Vitellogenin mRNA Levels Research Articles

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus

In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.

Purification of Sinus Gland Peptides Having Vitellogenesis-Inhibiting Activity from the Whiteleg Shrimp Litopenaeus Vannamei

Vitellogenesis-inhibiting hormone (VIH) in Crustacea belongs to the crustacean hyperglycemic hormone (CHH)-family. To characterize multiple VIH molecules in the whiteleg shrimp Litopenaeus vannamei, seven CHH-family peptides designated as Liv-SGP-A, -B, -C, -D, -E, -F, and -G were purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. The dose-response effects of these peptides on vitellogenin mRNA levels were examined using in vitro incubation of ovarian fragments of the kuruma prawn Marsupenaeus japonicus. Liv-SGP-D showed no significant inhibitory activities, while the other six peptides significantly reduced vitellogenin mRNA levels, however, with differing efficacies, in the order of Liv-SGP-C, -F, -G > -A, -B > -E. Liv-SGP-G was the most abundant CHH-family peptide in the sinus gland and showed strong vitellogenesis-inhibiting activity. As a result of detailed structural analysis, its complete primary structure was determined; it consisted of 72 amino acid residues and possesses an amidated C-terminus.

Effects of Bilateral and Unilateral Eyestalk Ablation on Vitellogenin Synthesis in Immature Female Kuruma Prawns, Marsupenaeus japonicus

In penaeid shrimp species, vitellogenin (VTG) synthesis in the ovary and hepatopancreas is under the inhibitory regulation of a neuroendocrine system, the X-organ/sinus gland complex in the paired eyestalks, and eyestalk ablation (removal of the X-organ/sinus gland complex) is widely used for inducing ovarian development. However, the difference in effects of bilateral and unilateral ablation on VTG gene expression has not been clarified so far. In the present study, VTG synthesis was monitored over a 16-day period after ablation and compared between replicates of immature female kuruma prawns, Marsupenaeus (Penaeus) japonicus, that had been bilaterally or unilaterally ablated and control specimens. After bilateral ablation, ovarian development was induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary increased significantly. Significant VTG mRNA increase was detected 12 h after bilateral ablation. In contrast, after unilateral ablation, ovarian development was not induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary did not change significantly from the control. These results indicate that in immature female prawns, unilateral ablation does not induce VTG gene expression, whereas bilateral ablation induces rapid VTG gene expression (<12 h). The ineffectiveness of unilateral ablation suggests that the remaining X-organ/sinus gland complex in the unilaterally ablated female prawns may secrete sufficient VIH to suppress VTG synthesis.

An in vitro/ in vivo screening assay as a sensitive tool to assess endocrine disruptive activity in surface water

Adult male fathead minnow were exposed for 14 or 28-days under flow-through conditions to undiluted filtered water samples from the rivers Meuse and Rhine in the Netherlands. The experiment included two vessels per treatment each containing 10 fish and samples of five fish were taken after 14 and 28 days. Additional groups were exposed to 17α-ethinylestradiol (EE2) as a reference and untreated drinking water as a negative control. Major endpoints examined included induction of vitellogenin (VTG) synthesis, VTG mRNA activity, hepato- and gonadosomatic indices (HSI and GSI) and gonadal histology. No significant difference was recorded in body weight or mean GSI values between the various treatments. Only exposure to Meuse water resulted in significantly higher HSI means after 14 days. Histological examination showed no apparent effects on gonadal tissue except for eosinophilic blood plasma in fish exposed to Meuse water or EE2. After 14 and 28 days, elevated VTG and VTG mRNA levels were measured in most livers of the fish exposed to Meuse water, but not in the fish exposed to Rhine water. This was confirmed by measuring estrogenic responses in the in vitro ER CALUX® assay. Induction of VTG synthesis proved to be the most sensitive endpoint in the Non Spawning Male Fish Assay for in vivo detection of bio-available estrogenic activity supplementary to a sensitive in vitro assay. The other endpoints examined varied too much and required a higher number of fish or replicates to achieve sufficient power for statistical testing making them less animal friendly.

