Acute toxicity assessment of arsenic, chromium and almix 20WP in Euphlyctis cyanophlyctis tadpoles.

Heavy metals and herbicide are gaining serious environmental concern in aquatic toxicology due to its adverse effects on aquatic organisms especially amphibians. Accordingly, present study first time evaluated the acute toxicity of two heavy metals [arsenic (As3+) and chromium (Cr6+)] and a herbicide (Almix) to Indian skittering frog tadpole, Euphlyctis cyanophlyctis. The LC50 values of As, Cr and Almix for 24, 48, 72 and 96 h were 73.58, 56.31, 43.58 and 32.58 mg L-1; 326.68, 224.31, 171.92 and 141.99 mg L-1; and 1297.85, 1148.22, 1033.62 and 955.17 mg L-1, respectively. It also revealed the concentration- and time-dependent increased mortality rate under these toxicants. The safety concentrations (SC) of As, Cr and Almix to tadpoles were 3.26, 14.20 and 95.52 mg L-1, respectively. The findings disclosed that As is highly toxic to E. cyanophlyctis than Cr and Almix. Alkaline phosphatase (ALP) activity showed varied responses to exposed chemicals. In particularly, ALP activity reduced significantly for Cr treatment. Glutathione S-transferase (GST) activity in E. cyanophlyctis was significantly inhibited by As treatment (p < 0.05); however, GST activity was remain unchanged for Cr and Almix (p > 0.05). The As toxicity correlates positively with GST inhibition (r = 0.779, p < 0.01); contrarily, Cr and Almix revealed negative correlation with GST induction (r = -0.461 and -0.19, respectively; p > 0.05). This result indicated that GST play a crucial role for regulating the tadpole mortality and intoxication by As, Cr and Almix. Overall, our findings demonstrate the different levels of toxic sensitivity (adverse effects) under different toxicants on E. cyanophlyctis tadpoles. Finally, the present findings could be used as baseline information of toxicosis for metalloid, heavy metal and herbicide exposures in wild frog populations.

Overexpression of PGE2 synthase by in vivo transient expression enhances immunocompetency along with fitness cost in a lepidopteran insect.

Prostaglandins (PGs) mediate various physiological functions in insects. Specifically, PGE2 is known to mediate immunity and egg-laying behavior in the beet armyworm, Spodoptera exigua A PGE2 synthase 2 (Se-PGES2) has been identified to catalyze the final step to produce PGE2 in S. exigua Its expression is inducible in response to immune challenge. Inhibition of the gene expression results in immunosuppression. In contrast, any physiological alteration induced by its uncontrolled overexpression was not recognized in insects. This study used the in vivo transient expression (IVTE) technique to induce overexpression and assessed subsequent physiological alteration in S. exiguaSe-PGES2 was cloned into a eukaryotic expression vector and transfected to Sf9 cells to monitor its heterologous expression. The Sf9 cells expressed the recombinant Se-PGES2 (rSe-PGES2) at an expected size (∼47 kDa), which was localized in the cytoplasm. The recombinant expression vector was then used to transfect larvae of S. exigua Hemocytes collected from the larvae treated with IVTE expressed the rSe-PGES2 gene for at least 48 h. The larvae treated with IVTE exhibited an enhanced competency in cellular immune response measured by hemocyte nodule formation. In addition, IVTE treatment of Se-PGES2 induced gene expression of antimicrobial peptides without any immune challenge. The larvae treated with IVTE became significantly resistant to infection of an entomopathogenic nematode, Steinernema monticolum, or to infection to its symbiotic bacterium, Xenorhabdus hominickii However, IVTE-treated S. exigua larvae suffered from reduced pupal size and fecundity.

Integrated assessment of biochemical markers in premetamorphic tadpoles of three amphibian species exposed to glyphosate- and methidathion-based pesticides in single and combination forms.

In this study, we evaluated the toxic effects of a glyphosate-based herbicide (GBH) and a methidathion-based insecticide (MBI), individually and in combination, on premetamorphic tadpoles of three anuran species: Pelophylax ridibundus, Xenopus laevis, and Bufotes viridis. Based on the determined 96-h LC50 values of each species, the effects of a series of sublethal concentrations of single pesticides and their mixtures after 96-h exposure and also the time-related effects of a high sublethal concentration of each pesticide were evaluated, with determination of changes in selected biomarkers: glutathione S-transferase (GST), glutathione reductase (GR), acetylcholinesterase (AChE), carboxylesterase (CaE), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Also, the integrated biomarker response (IBR) was used to assess biomarker responses and quantitatively evaluate toxicological effects. Isozyme differences in CaE inhibition were assessed using native page electrophoresis; results showed that GBH to cause structural changes in the enzyme but not CaE inhibition in P. ridibundus. In general, single MBI and pesticide mixture exposures increased GST activity, while single GBH exposures decreased GST activity in exposed tadpoles. The AChE and CaE activities were inhibited after exposure to all single MBI and pesticide mixtures. Also, higher IBR values and GST, GR, AST, and LDH activities were determined for pesticide mixtures compared with single-pesticide exposure. This situation may be indicative of a synergistic interaction between pesticides and a sign of a more stressful condition.

Chitin synthase A: a novel epidermal development regulation gene in the larvae of Bombyx mori.

