Insulin signaling in the long-lived reproductive caste of ants.

In most organisms, reproduction is correlated with shorter life span. However, the reproductive queen in eusocial insects exhibits a much longer life span than that of workers. In Harpegnathos ants, when the queen dies, workers can undergo an adult caste switch to reproductive pseudo-queens (gamergates), exhibiting a five-times prolonged life span. To explore the relation between reproduction and longevity, we compared gene expression during caste switching. Insulin expression is increased in the gamergate brain that correlates with increased lipid synthesis and production of vitellogenin in the fat body, both transported to the egg. This results from activation of the mitogen-activated protein kinase (MAPK) branch of the insulin signaling pathway. By contrast, the production in the gamergate developing ovary of anti-insulin Imp-L2 leads to decreased signaling of the AKT/forkhead box O (FOXO) branch in the fat body, which is consistent with their extended longevity.

RNAi silencing of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene inhibits vitellogenesis in Chinese mitten crab Eriocheir sinensis.

The sesquiterpenoid methyl farnesoate (MF), a de-epoxide form of insect juvenile hormone III (JH III), plays an essential role in regulating many crucial physiological processes in crustaceans including vitellogenesis and reproduction. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of JH III and MF. In the present study, a full-length cDNA encoding HMGR (EsHMGR) in Eriocheir sinensis was isolated and characterised. Sequence analysis of EsHMGR revealed that it belongs to Class I HMGR family proteins with HMG-CoA-binding and NADPH-binding domains, both important for HMGR activity. In addition to its ubiquitous tissue expression, expression of EsHMGR was highly specific to the ovary, the main site of Vg synthesis. During ovarian development, EsHMGR expression in ovary displayed a stage-specific pattern, and was correlated with expression of vitellogenin (EsVg) in hepatopancreas, which suggests that EsHMGR possibly involved in vitellogenesis. To further investigate the functional role of EsHMGR in vitellogenin biosynthesis in E. sinensis, RNA interference-mediated gene silencing was carried out both in vitro and in vivo. Quantitative PCR results showed that injection of EsHMGR double-stranded RNA (dsRNA) led to a significant decrease in EsVg expression levels in ovary and hepatopancreas both in vitro and in vivo. Taken together, the results suggest that EsHMGR is involved in vitellogenin biosynthesis in female E. sinensis, which may provide a new resource for HMGR enzymes participating in reproduction in crustaceans.

Characterization of the foxl2 gene involved in the vtg expression in mud crab (Scylla paramamosain).

Forkhead box protein L2 (Foxl2) is involved in multiple physiological processes, such as ovarian development, granulosa cell differentiation, ovarian follicle development, and oocyte growth. In this study, a Spfoxl2 gene encoded 530 amino acid protein with characteristic forkhead (FH) domain was identified from transcriptome data of mud crab Scylla paramamosain and validated the accuracy by PCR technology. Meanwhile, the orthologues of the Spfoxl2 gene in other 14 crustacean species were identified with the same method. Further multiple sequence alignment analysis revealed the Foxl2 was highly conserved, especially in the FH domain, even completely identical in several species. Besides, the semi-quantitative PCR (Sq-PCR) result showed Spfoxl2 gene was mainly expressed in the gonad (testis and ovary). Further quantitative real-time PCR (qRT-PCR) result demonstrated its expression level in the testis was significantly higher than that in the ovary (p < 0.01). In addition, the qRT-PCR result showed that in zoea V, megalopa, and larval I, the expression level of Spfoxl2 in megalopa is the highest. In addition, a putative Foxl2 binding site was identified on the promoter region of Spvtg, and knockdown of Spfoxl2 mediated by RNAi technology increased the expression of Spvtg in the ovary, suggesting Spfoxl2 might be the upstream negative regulator of Spvtg. Overall, this study provided new insights into the role of Spfoxl2 in ovary development through regulating Spvtg expression in S. paramamosain.

Does estrogen regulate vitellogenin synthesis in corals?

