Panonychus citri is one of the most destructive pests in citrus orchards, exhibiting varying degrees of tolerance to numerous insecticides, such as spirodiclofen. To effectively manage pests, this study explores the response of P. citri to spirodiclofen stress from the perspectives of life history, enzymatic parameters, and reproduction. The effects of two concentrations (LC30 and LC50) of spirodiclofen on the biological parameters of P. citri were evaluated by the life table method. The results showed that the development duration, fecundity, oviposition days, and lifespan were shortened, though the pre-oviposition period of two treatments was prolonged in comparison with the control. A significant decrease was recorded in the net reproductive rate (R0) and the mean generation time (T) for the two treatments. Nevertheless, the intrinsic rate of increase (r) and the rate of increase (λ) were not significantly affected in the LC30 treatment, whereas they declined in the LC50 treatment. The enzyme activity assay resulted in higher activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and carboxylesterase (CarE), among the treatments than the control. In contrast, the treatments recorded lower cytochromeP450 (CYP450) and Glutathione S-transferase (GST) activities than the control. Furthermore, the study detected that relative mRNA expression of Vitellogenin (Vg) and Vitellogenin receptor (VgR) for two treatments were lower than the control. In summary, two concentrations of spirodiclofen inhibited progeny growth and fecundity of P. citri. Additionally, the results of this study may support further research on tolerance of P. citri in response to spirodiclofen stress.

Female crabs enter a stage of rapid ovarian development after mating, and cholesterol is a substrate for steroid hormone synthesis. Therefore, in this experiment, an 8-week feeding trial was conducted to investigate the effects of mating treatments (mated crab and unmated crab) and three dietary cholesterol levels (0·09 %, 0·79 % and 1·40 %) on ovarian development, cholesterol metabolism and steroid hormones metabolism of adult female swimming crab (Portunus trituberculatus). The results indicated that crabs fed the diet with 0·79 % cholesterol significantly increased gonadosomatic index (GSI) and vitellogenin (VTG) content than other treatments in the same mating status. Moreover, mated crabs had markedly increased GSI and VTG content in the ovary and hepatopancreas than unmated crabs. The histological observation found that exogenous vitellogenic oocytes appeared in the mated crabs, while previtellogenic oocytes and endogenous vitellogenic oocytes were the primary oocytes in unmated crabs. The transmission electron microscopy analysis showed that when fed diet with 0·79 % cholesterol, the unmated crabs contained more rough endoplasmic reticulum and mated crabs had higher yolk content than other treatments. Furthermore, mating treatment and dietary 0·79 % cholesterol level both promoted cholesterol deposition by up-regulation of the mRNA and protein expression levels of class B scavenger receptors 1 (Srb1), while stimulating the secretion of steroid hormones by up-regulation of the mRNA and protein expression of steroidogenic acute regulatory protein (Star). Overall, the present results indicated that mating behaviour plays a leading role in promoting ovarian development, and dietary 0·79 % cholesterol level can further promote ovarian development after mating.

Neoseiulus barkeri (HUGHES) is the natural enemy of spider mites, whiteflies and thrips. Screening for chemically-resistant predatory mites is a practical way to balance the contradiction between the pesticide using and biological control. In this study, the number of eggs laid by fenpropathrin-susceptible and resistant strains of N. barkeri was compared. Additionally, we cloned three N. barkeri vitellogenin (Vg) genes and used quantitative real-time polymerase chain reaction to quantify Vg expression in susceptible and resistant strains. The total number of eggs significantly increased in the fenpropathrin-resistant strain. The full-length cDNA cloning of three N. barkeri Vg genes (NbVg1, NbVg2 and NbVg3) revealed that the open reading frames of NbVg1, NbVg2 and NbVg3 were 5571, 5532 and 4728 bp, encoding 1856, 1843 and 1575 amino acids, respectively. The three N. barkeri Vg possessed the Vitellogenin-N domain (or lipoprotein N-terminal domain (LPD_N)), von Willebrand factor type D domain (VWD) and the domain with unknown function 1943 (DUF1943). The NbVg1 and NbVg2 expression levels were significantly higher in the resistant strain than in the susceptible strain, while the NbVg3 expression level was lower in the resistant strain. Thus, we speculate that the increased number of eggs laid by the fenpropathrin-resistant strain of N. barkeri may be a consequence of changes in Vg gene expression.