Chitin synthase is the key regulatory enzyme for chitin synthesis and excretion in insects, as well as a specific target of insecticides. The chitin synthase A gene (BmChsA) cloned from Bombyx mori, the model species of lepidopteran, is an epidermis-specific expressed gene during the molting stage. Knockdown BmChsA gene in 3rd instar larvae increased the number of non-molting and abnormal molting larvae. Exposure to nikkomycin Z, a chitin synthase inhibitor downregulated the expression of BmChsA and decreased the amount of epidermis chitin during the molting process. The thickness of the new epidermis and its dense structure varied greatly. The exogenous hormones significantly upregulated the expression of BmChsA with low levels of endogenous MH and high levels of endogenous JH immediately after molting. With low levels of endogenous hormones during the mulberry intake process, BmChsA was rarely upregulated by exogenous hormones. With high levels of endogenous MH and low levels of endogenous JH during the molting stage, we did not detect the upregulation of BmChsA by exogenous hormones. The expression of BmChsA was regulated by endocrine hormones, which directly affected the chitin synthesis-dependent epidermal regeneration and molting process.

Chitin synthase B: a midgut-specific gene induced by insect hormones and involved in food intake in Bombyx mori larvae.

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third-instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20-hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry-leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin-synthesis-dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.

Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect.

1. When parasitized, both vertebrates and invertebrates can enhance the immune defence of their offspring, although this transfer of immunity is achieved by different mechanisms. In some insects, immune-challenged males can also initiate trans-generational immune priming (TGIP), but its expressions appear qualitatively different from the one induced by females similarly challenged. 2. The existence of male TGIP challenges the traditional view of the parental investment theory, which predicts that females should invest more into their progeny than males. However, sexual dimorphism in life-history strategies and the potential costs associated with TGIP may nevertheless lead to dissymmetric investment between males and females into the immune protection of the offspring. 3. Using the yellow mealworm beetle, Tenebrio molitor, we show that after parental exposure to a bacterial-like infection, maternal and paternal TGIP are associated with the enhancement of different immune effectors and different fitness costs in the offspring. While all the offspring produced by challenged mothers had enhanced immune defence, only those from early reproductive episodes were immune primed by challenged fathers. 4. Despite the fact that males and females may share a common interest in providing their offspring with an immune protection from the current pathogenic threat, they seem to have evolved different strategies concerning this investment.

Activity levels of B-esterases in the tadpoles of 11 species of frogs in the middle Paraná River floodplain: implication for ecological risk assessment of soybean crops.

Soybean fields provide habitats for many species of amphibians. However, the persistence and health of amphibian populations may be at risk from the increasing use of pesticides and other agricultural chemicals. We examined the activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterases (CbEs) in 11 syntopic species of larval anurans. In vitro effects of malaoxon causing 50% BChE inhibition (IC(50)) were also studied. In addition, we calculated a relative risk index (RI) based on the geographic distributions of the anurans, the phenology of soybean cultivation, and basal enzymatic values related to potential pesticide detoxification. Among the 11 species, AChE activity varied from 17.5 ± 1.6 to 68.2 ± 4.7 nmol min(-1) mg(-1) protein (PT). BChE activity also varied significantly, ranging from 3.3 ± 0.4 to 7.5 ± 0.4 nmol min(-1) mg(-1)PT. Both measures of CbE activities varied widely (CbE α-NA: 2.1 ± 0.5-12.4 ± 1.1 nmol min(-1) mg(-1) PT; CbE-4NPV: 21.8 ± 1.8-102.6 ± 7.9 nmol min(-1) mg(-1) PT). We also corroborate that lower BChE activity levels for the tadpoles were associated at minor IC(50) values. The results of this study demonstrate significant variation in enzymatic levels among several tadpole species and intermediate to high RI values for 7 species. Based on these results, it appears that a conversion of native ecosystems to soybean crops may lead to increased ecological risk for anuran amphibians.

Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata.

We have isolated the cDNAs of all enzymes involved in the mevalonate pathway portion of the juvenile hormone (JH) biosynthetic pathway in Bombyx mori, i.e., those responsible for the formation of farnesyl diphosphate from acetyl-CoA. There is a single gene encoding each enzyme of this pathway, with the exception of farnesyl diphosphate synthase (FPPS), for which we identified three homologs. All but two of these enzymes are expressed almost exclusively in the corpora allata (CA), as indicated by quantitative RT-PCR analyses. Phosphomevalonate kinase (MevPK) was expressed in many tissues, including the CA. In day 2 4th instars, FPPS1 expression was detected primarily in the Malpighian tubules, but expression of the structurally related FPPS2 and FPPS3 occurred mainly in the CA. Since FPPS3 transcripts were 55 times less abundant than those of FPPS2, the latter is expected to play a major role in JH biosynthesis at this stage. Studies on the developmental expression of these enzymes in the CA showed that the levels of all transcripts were high during the 4th instar larvae, a stage at which in vitro JH biosynthesis was high. However, the transcripts of all the mevalonate enzymes declined to low levels and JH acid O-methyltransferase (JHAMT) transcript disappeared by day 3 when CA ceased JH production after the final larval molt. The CA did not synthesize JH during the pupal stage, coincident with the limited expression of mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate kinase and isopentenyl diphosphate isomerase, and the inactivation of the JHAMT gene. Only female CA produced JH in the adult stage, a feature associated with the re-expression of JHAMT in female but little in male adult CA. Altogether, our results point to a relationship between JH biosynthesis and expression of most JH biosynthetic enzymes in the CA.