Laboratory exposure to 17β-estradiol fails to induce vitellogenin and estrogen receptor gene expression in the marine invertebrate Mytilus edulis

To investigate the effect of estrogenic compounds on the marine mussel Mytilus edulis, an assay was developed to measure the expression of two vertebrate estrogen responsive genes—estrogen receptor ( ER) and vitellogenin ( VTG) genes. Expression was measured in M. edulis gonads following a 10-day exposure to 200 ng/l 17β-estradiol (estradiol). The concentrations of esterified estradiol in mussel tissue increased 15-fold in a time-dependent manner—confirming uptake of the compound by the mussels, however there was no significant increase of free estradiol in mussel tissues during the exposure period. The ER and VTG mRNA levels in the gonads of both sexes were measured at days 1–3, 5, and 10 in control and exposed mussels. However, no significant change in the expression of either the ER or VTG genes was recorded at any of the sampled time points. The results suggest that either a regulatory mechanism exists in a mussel that is able to maintain constant levels of free estradiol by converting the excess estradiol into esterified products which may have reduced affinity for the estrogen receptor, or alternatively, that the ER and VTG genes are unresponsive to estrogens in these organisms. The significance of these findings in terms of the utility of ER and VTG as biomarkers of endocrine disruption in bivalve species is discussed.

Protein and gene expression of VTG in response to 4-nonylphenol in rockfish ( Sebastes schlegeli)

To investigate the in vivo estrogenic activity of 4-nonylphenol (4-NP) on reproductive function, we have measured the expression level of rockfish vitellogenin (rVTG) mRNA, concentration of plasma VTG and levels of plasma estradiol-17β (E 2) and testosterone (T) by radioimmunoassay (RIA). An rVTG mRNA transcript of approximately 4.0 kb was extracted from hepatic tissue for Northern blot analysis, and the effects of 4-NP on rVTG mRNA expression in vivo with both male and female juvenile rockfish were examined. The level of rVTG mRNA expression was increased 48 h after injection with 4-NP of 10 mg/kg body mass and after injection in both male and female rockfish. The level of rVTG mRNA expression was increased 24 h after injection in male rockfish injected with 4-NP of 25 mg/kg body mass, while the level of rVTG mRNA expression was increased 12 h after injection in female injected with same dosage. Concentrations of plasma rVTG in female injected with 4-NP of 10 and 25 mg/kg b.w. were increased 72 h after injection and reached 35 and 66 mg/mL, respectively. In male, rVTG concentrations with the dosage of 25 mg/kg body mass were increased 72 h after injection and reached to 14 and 65 mg/mL at 168 h, respectively. Plasma concentrations of T and E 2 in female and male injected with 4-NP were not significantly different between two 4-NP dosages. These results suggest that 4-NP may disrupt the reproductive system of immature rockfish by acting directly on vitellogenesis. Immature females injected with 4-NP were more sensitive than immature males, but VTG in immature males is likely to return to normal conditions slowly. There are obviously large interspecies differences in response of fish to 4-NP.

Production and characterization of recombinant vitellogenesis-inhibiting hormone from the American lobster Homarus americanus

Recombinant peptides related to vitellogenesis-inhibiting hormone (VIH) of the American lobster Homarus americanus were expressed in bacterial cells, and then purified after being allowed to refold. Biological activities of the recombinant VIHs having an amidated C-terminus (rHoa-VIH-amide) and a free carboxyl-terminus (rHoa-VIH-OH) were examined using an ovarian fragment incubation system derived from the kuruma prawn, Marsupenaeus japonicus. The rHoa-VIH-amide significantly reduced vitellogenin mRNA levels in the ovary, while rHoa-VIH-OH had no effect. This is the first report that describes the production of a crustacean VIH having biological activity and the importance of the C-terminal amidation for its vitellogenesis-inhibiting activity.

Expression of gonadotropin subunit genes following 4-nonylphenol exposure in masu salmon: Effects on transcript levels and promoter activities via estrogen receptor alpha

The 4-nonylphenol (NP) group is classified as some of the most potent endocrine disrupting chemicals (EDCs) reported to have estrogenic effects on reproductive endocrine system in vertebrates. In the present study, we investigated the effect of NP on expression of gonadotropin (GTH) subunit genes in masu salmon ( Oncorhynchus masou) to clarify pituitary-based reproductive impact by EDCs. Female juvenile fish were injected with NP (a mixture of ring and chain isomers; 10 or 50 mg kg − 1 body weight) and maintained for 3 days post-injection. A semi-quantitative reverse transcription–polymerase chain reaction was used to analyze the pituitary GTHα, follicle-stimulating hormone β (FSHβ), and luteinizing hormone β (LHβ), and hepatic vitellogenin (VTG) mRNA levels. A low dose of NP induced the GTHα and LHβ mRNA levels. High dose of NP slightly reduced FSHβ mRNA levels in contrast to increased VTG mRNA levels. In a promoter study, NP (1–10 nM) increased the activities of luciferase reporter gene located downstream of masu salmon GTHα or LHβ 5′-flanking region depending on the estrogen receptor α (ERα) in transiently transfected mammalian cells. In contrast, the luciferase activity of FSHβ was elevated by NP in an ERα-independent manner. These results suggest that GTH subunit gene expression of masu salmon may be affected by EDCs at the transcription level and that the genes are useful markers for pituitary effects of xenoestrogens.

