutations That Cause Human Disease Are Associated with Intracellular Mistrafficking of NT5E Protein

Ecto-5′-nucleotidase/CD73/NT5E, the product of the NT5E gene, is the dominant enzyme in the generation of adenosine from degradation of AMP in the extracellular environment. Nonsense (c.662C→A, p.S221X designated F1, c.1609dupA, p.V537fsX7 designated F3) and missense (c.1073G→A, p.C358Y designated F2) NT5E gene mutations in three distinct families have been shown recently to cause premature arterial calcification disease in human patients. However, the underlying mechanisms by which loss-of-function NT5E mutations cause human disease are unknown. We hypothesized that human NT5E gene mutations cause mistrafficking of the defective proteins within cells, ultimately blocking NT5E catalytic function. To test this hypothesis, plasmids encoding cDNAs of wild type and mutant human NT5E tagged with the fluorescent probe DsRed were generated and used for transfection and heterologous expression in immortalized monkey COS-7 kidney cells that lack native NT5E protein. Enzyme histochemistry and Malachite green assays were performed to assess the biochemical activities of wild type and mutant fusion NT5E proteins. Subcellular trafficking of fusion NT5E proteins was monitored by confocal microscopy and western blot analysis of fractionated cell constituents. All 3 F1, F2, and F3 mutations result in a protein with significantly reduced trafficking to the plasma membrane and reduced ER retention as compared to wild type protein. Confocal immunofluorescence demonstrates vesicles containing DsRed-tagged NT5E proteins (F1, F2 and F3) in the cell synthetic apparatus. All 3 mutations resulted in absent NT5E enzymatic activity at the cell surface. In conclusion, three familial NT5E mutations (F1, F2, F3) result in novel trafficking defects associated with human disease. These novel genetic causes of human disease suggest that the syndrome of premature arterial calcification due to NT5E mutations may also involve a novel “trafficking-opathy”.     

Genetische Analyse zweier Mutanten vonDinophilus gyrociliatus (Archiannelida) mit veränderter Eigröße

D. gyrociliatus has two types of eggs: a smaller type giving rise to ♂♂, and a larger type developing into ♀♀. Two mutants, which originated in laboratory cultures derived from animals collected at Helgoland, produced eggs of an abnormal size; the first, “Gr”, yielding larger-than-normal male-type eggs, the second, “Kl”, smaller male-type eggs. Size of eggs is determined by the genotype of the mother, the genotype of the eggs being without influence. Both mutants show a dominant monofactorial inheritance. In crosses withGr/Gr-individuals an exceptionally high rate of reverse mutations to wildtype (1,4 to 5,0 %) has been observed. Results indicate thatGr is either an unstable gene or a chromosome mutation in the form of a duplication or trisomy of one chromosome.