Most broadcast spawner corals have a vitellogenic phase that lasts at least 6 months. It is established that estrogen regulates vitellogenin synthesis in vertebrates. Although some research have been conducted on the physiological role of sex steroids in corals, little is known about their involvement in oocyte development. This study aimed to detect steroid hormones - progesterone, testosterone, and estradiol-17ß (E2) - in Acropora tenuis and study the relationships between vitellogenesis/vitellogenin synthesis and these steroids. This study also investigated the effect of E2 on vitellogenin synthesis in corals and identified steroidogenic enzymes in A. tenuis genome. Branches from tagged coral colonies were collected monthly from March to November. Histological observations showed that oocytes were vitellogenic from March to May (Stage IV and V), but not in June, and that gonads were occupied by immature oocytes in September (Stage I). Real-time qPCR revealed that vitellogenin (vg1 and vg2) transcript levels in coral branches were high in April and May, implying that corals actively underwent vitellogenesis during these months, and spawned before June. Liquid chromatography-mass spectrometry revealed that E2 could be detected in coral branches in March, April, and May, but not in June, whereas testosterone and progesterone did not fluctuate much in the same months. Immersing branches in E2-containing seawater failed to increase vitellogenin transcript levels. The results indicate that E2 is involved in oogenesis but does not positively regulate vitellogenin synthesis. Steroidogenic enzymes (except CYP19A) were identified in A. tenuis, suggesting that corals may endogenously synthesize progestogens and androgens from cholesterol.

Retinoid X receptor modulates vitellogenin gene expression in black tiger shrimp, Penaeus monodon.

Effective inducing of ovarian maturation in female shrimp broodstock is important for successful breeding programs. Vitellogenesis is a biochemical process during which a yolk protein precursor vitellogenin (Vg) is synthesized and thus, can be used to indicate ovarian maturation stage. In this study, transcriptional regulation of Vg synthesis in the black tiger shrimp, Penaeus monodon was investigated. Genome walking on 5' upstream sequence of Vg gene revealed several putative binding sites of lipophilic retinoic acid response elements (RARE), and nuclear hormone responsive elements. Deletion of RARE significantly reduced the promoter activity to drive the expression of luciferase reporter gene in Sf-9 cells. To validate the trans-factor that potentially controls Vg expression through RARE, a cDNA encoding retinoid X receptor (PmRXR), one of the RARE-bound transcription factors was cloned from P. monodon's ovary. PmRXR expression was detected in various shrimp tissues, and was up-regulated during ovary development in a similar way to Vg expression. The DNA-binding domain of PmRXR protein showed specific binding to RARE-containing region on Vg 5' upstream sequence as determined by Electrophoretic Mobility Shift Assay (EMSA). Furthermore, dsRNA-mediated PmRXR silencing in previtellogenic and vitellogenic shrimp revealed that suppression of PmRXR could reduce Vg transcript in both stages. Taken together, the results presented in this study indicate that RXR is possibly an activator protein that modulates Vg expression in shrimp ovary through the binding to RARE.

Insulin/IGF signaling and TORC1 promote vitellogenesis via inducing juvenile hormone biosynthesis in the American cockroach.

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient sensors insulin/IGF signaling (IIS) and target of rapamycin complex1 (TORC1), was investigated to elucidate the molecular mechanisms of vitellogenesis regulation in the American cockroach, Periplaneta americana Our data showed that a block of JH biosynthesis or JH action arrested vitellogenesis, in part by inhibiting the expression of doublesex (Dsx), a key transcription factor gene involved in the sex determination cascade. Depletion of IIS or TORC1 blocked both JH biosynthesis and vitellogenesis. Importantly, the JH analog methoprene, but not bovine insulin (to restore IIS) and amino acids (to restore TORC1 activity), restored vitellogenesis in the neck-ligated (IIS-, TORC1- and JH-deficient) and rapamycin-treated (TORC1- and JH-deficient) cockroaches. Combining classic physiology with modern molecular techniques, we have demonstrated that IIS and TORC1 promote vitellogenesis, mainly via inducing JH biosynthesis in the American cockroach.

JNK signaling pathway regulates the development of ovaries and synthesis of vitellogenin (Vg) in the swimming crab Portunus trituberculatus.