The effects of crustacean hyperglycemic hormone-family peptides on vitellogenin gene expression in the kuruma prawn, Marsupenaeus japonicus

In crustaceans, eyestalk ablation induces gonadal maturation of which vitellogenin gene expression is an essential step. However, the molecular mechanisms by which the hormones produced by the X-organ/sinus gland complex in the eyestalk regulate vitellogenesis remain poorly understood. We therefore investigated the effects of sinus gland extracts and certain sinus gland peptides belonging to the crustacean hyperglycemic hormone peptide family on vitellogenin gene expression in ovarian fragments of immature kuruma prawn, Marsupenaeus japonicus. Vitellogenin mRNA levels in incubated ovarian fragments were significantly higher than those in unincubated ovarian fragments prepared from the same animal. Sinus gland extracts and sinus gland peptide-III (type I peptide) both reduced vitellogenin mRNA levels in a dose-dependent manner. In contrast, neither molt-inhibiting hormone (sinus gland peptide-IV) nor molt-inhibiting hormone B, both of which are type II peptides, exerted significant effects on vitellogenin mRNA levels. These results suggest that, in the immature ovary, sinus gland peptide-III is involved in the suppression of vitellogenin gene expression. The existence of such a peptide in the X-organ/sinus gland complex provides a rationale for the significant increase in vitellogenin mRNA levels in the ovaries of eyestalk-ablated prawns.

Starvation affects vitellogenin production but not vitellogenin mRNA levels in the lubber grasshopper, Romalea microptera

The interaction of juvenile hormone (JH) and nutrition was studied during the oviposition cycle of the Eastern Lubber grasshopper ( Romalea microptera). Starvation of females early or in the middle of the cycle inhibited oocyte growth. Starvation for 4 days also reduced hemolymph levels of JH III and vitellogenesis (Vg) to 25% and 15%, respectively, of the levels in fed animals. Likewise, Vg production by fat body fragments incubated in vitro was reduced to 2% of the levels in fed animals and total protein synthesis was reduced to 25%, suggesting that starvation had a stronger effect on Vg synthesis than on protein synthesis. These effects were reversed when starved animals were fed again. However, fat body levels of Vg-mRNA were similar in fed and starved animals, indicating that starvation did not affect transcript levels. We tested whether the decline in JH levels mediated the other starvation effects by infusing animals with JH III or vehicle for 2 days at the onset of starvation. Infusion of JH elevated JH and Vg-mRNA levels 670% and 103%, respectively, above the levels in vehicle-infused animals. However, Vg production and hemolymph levels of Vg were similar to the levels in vehicle-infused animals. These data suggest that JH alone is insufficient to stimulate Vg production.

The dynamics of vitellogenin gene expression differs between intact and eyestalk ablated kuruma prawn Penaeus (Marsupenaeus) japonicus

In order to compare the dynamics of vitellogenin gene expression between naturally maturing prawns and prawns induced to mature artificially by eyestalk ablation, a cDNA encoding vitellogenin was cloned from a cDNA library prepared from the hepatopancreas of the kuruma prawn Penaeus (Marsupenaeus) japonicus, and a quantitative real-time reverse transcription — polymerase chain reaction (RT-PCR) system was developed. Sequence analysis revealed the likely possibility that vitellogenin cDNA from the hepatopancreas was identical to that from the ovary which had been isolated in a previous study. Based on this information, a quantitative real-time RT-PCR system was established and the dynamics of vitellogenin mRNA levels were examined. In naturally maturing prawns, vitellogenin mRNA levels were maintained at low levels during the previtellogenic stage, and thereafter, levels increased as vitellogenesis progressed but decreased during the latter stages of maturation in the hepatopancreas and ovary. In contrast, in eyestalk-ablated prawns, changes in mRNA levels differed in both tissues; an obvious increment of mRNA levels was revealed in the ovary, whereas mRNA levels were negligible in the hepatopancreas. This suggests that eyestalk ablation cannot be used to accurately simulate the natural process of vitellogenin gene expression during vitellogenesis, and that vitellogenin gene expression is regulated in a tissue-specific manner.