There is an ongoing relationship between host plants and herbivores. The nutrient substances and secondary compounds found in the host plant can not only impact the growth and development process of herbivores, but, more importantly, may also affect their survival and reproductive fitness. Vitellogenesis is the core process of reproductive regulation and is generally considered as a reliable indicator for evaluating the degree of ovarian development in females. Vitellogenin (Vg) plays a critical role in the synthesis and secretion of yolk protein. In this study, the full-length cDNA of the Vg gene in an alien invasive species, the nipa palm hispid beetle Octodonta nipae Maulik (Coleoptera: Chrysomelidae) (OnVg) was cloned and, the effect of host plant on the OnVg expression level and ovarian development was investigated. The results revealed that the OnVg was highly and exclusively expressed in adult females, but barely detectable in larvae, pupae and adult males. The relative expression level of OnVg and egg hatchability were much higher in females fed on Phoenix canariensis (their preferred host) than those fed on Phoenix roebelenii. A positive correlation relationship between OnVg expression and egg hatchability was also detected. Additionally, the anatomy of the female reproductive system showed that the ovaries of individuals fed on P. canariensis were considerably more developed than in females fed on P. roebelenii. The results may be applicable to many pest management situations through reproductive disturbance by alternating host plant species or varieties or by reproductive regulation through vitellogenesis mediated by specific endocrine hormones.

Evolution of resistance to insecticides usually has fitness tradeoffs associated with adaptation to the stress. The basic regulation mechanism of tradeoff between reproduction and resistance evolution to Bacillus thuringiensis (Bt) toxin in the cotton bollworm, Helicoverpa armigera (Ha), based on the vitellogenin (Vg) gene expression was analyzed here. The full-length cDNA of the Vg gene HaVg (JX504706) was cloned and identified. HaVg has 5704 base pairs (bp) with an open reading frame (ORF) of 5265 bp, which encoded 1756 amino acid protein with a predicted molecular mass of 197.28 kDa and a proposed isoelectric point of 8.74. Sequence alignment analysis indicated that the amino acid sequence of HaVg contained all of the conserved domains detected in the Vgs of the other insects and had a high similarity with the Vgs of the Lepidoptera insects, especially Noctuidae. The resistance level to Cry1Ac Bt toxin and relative HaVg mRNA expression levels among the following four groups: Cry1Ac-susceptible strain (96S), Cry1Ac-resistant strain fed on artificial diet with Bt toxin for 135 generations (BtR stands for the Cry1Ac Bt resistance), progeny of the Cry1Ac-resistant strain with a non-Bt-toxin artificial diet for 38 generations (CK1) and the direct descendants of the 135th-generation resistant larvae which were fed on an artificial diet without the Cry1Ac protein (CK2) were analyzed. Compared with the 96S strain, the resistance ratios of the BtR strain, the CK1 strain and the CK2 strain were 2917.15-, 2.15- and 2037.67-fold, respectively. The maximum relative HaVg mRNA expression levels of the BtR strain were approximately 50% less than that of the 96S strain, and the coming of maximum expression was delayed for approximately 4 days. The overall trend of the HaVg mRNA expression levels in the CK1 strain was similar to that in the 96S strain, and the overall trend of the HaVg mRNA expression levels in the CK2 strain was similar to that in the BtR strain. Our results suggest that the changes in reproduction due to the Bt-toxin resistance evolution in the BtR strain may be regulated by the Vg gene expression. The down-regulation of HaVg at the early stages resulted in a period of delayed reproduction and decreased fecundity in the BtR strain. This performance disappeared when the Bt-toxin selection pressure was lost.

Reproductive parameters of Ligula intestinalis-infected roach (Rutilus rutilus) which were held under long-tem laboratory conditions with unlimited food supply were investigated. Although uninfected and infected roach showed no difference in condition factor and both groups deposited perivisceral fat, the gonadosomatic-index was significantly lower in infected female and male roach. Quantitative histological analysis revealed that gonad development was retarded upon parasitization in both genders. In contrast to the phenotype described in the field, infected females were able to recruit follicles into secondary growth, but a high percentage of secondary growth follicles underwent atresia. In both genders, the histological data corresponded well with reduced expression of pituitary gonadotropins and lowered plasma concentrations of sex steroids, as revealed by real-time RT-PCR and ELISA, respectively. Furthermore, a reduction of vitellogenin mRNA and modulated expression of sex steroid receptors in the liver was demonstrated. Like in the field, there was a significant adverse impact of L. intestinalis on host reproductive physiology which could not be related to parasite burden. Our results show, for the first time, that maintenance under laboratory conditions can not abolish the deleterious effect of L. intestinalis on gametogenesis in roach, and indicate a specific inhibition of host reproduction by endocrine disruption.