The development of Portunus trituberculatus egg cells is directly related to the nutritional status of the fertilized egg, which affects the key production stages of offspring hatching. Vitellogenin plays a key role in the nutrient supply required for the development of the egg cells. The c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase (MAPK) superfamily and plays an important role in cell proliferation, transformation, differentiation, and apoptosis. At present, there are no reports on the involvement of the JNK signaling pathway in the reproductive regulation of P. trituberculatus. In this study, rapid amplification of complementary DNA ends amplification technology was used to clone the full length of JNK complementary DNA, which has a length of 2094 bp, including an open reading frame (ORF) of 1266 bp encoding a 421-amino acid protein. The protein includes the S_TKC conserved domain with a TPY phosphorylation site, which is a typical feature of the JNK gene family. Observing tissue sections found the oocytes in the inhibitor group developed slowly, while the oocytes in the activated group showed accelerated development. Meanwhile, Portunus trituberculatus JNK and vitellogenin (Vg) genes exhibited the same trend in the hepatopancreas and ovaries, and the expression of the SP600125 group was downregulated (P < 0.05), while the anisomycin group was upregulated (P < 0.05). In addition, JNK enzyme activity and vitellin (Vn) content in the ovarian tissue showed that the JNK activity of the SP600125 group decreased, while activity increased in the anisomycin group. The accumulation of Vn content in the SP600125 group decreased, and that in the anisomycin group increased. In summary, after injection with inhibitor or activator, the JNK signaling pathway of P. trituberculatus was inhibited or activated, the accumulation of Vn in the ovary was reduced or increased, and ovarian development was inhibited or accelerated, respectively. These results indicated that the JNK signaling pathway is involved in the regulation of Vg synthesis and ovarian development in P. trituberculatus. The results of this study further add to the knowledge of the breeding biology of P. trituberculatus and provide a theoretical reference for the optimization of breeding techniques in aquaculture production systems.

Choriogenin transcription in medaka embryos and larvae as an alternative model for screening estrogenic endocrine-disrupting chemicals.

This study assessed the transcription levels of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL, ChgH, and ChgHm), cytochrome P450 aromatase (CYP19a1b), and ER subtypes (ERα, ERß1, and ERß2), in 7 days-post-fertilization (dpf) embryos and 9 and 12 dpf larvae of medaka (Oryzias latipes) exposed to estrogenic endocrine-disrupting chemicals (EDCs). The <5 h-post-fertilization embryos were exposed to EDCs such as 17ß-estradiol (E2), p-n-nonylphenol (NP), and bisphenol A (BPA). In E2 (0.10-222 nM)-treated 7 dpf embryos and 9 or 12 dpf larvae, ChgL, ChgH, and ChgHm expression was up-regulated in a concentration-dependent manner. By contrast, interestingly, Vtg1 and Vtg2 expression was not induced in E2-treated 7 dpf embryos but was significantly induced in 9 and 12 dpf larvae, suggesting a developmental-stage-specific regulatory mechanism underlying Vtg expression. The maximum concentrations of NP (0.09-1.5 µM) and BPA (1.8-30 µM) up-regulated Chg expression in 9 or 12 dpf larvae, and the relative estrogenic potencies (REPs) of E2, NP, and BPA were 1, 2.1 × 10-4, and 1.0 × 10-5, respectively. Chg messenger RNA (mRNA) in medaka embryos and larvae can be used as a sensitive biomarker for screening potential estrogenic EDCs. Our assay system using embryos and larvae can be used as an in vivo alternative model because independent feeding stages (e.g., embryonic and early larval stages) are suitable alternatives.

Does hepatotoxicity interfere with endocrine activity in zebrafish (Danio rerio)?

Vitellogenin (VTG), a well-established biomarker for the diagnosis of endocrine activity in fish, is used in multiple OECD test guidelines (TG) to identify activities of chemicals on hormonal pathways. However, the synthesis of VTG may not only be modified by typical endocrine-related pathways, but also through non-endocrine-mediated processes. In particular, hepatotoxicity, i.e. toxicant-induced impairment of liver structure and function, might influence VTG as a biomarker, since VTG is synthesized in hepatocytes. An intimate understanding of the interplay between endocrine-related and non-endocrine-related pathways influencing VTG production is crucial for the avoidance of erroneous diagnoses in hazard assessment for regulatory purposes of chemical compounds. In order to investigate whether hepatotoxicity may interfere with hepatic VTG synthesis, adult zebrafish (Danio rerio) were exposed to three well-known hepatotoxicants, acetaminophen, isoniazid and acetylsalicylic acid, according to OECD TG 230. Various hepatotoxicity- and endocrine system-related endpoints were recorded: mRNA expression of selected endocrine- and hepatotoxicity-related marker genes in the liver; VTG levels in head/tail homogenates; and liver histopathology. All three test compounds induced significant, but mild single cell necrosis of hepatocytes and transcriptional changes of hepatotoxicity-related marker genes, thus confirming hepatotoxic effects. A positive correlation between hepatotoxicity and reduced hepatic VTG synthesis was not observed, with the single exception of a weak increase in female zebrafish exposed to APAP. This suggests that - in studies conducted according to OECD TG 229 or 230 - it is unlikely that hepatotoxic chemicals will interfere with the hepatic capacity for VTG synthesis.