Vitellogenesis in the Red Crab, Charybdis feriatus: Contributions from Small Vitellogenin Transcripts (CfVg) and Farnesoic Acid Stimulation of CfVg Expression

During reproductive maturation of the female red crab, Charybdis feriatus, the oocytes rapidly accumulate 110- and 78-kDa major polypeptides. Although the hepatopancreas expresses a high level of vitellogenin (CfVg) mRNA, tissue proteins and secreted proteins of the hepatopancreas consist of only small polypeptides. In addition to the 8.0-kb transcripts, many smaller mRNAs specific to the CfVg gene can be detected. These results suggest that the hepatopancreas also produces smaller CfVg transcripts for small CfVg subunits. Using an RT-PCR cloning approach, a population of the small cDNA clones were isolated. Determining the DNA sequence of these clones revealed that these transcripts were most likely the result of alternative splicing and/or alternative expression of the CfVg gene. In vitro treatment of the hepatopancreas fragments with low levels of farnesoic acid stimulated the expression of CfVg.

Cadmium: An Endocrine Disrupter That Affects Gene Expression in the Liver and Brain of Juvenile Rainbow Trout1

An inhibition of vitellogenesis is observed in fish exposed to cadmium (Cd), either in natural or in experimental conditions. To investigate whether this correlates or not with modifications in the expression of several genes involved in reproduction, we have performed a study on juvenile rainbow trout (Oncorhynchus mykiss) exposed to waterborne Cd in combination with estradiol (E2). A relative reverse transcription-PCR protocol was used to evaluate the effect of Cd exposure on the expression of several genes. We quantified vitellogenin, rainbow trout estradiol receptor alpha (rtERalpha), short and long isoforms (rtERalphaS and rtERalphaL), mRNA levels in liver, and salmon GnRH1, salmon GnRH2, rtERalphaS, and rtERalphaL mRNA levels in the brain. In liver, Cd reduced the E2-stimulated mRNA levels of vitellogenin as well as these of both rtERalpha isoforms in a dose-dependent manner. In brain tissue, our results indicate that rtERalpha mRNA levels are not enhanced by E2. Cd treatments did not modify rtERalphaS isoform expression but reduced rtERalphaL expression in the brain. Focusing on the expression of salmon GnRH (sGnRH) genes, E2 did not affect mRNA levels, but experiments with Cd alone greatly enhanced sGnRH 1 as well as sGnRH 2 gene expression in a dose-dependant manner. This study supports the idea that Cd is an important endocrine disrupter that could act through an inhibition of E2-stimulated genes in the liver and also through a central effect on sGnRH gene expression. Cd may affect a number of E2 signaling pathways but could also affect the reproductive axis by nonestrogenic mechanisms.

Consequences of xenoestrogen exposure on male reproductive function in spottail shiners (Notropis hudsonius).

There is limited information on the physiological consequences associated with exposure to xenoestrogens under field conditions. The objectives of this study were to determine the presence of estrogenic chemicals in the St. Lawrence River and their effects on male reproduction in the spottail shiner (Notropis hudsonius). Hepatic vitellogenin (VTG) mRNA levels in immature shiners indicate extensive estrogenic contamination spanning almost 50 km both upstream and downstream from the island of Montreal. Stages of spermatogenesis were assessed in fish captured at sites having varying levels of estrogenic contamination. In control fish, 95% had testis of either stage IV (50%) or stage V (45%) of spermatogenesis. At Ile Dorval, where VTG mRNA levels are moderate, fish had testes of stage III (38%) and IV (45%) and only 15% of fish were at spermatogenic stage V. In contrast, at Ilet Vert and Ile Beauregard, located in the sewage effluent plume from the City of Montreal and where hepatic VTG mRNA levels are high in fish, none of the fish were at stage V and 8% of fish at Ilet Vert were at stage II of development. Sperm concentration and various motility parameters were significantly lower in shiners from Ilet Vert as compared with those from Iles de la Paix (reference). Histological analyses of testes revealed that more than one-third of the fish captured at sites with the highest estrogenic contamination displayed intersex, a condition in which ovarian follicles were developing within the testis. These data indicate that there is significant estrogenic contamination in the St. Lawrence River that is associated with impaired reproductive function in male fish.

Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish.

The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17 beta-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.

Effects of progesterone and estradiol on the reproductive axis in immature diploid and triploid rainbow trout

In fish species, many studies demonstrated the crucial role of estradiol (E2) in the development of the reproductive axis, but progesterone (P) has been described mainly as a precursor steroid and no clear role by itself has been reported. Moreover, a cooperative effect of P (or another progestin) and E2 in fish has never been reported to our knowledge. In the present work, we investigated the effects of P, alone or in combination with E2, on the reproductive-axis of immature rainbow trout ( Oncorhynchus mykiss). Liver vitellogenin and estradiol receptor (rtER) mRNA levels increased after E2 treatment, but were unchanged by P treatments as a reflection of peripheral action of steroids. In contrast, at the pituitary level, LH contents increased after E2 and/or P treatments. Focusing on the brain level, we confirmed a clear up regulation of rtER expression by E2 in sterile triploid females, and we also demonstrated a similar stimulating effect of P alone but no cooperative effect together with E2. In conclusion, our data demonstrate that in immature trout, prior to the beginning of the first reproductive cycle, unlike E2, P is able to stimulate the reproductive brain-pituitary axis without affecting vitellogenin synthesis in the liver.