The effects of parasite infection by the cestode Ligula intestinalis on the reproductive function and endocrine system of wild roach Rutilus rutilus were evaluated. Gonad maturation, plasma vitellogenin, plasma steroid concentrations (i.e. progesterone, 11-keto-testosterone and 17-β-estradiol) and brain aromatase activity were investigated in relation with parasitization. A low prevalence (8%) of ligulosed roach and a moderate impact of parasitization (mean parasitization index of 8·8%) were found in the studied population. Inhibition of gonad maturation generally resulted from infestation but 5% of the ligulosed roach nevertheless reached maturity. Main sex steroid plasma content was depleted in both genders. Male 11-keto-testosterone, female 17-β-estradiol and progesterone plasma concentrations of both genders were, respectively, 27, 5 and 3 times lower in ligulosed fish when compared to their non-infected counterparts. Progesterone levels were negatively correlated with the parasitization index in females. Brain aromatase activity of infected roach was reduced to 50% of that of the non-infected fish. These results demonstrate significant negative effects on the reproductive function of wild roach infected by the tapeworm L. intestinalis collected from a site with low contamination.

Vitellogenin synthesis by the fat body has been monitored using in vitro culture and immunoprecipitation. This system was found to be efficient for measuring vitellogenin production in both non-infected Tenebrio molitor and those infected with Hymenolepis diminuta. In fat bodies from infected beetles, vitellogenin production was decreased by up to 75% (day 24 post-infection) and, at all times investigated, vitellogenin synthesis was significantly below control levels (days 3–30 post-infection). Incubating fat bodies from control insects with isolated metacestodes indicated that this may be a direct effect by the parasite which is developmental stage-specific. Stage II, but not stage III–IV, nor heat-killed parasites could bring about this decrease in vitellogenin. In addition, these effects may be density dependent within the range of 2–20 parasites per fat body; only 2 metacestodes were necessary to cause a significant decrease. Since metacestodes do not take up vitellogenin, nor limit the amount of [14C] leucine available to the fat body for vitellogenin production, it is conceivable that the parasite produces a potent inhibitor of vitellogenin synthesis, or a molecule which induces cells within the fat body to do so.

We have previously described the major yolk protein, vitellin, in the British blackfly Simulium ornatum Meigen. Here we demonstrate that vitellogenin, synthesized in the fat body and secreted into the haemolymph, is composed of subunits with the same approximate molecular weight as vitellin, namely 200 and 68 kDa. Simulium ornatum is the natural vector for the cattle filarial nematode Onchocerca lienalis Stiles, which induces host fecundity depletion. A significant reduction in ovarian vitellin content was associated with infection by intrathoracic injection of 20 O. lienalis microfilariae immediately after blood-feeding. Fat body synthesis of vitellogenin was significantly reduced as early as 8 h post-infection in comparison with sham-injected flies. When total haemolymph protein from infected and sham injected flies was compared, titres were significantly depressed 6 h post-infection. However, later in the infection, titres were elevated by 30%, the major component being vitellogenin. The injection of dead microfilariae had no effect. An infection burden of a single parasite caused a significant reduction in ovarian protein content in comparison with shams, but no further significant decrease was observed as the parasite burden was increased from 5 to 20. Possible mechanisms underlying the disturbance of Simulium reproductive physiology are proposed.

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.

During the past four decades, research efforts directed toward reducing shell egg cholesterol content have centred on genetic selection or alteration of the laying hens' diet with various nutrients, natural products, non-nutritive factors, or pharmacological agents. However, the vast majority of these experimental approaches elicited only minimal changes (<10%) at best or, as in the case of dietary azasterols and triparanol, resulted in the unacceptable replacement of yolk cholesterol by desmosterol. In contrast, when 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (‘statins’), garlic paste, or pharmacological amounts of copper were orally administered to chickens, yolk cholesterol levels were reduced by up to 46%, 32%, or 34%, respectively. The previous paper in this two-part series provided a justification for low-cholesterol egg production and reviewed cholesterol metabolism in the laying hen, avian embryonic cholesterol needs, and genetic and nutritional approaches to chicken egg cholesterol reduction. The present paper provides a comprehensive overview of the use of non-nutritive dietary factors and select pharmacological compounds as egg cholesterol-lowering agents, and discusses emerging strategies for lowering the cholesterol content of shell eggs. With the recent advances in avian genomics and transgenesis, it is anticipated that the greatest advances in the area of yolk cholesterol reduction will be attained through manipulation of key genes whose protein products mediate intestinal sterol absorption, hepatic cholesterol and lipoprotein synthesis, and/or lipoprotein uptake by growing oocytes. However, the future commercial availability of low-cholesterol eggs produced by transgenic hens ultimately will be influenced by the nature of the regulatory environment for bioengineered foods, public acceptance, and economics.