A cross-talk between leptin and 17ß-estradiol in vitellogenin synthesis in rainbow trout Oncorhynchus mykiss liver.

The existence of nutritional and energy reserves is fundamental for fish female fertility, so that the existence of a correlation between metabolic reserves and reproductive capacity is suggested. Leptin regulates body weight and energy homeostasis. Estradiol induces the synthesis of vitellogenin, a phospholipoglycoprotein produced by the liver and taken up by the growing oocytes. The objective of this study was to investigate the possible existence of a crosstalk between 17ß-estradiol (E2) and leptin in the modulation of E2-induced vtg in the rainbow trout Oncorhynchus mykiss. Liver slices were incubated with recombinant trout leptin (rt-lep) at three different concentrations (1-10-100 ng/ml). rt-lep brought about the decrease of E2-induced vtg secretion in the medium and the down-regulation of vtg mRNA expression. Moreover, rt-lep stimulated the lipase activity and diminished the liver fatty acid content. The combined employment of signal transduction inhibitors and the analysis of signal transduction phosphorylated factors revealed that rt-lep effect on E2-induced vtg occurred through the activation of phosphodiesterase, protein kinase C, MAP kinases, and protein kinase A. In conclusion, our study suggests that leptin influences E2-induced vtg synthesis in the rainbow trout Oncorhynchus mykiss by modifying both the protein and the lipid components.

Caste-based differential transcriptional expression of hexamerins in response to a juvenile hormone analog in the red imported fire ant (Solenopsis invicta).

The reproductive ground plan hypothesis proposes that gene networks regulating foraging behavior and reproductive female physiology in social insects emerged from ancestral gene and endocrine factor networks. Expression of storage proteins such as vitellogenins and hexamerins is an example of this co-option. Hexamerins, through their role modulating juvenile hormone availability, are involved in caste determination in termites. The genome of the fire ant (Solenopsis invicta) encodes four hexamerin genes, hexamerin-like (LOC105192919, hereafter called hexamerin 1), hexamerin (LOC105204474, hereafter called hexamerin 2), arylphorin subunit alpha-like, and arylphorin subunit beta. In this study, a phylogenetic analysis of the S. invicta hexamerins determined that each predicted protein clustered with one of the orthologous Apis mellifera hexamerins. Gene expression analyses by RT-qPCR revealed differential expression of the hexamerins between queens and workers, and between specific task-allocated workers (nurses and foragers). Queens and nurses had significantly higher expression of all genes when compared to foragers. Hexamerin 1 was expressed at higher levels in queens, while hexamerin 2 and arylphorin subunit beta were expressed at significantly higher levels in nurses. Arylphorin subunit alpha-like showed no significant difference in expression between virgin queens and nurses. Additionally, we analyzed the relationship between the expression of hexamerin genes and S-hydroprene, a juvenile hormone analog. Significant changes in hexamerin expression were recorded in nurses, virgin queens, and foragers 12 h after application of the analog. Hexamerin 1 and arylphorin subunit alpha-like expression were significantly lower after analog application in virgin queens. In foragers, hexamerin 2 and arylphorin subunit beta were significantly lower after analog application, while in nurses expression of all genes were significantly lower after analog application. Our results suggest that in S. invicta hexamerin genes could be associated with reproductive division of labor and task-allocation of workers.

A putative direct repeat element plays a dual role in the induction and repression of insect vitellogenin-1 gene expression.

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling.

Transcriptional and physiological effects of the pyrethroid deltamethrin and the organophosphate dimethoate in the brain of honey bees (Apis mellifera).