The presence of morphologically intermediate papilla syndrome in United Kingdom populations of sand goby (Pomatoschistus spp.): Endocrine disruption?

The sand goby (Pomatoschistus spp.) is a small estuarine fish. Its abundance, life history, and sedentary nature lead to its adoption as a key species in the U.K. Endocrine Disruption in the Marine Environment (EDMAR) Program. This study investigated the presence of classic markers of estrogenic exposure by determining vitellogenin (VTG) and zona radiata protein (ZRP) mRNA levels and ovotestis in estuarine-caught male gobies and investigated morphological changes in the urogenital papilla (UGP). Laboratory exposures to estrogens were also conducted to ascertain the responses of these markers. Wild-caught male fish showed no evidence of ovotestis, VTG, or ZRP mRNA induction. Laboratory exposures suggested that sensitivity of the goby to VTG/ ZRP mRNA induction was similar to flounder. The UGP inspection of wild-caught specimens revealed evidence of feminization of male papillae, a condition denoted as morphologically intermediate papilla syndrome (MIPS). Morphologically intermediate papilla syndrome was more prevalent at estrogenically contaminated sites. Juvenile goby experimentally exposed to 17beta-estradiol for 11 to 32 weeks exhibited signs of the MIPS condition, showing that it was inducible by estrogenic exposure and could therefore be a form of estrogenic endocrine disruption. The estuaries where the MIPS condition was most prevalent (>50% at certain sites) were the Tees, Mersey, and Clyde. The potential of the MIPS condition to significantly interfere with reproductive performance is discussed as well as its use as a monitoring tool for endocrine disruption in the estuarine environment.

Effect of Endocrine Disrupters on mRNA Expression of Vitellogenin (VTG) II and Very Low Density Lipoprotein (apoVLDL) II in the Liver of Quail Embryos.

The present study was conducted to assess estrogenic activity of nonylphenol (NP) and octylphenol (OP) in quail embryos by determining mRNA levels of liver vitellogenin (VTG) II and very low density lipoprotein (apoVLDL) II. The fertile eggs were treated with a single injection of either NP, OP or ethynyl estradiol (EE) at doses of 10 and 100 nmole/egg in 20μl on day 13 of incubation. In the control group the eggs were treated with the vehicle (corn oil, 20μl/egg). On day 15 of incubation the liver was collected and total RNA was extracted. Both mRNA levels were determined by RT-PCR assay and were expressed in relation to β actin mRNA levels. No expression of VTG II mRNA was detected in the control group, whereas a marked induction of VTG II mRNA was revealed in the EE treatment. A weak but distinct expression of VTG II mRNA was evident in the NP and OP treatment groups. ApoVLDLII transcripts were detected in the control group and induced markedly by the injection of EE with higher expression in females. NP also induced considerable expression in females, whereas no transcripts were detected in males. OP also induced the transcript in females but in males OP at 10 nmole was effective. This study indicates that NP and OP possess estrogenic activity in terms of liver VTG II and apoVLDLII mRNA expression in the quail embryo, and that apoVLDLII expression in female embryo is more sensitive to estrogenic substances.

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA.

BackgroundThe ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees.ResultsThe vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96 %) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days.ConclusionInjection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

Preliminary investigations on vitellogenin m-RNA induction in some bioindicator Mediterranean fish species

This paper describes the development of a RT-PCR method for assaying Vtg gene expression in different marine fish as a potentially valuable and sensitive biomarker of exposure to estrogenic chemicals. The levels of Vtg mRNA have been analyzed using primers specifically designed for the various species and the procedures have been standardized relative to actine mRNA expression levels. Different species were analyzed including organisms with a great potential as bioindicators in the Mediterranean (i.e. the red mullet Mullus barbatus, the striped mullet Mugil cephalus, the European eel Anguilla anguilla) or exposed to biomagnification of halogenated hydrocarbons and with elevated commercial value (the bluefin tuna Thunnus thynnus). The analysis of vitellogenin mRNA levels has been standardized in feral fish providing suitable indications for a future development of this approach.