In laying hens, cholesterol is primarily biosynthesized in the liver and incorporated into vitellogenin (VTG) and triglyceride-rich very low density lipoprotein (VLDL) particles, which are secreted into the bloodstream and subsequently taken up by growing oocytes via receptor-mediated endocytosis. VLDL and VTG are then intracellularly transformed into yolk, constituting ∼60% and 24% of yolk dry matter and ∼95% and 5% of yolk cholesterol, respectively. During the past four decades, research efforts directed toward reducing shell egg cholesterol content have centred on genetic selection or alteration of the laying hens’ diet with various nutrients, natural products, non-nutritive factors, or pharmacological agents. The present paper will provide a justification for low-cholesterol egg production, primarily in the context of the heterogeneity of response to dietary cholesterol in humans, and will review cholesterol metabolism in the laying hen, avian embryonic cholesterol needs, and genetic and nutritional approaches to chicken egg cholesterol reduction. A subsequent paper will provide a comprehensive overview of the use of non-nutritive dietary factors and select pharmaceuticals as egg cholesterol-lowering agents, and will discuss emerging strategies for lowering the cholesterol content of shell eggs.

Cyphocarax gilbert (Szidat, L., 1948) is a fish commonly found in coastal drainage of eastern Brazil. This fish is sometimes caught with signs of infection by the crustacean Riggia paranensis, a haematophagous parasite. A remarkable feature of infected fish is that they lack gonads. In this paper we have analysed the frequency of parasitism, the gonadal development of non-infected fish and the profile of plasma proteins in both infected and non-infected specimens. Two reproductive periods/year were observed, beginning in February and August. On average, 40% of fish were infected, in the Itabapoana River (Brazil). Sex-specific proteins were identified by electrophoresis. SDS-PAGE analysis demonstrated that a 143 kDa female-specific glycolipoprotein (FSP) is a calcium-binding phosphoprotein. FSP was isolated through ultracentrifugation and SDS-PAGE analysis showed that the native protein is composed of three polypeptides of 143, 100 and 70 kDa. Both FSP and a 33 kDa male-specific protein (MSP) are absent from infected fish plasma. FSP levels in female plasma changes with the developmental stage of gonads. Altogether these data suggest that the FSP corresponds to fish vitellogenin. Furthermore, the absence of the above-mentioned proteins in infected fish suggests that R. paranensis might interfere with the regular hormonal process of fish vitellogenesis.

Vitellogenin (VTG) levels were measured in the plasma of 143 female skipjack tuna, Katsuwonus pelamis caught in the Western Indian Ocean. These levels were correlated with the gonadosomatic index (GSI) and with plasma 17β-estradiol (E2) levels during the reproductive cycle. VTG and E2 levels were measured using a competitive enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA), rcspectively. VTG was purified from a pool of plasma obtained from females with high GSI using a double chromatography method (gel filtration and ion exchange). A specific antibody was obtained in rabbits. The VTG immunoenzymeassay developed gave an assay detection limit (90% binding) of 15 ng.ml-1. Mean VTG levels ranged from 1.2 mg.ml−1 to 4 mg.ml−1. Mean monthly VTG values remained relatively elevated during the resting periods (1.2 mg.ml−1 in April and 1.9 mg.ml-1 in September), while maximum levels reached only 4 mg.ml−1 during the reproductive season. The highest GSI values were observed from November to March during the major reproductive season corresponding to the North monsoon and from early June to late August during the minor reproductive season corresponding to the East monsoon. Lowest GSI values were found in April-May and September-October. Mean E2 levels exhibited considerable variability among all females sampled ranging from 700 pg.ml−1 to 9 ng.ml−1. This study demonstrated that the sexual maturation was correlated with the monsoon seasons and that there was a positive correlation between monthly variations of GSI, VTG and E2 levels in the skipjack tuna population from the Western Indian Ocean.