The pyrethroid deltamethrin and the organophosphate insecticide dimethoate are widely used in agriculture and in urban areas. Both plant protection products (PPPs) unintendedly result in adverse effects in pollinators. Currently, the sublethal effects of both compounds are poorly known, particularly on the molecular and biochemical level. Here we analysed effects of deltamethrin and dimethoate at environmental and sublethal concentrations in honey bee workers by focusing on transcriptional changes of target genes in the brain. In addition, expression of vitellogenin protein and activity of acetylcholinesterase were assessed upon dimethoate exposure to assess physiological effects. Deltamethrin resulted in induction of the cyp9q2 transcript at 0.53 ng/bee, while dimethoate led to induction of vitellogenin on the mRNA and protein level at 2 ng/bee. Transcripts of additional cytochrome P450-dependent monooxygenases (cyps) and genes related to immune system regulation were not differentially expressed upon PPP exposure. Dimethoate but not deltamethrin led to a strong and concentration-related inhibition of the acetylcholinesterase at 2 and 20 ng/bee. Our data demonstrate that deltamethrin and dimethoate exhibit transcriptional effects at environmental concentrations in the brain of honey bees. Dimethoate also strongly affected physiological traits, which may translate to adverse effects in forager bees.

Mechanisms for type-II vitellogenesis-inhibiting hormone suppression of vitellogenin transcription in shrimp hepatopancreas: Crosstalk of GC/cGMP pathway with different MAPK-dependent cascades.

Vitellogenesis is the process of yolk formation via accumulating vitellin (Vn) with nutrients in the oocytes. Expression of vitellogenin (Vg), the precursor of Vn, is one of the indicators for the start of vitellogenesis. In Pacific white shrimp (Litopenaeus vannamei), the type-II vitellogenesis-inhibiting hormone (VIH-2) effectively suppresses hepatopancreatic Vg mRNA expression. In this study, we demonstrate the increasing transcript levels of hepatopancreatic Vg during L. vannamei ovarian development, suggesting that the hepatopancreas-derived Vg/Vn may also contribute to vitellogenesis in this species. Using a combination of in vivo injections and in vitro primary cell cultures, we provide evidences that the inhibition of VIH-2 on hepatopancreatic Vg gene expression is mediated through a functional coupling of the GC/cGMP pathway with different MAPK-dependent cascades in female shrimp. In VIH-2 signaling, the NO-independent GC/cGMP/PKG cascades were upstream of the MAPKs. Activations of the MAPK signal by VIH-2 include the phosphorylation of JNK and the mRNA/protein expression of P38MAPK. Additionally, the cAMP/PKA pathway is another positive intracellular signal for hepatopancreatic Vg mRNA expression but is independent of its VIH-2 regulation. Our findings establish a model for the signal transduction mechanism of Vg regulation by VIH and shed light on the biological functions and signaling of the CHH family in crustaceans.

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish.

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17ß-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11ß-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20ß,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.

Lipophorin receptor regulates Nilaparvata lugens fecundity by promoting lipid accumulation and vitellogenin biosynthesis.

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens. The deduced amino acid sequence of NlLpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction.

Dietary amino acid and vitamin complex protects honey bee from immunosuppression caused by Nosema ceranae.

Microsporidium Nosema ceranae is well known for exerting a negative impact on honey bee health, including down-regulation of immunoregulatory genes. Protein nutrition has been proven to have beneficial effects on bee immunity and other aspects of bee health. Bearing this in mind, the aim of our study was to evaluate the potential of a dietary amino acid and vitamin complex "BEEWELL AminoPlus" to protect honey bees from immunosuppression induced by N. ceranae. In a laboratory experiment bees were infected with N. ceranae and treated with supplement on first, third, sixth and ninth day after emergence. The expression of genes for immune-related peptides (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) was compared between groups. The results revealed significantly lower (p<0.01 or p<0.001) numbers of Nosema spores in supplemented groups than in the control especially on day 12 post infection. With the exception of abacein, the expression levels of immune-related peptides were significantly suppressed (p<0.01 or p<0.001) in control group on the 12th day post infection, compared to bees that received the supplement. It was supposed that N. ceranae had a negative impact on bee immunity and that the tested amino acid and vitamin complex modified the expression of immune-related genes in honey bees compromised by infection, suggesting immune-stimulation that reflects in the increase in resistance to diseases and reduced bee mortality. The supplement exerted best efficacy when applied simultaneously with Nosema infection, which can help us to assume the most suitable period for its application in the hive.

Vitellogenin induction in caudal fin of guppy (Poecilia reticulata) as a less invasive and sensitive biomarker for environmental estrogens.

Guppy (Poecilia reticulata) is an ideal model for studying environmental estrogens, and its large caudal fin has a high capacity to regenerate. This study analyzed the feasibility of caudal fin for detecting vitellogenin (Vtg), the most commonly used biomarker of environmental estrogens. Firstly, a sandwich ELISA for guppy Vtg was developed using purified lipovitellin and its antibody and it had a working range of 7.8-1000 ng/mL and detection limit of 3.1 ng/mL. The ELISA was used to detect tissue distribution of Vtg. In male guppy exposed to 50 and 100 ng/L 17ß-estradiol (E2), Vtg concentration in caudal fin was higher than that in whole fish, brain, eyes, gonad, and skin, and was close to that in the liver. Furthermore, male guppies were exposed to environmental concentrations of 17a-ethinylestradiol (EE2) and bisphenol S (BPS) to validate the utility of caudal fin Vtg for detecting estrogenic activities. The lowest observed effect concentration of EE2 and BPS were lower than 2 ng/L and 1 µg/L, which were below or equal to the values reported for other species, demonstrating that caudal fin Vtg was highly sensitive to estrogenic chemicals. Therefore, caudal fins of guppies are suggested as alternative samples for Vtg biomarker detection.

Characterizing combined effects of antiestrogenic chemicals on vitellogenin production in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Fish are exposed to a complex mixture of endocrine disrupting compounds (EDC), some of which display antiestrogenic activity leading to suppression of estrogen receptor (ER)- mediated reproductive processes. Although the main mode of action (MoA) of these antiestrogens is to directly interfere with natural ligand binding of the ER, several other MoA have been proposed. The aim of the present study was to characterize single and combined antiestrogenic effects of the aryl hydrocarbon receptor (AhR)-agonist ß-naphthoflavone (BNF) and ER-antagonist 4-hydroxytamoxifen (OHT) on vitellogenin (Vtg) protein using primary rainbow trout (Oncorhynchus mykiss) hepatocytes. Supporting transcriptional analysis of ER-responsive genes (estrogen receptor-α (er-α), vitellogenin-1 (vtg-1), eggshell zona radiata protein (zrp)) and AhR-mediated genes (aryl hydrocarbon receptor-2ß, cytochrome p450-1a (cyp1a)) was performed by qPCR to characterize the antiestrogenic influence on ER- and AhR-mediated responses. Data demonstrated that both BNF and OHT significantly reduced 17ß-estradiol (E2)-induced Vtg protein expression in a concentration responsive manner, whereas exposure to a mixture of these produced an additive antiestrogenic effect. The results observed at the protein level were further supported by transcriptional analysis of ER-responsive genes (er-α, vtg-1, zrp), where only E2-induced vtg-1 gene expression was significantly decreased by OHT and the mixture of OHT and BNF. E2-induced er-α and zrp gene expression was not markedly altered. The significant reduction of E2-induced vtg-1 gene expression by OHT suggested that the antiestrogenic effect of this compound may be associated with ER signaling pathway. Specific genes involved in putative AhR-ER cross-talk were also investigated, however none were directly associated with the compound anti-estrogenic MoA. Although the MoA of the single compounds and mixture were not completely characterized, the present study enhanced our knowledge of the combined toxicity mediated by antiestrogens acting through different MoA.

Silencing of Two Vitellogenin Genes Inhibits Oviposition in the Chagas Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

RNA interference caused by exogenous double-stranded RNA (dsRNA) is used to downregulate crucial genes to control insects. The reproductive success of all oviparous species depends on vitellogenin (Vg) biosynthesis and its accumulation in the developing oocytes. Adult females of Triatoma infestans were independently injected with two Vg dsRNAs (Vg1 dsRNA or Vg2 dsRNA) or nuclease-free water (control) 24 hours before feeding, and a group of adult females not injected was also analyzed (control). Vg1 and Vg2 messenger RNAs silencing was verified by reverse transcription quantitative polymerase chain reaction. The transcript levels of the Vg1 and Vg2 genes were significantly reduced after dsRNA treatment in fat body and ovary of T. infestans in relation to those detected in individuals injected with nuclease-free water and not injected (controls). Moreover, the present study demonstrated that the silencing of the Vg1 or Vg2 genes inhibits oviposition in the Chagas disease vector T. infestans. These findings may have important implications for the development of novel vector control strategies.