Two specific Enzyme Linked lmmunosorbent Assays (ELISA) for vitellogenin were developed using two different antisera anti-plasma vitellogenin purified by SDS polyacrilamide gel electrophoresis and electroelution. Purified vitellogenins migrated as single bands under SDS conditions with an apparent molecular mass of 154 and 152 kDa for Chrysichthys nigrodigitatus and Heterobranchus longifilis respectively.The ELISA was performed on 96 well microtiter plates using specific antibodies raised on rabbits and a second antibody (Goat anti-rabbit) labeled with peroxidase. Parallel displacement curves were obtained between purified vitellogenin and female plasma as well as plasma from estradiol treated fish. Both antibodies cross-reacted with the heterologous vitellogenin but the curves were not parallel, indicating different antigenic structures of the two vitellogenin molecules from the two catfish species. The reliability and the specificity of these assays allowed us to quantify vitellogenin in plasma samples during vitellogenesis of the two species. The annual vitellogenin plasma levels presented a good correlation with mean oocyte diameter and reproduction period in Chrysichthys nigrodigitatus. In Heterobranchus longifilis the mean oocyte diameter is almost constant all year round, but vitellogenin levels presented significant variations with higherlevels corresponding mainly to the low gonado-somatic index values observed during the dry seasons.

Adult two-year-old broodstock sea bass (Dicentrarchus labrax), raised in captivity, were fed ad libitum a natural diet (trash fish) until the end of their first spawning, and maintained under natural conditions of light and temperature. Six months before the second spawning, fish were randomly separated in two tanks with running sea water (salinity 37.80‰, pH 8.3) and maintained under similar environmental conditions. Fish were fed two different ration levels: group F, 1.04%, and group H, 0.45% body weight/day. The Jow ration supplied affected broodstock growth as indicated by decreased growth rates in weight and condition factor in group H, when compared to group F. The mean fecundity per female (No. eggs/female) in group H fish was similar to that from F group, while the relative fecundity (No. eggs/kg female) was more elevated in the H group. However, group H showed a delay in spawning time and slightly higher number of spawnings per female and larger spawning period, when compared to group F fish. In addition, group H females had reduced 17β-estradiol (E2) plasma levels and presented vitellogenic oocytes in the ovary a month later than group F females. By contrast, the vitellogenin (VGT) plasma levels remained similar between groups over the reproductive cycle. The egg quality (i.e. percentage of buoyant eggs), the egg biochemical composition and larval survival at hatching were similar between the two groups. The larvae from both groups also exhibited the same survival after 40 days of culture, although group H produced smaller eggs that yielded smaller newly-hatched larvae. Although the low ration supplied significantly reduced the growth and E2 plasma levels of sea bass, these preliminary results suggest that these changes do not dramatically affect the egg viability or the survival of larvae 46 days after hatching.

Vitellogenin (VTG) was isolated from the haemolymph of Penaeus vannamei by a three stepprocedure including ultracentrifugation, gel filtration and ion exchange chromatography. VTG wasused to raise polyclonal antibodies that were purified by ion exchange chromatography. A two stepcompetitive assay was developed in which VTG could be quantitated by its capacity to inhibit thebinding of antibody to the VTG previously adsorbed onto a solid phase. Sensitivity from equilibriumand from non-equilibrium assays was 41 and 2.3 ng/ml respectively. Estimates of within-assay andbetween-assay variabilities of standard curves were 4.2 and 9.6% respectively. The inhibition curves for dilutions of haemolymph from vitellogenic females, egg yolk extracts and purified vitellin (VTL) were parallel to the standard VTG curve, haemolymph from immature females and males showed no cross-reactivity. The antibodies directed against VTG recognize but partially VTL as shown by displacement curves. Recovery tests were near 100%. Thus the procedure was considered to be suitable for the measurement of haemolymphatic VTG. The VTG enzyme-linked immunosorbent assay (ELISA) developed in this study was validated by detecting physiological VTG changes in female shrimps after being fed squid extracts.

Sex steroid levels were determined in various commercial fish meals and complete diets. The results obtained demonstrate that estrone (E1), 17 β-estradiol (E2) and androgens (A) (testosterone and 11-ketotestosterone), are present in diets in high quantities (up to 935±350 ng/100 g for E2, 615±190 ng/100 g for E1 and 1100±120 ng/100 g for A) and with considerable variation both between different diets, and between different batches of the same diet. These high levels of sex steroids are thought to influence the sex steroid plasmatic levels and plasma vitellogenin content in fish. Plasma sex steroid assays in the Siberian sturgeon Acipenser baeri show that plasmatic 17 β-estradiol levels are never significantly different between males and females at various stages of oogenesis and spermatogenesis, except in 6 year old females in stage IV, which corresponds to the maximum incorporation of vitellogenin. In males the plasma 17 β-estradiol levels are exceptionally high and induce vitellogenin synthesis, as demonstrated by various chemical and immunological techniques.