Holometabolous Research Articles

Asymmetrical Competition between Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) Coexisting in Breeding Sites.

Aedes aegypti and Culex quinquefasciatus are mosquito vectors for several tropical diseases that represent a current public health problem. The ecological requirements for each species are different, however, both species show high biological adaptability, which promotes their coexistence in the same breeding sites. The purpose of this study was to assess the effect of larval association between Ae. aegypti and Cx. quinquefasciatus under different laboratory conditions of food supply and temperature, and under field simulated conditions like peridomestic containers. Our findings showed that under field simulated conditions there was no asymmetrical competition in mixed cultures with the different Cx. quinquefasciatus/Ae. aegypti ratios tested. However, under laboratory conditions in which different doses of food supply were evaluated, it was observed that competition between the two species takes place. Larval coexistence under food scarcity conditions (0.95 mg/larva) showed that Ae. aegypti had a greater adult emergence than Cx. quinquefasciatus and was capable of depriving Cx. quinquefasciatus of the food needed to complete metamorphosis. In an intermediate dose of food (1.9 mg/larva), the dry weight of Cx. quinquefasciatus adults decreased, and their larval development time increased when Cx. quinquefasciatus/Ae. aegypti ratio was low. Also, a temperature effect was assessed demonstrating that Cx. quinquefasciatus was more vulnerable to changes in temperature. We suggest that Ae. aegypti is more successful in exploiting microhabitats when food is scarce, due to its scrape active feeding habitats and fast larval development times. Therefore, in conditions of food paucity both species will compete, and Ae. aegypti larvae will prevail.

Tetrabromoethylcyclohexane affects gonadal differentiation and development in the frog Pelophylax nigromaculatus

Tetrabromoethylcyclohexane (TBECH), an additive brominated flame retardant, has been shown to have an androgenic activity in vitro. In the present study, we aimed to investigate the effects of TBECH on gonadal differentiation and development in the frog Pelophylax nigromaculatus, an amphibian species sensitive to androgenic chemicals, and to assess the androgenic activity of TBECH in vivo. P. nigromaculatus tadpoles were exposed to TBECH (1, 10, 100nM) from Gosner stage 24 to complete metamorphosis, and to 5α-dihydrotestosterone (DHT) as a positive control. We found that 1nM DHT resulted in 100% males, while the sex ratio in the solvent control group was close to 1:1. In all the TBECH treatment groups, sexually ambiguous gonads based on gross morphology and intersexualities with testicular and ovarian histological structures were found, but no abnormality occurred in the solvent control. In the 1, 10, 100nM TBECH treatment groups, the female percentages were 52%, 31%, 17%, with 36%, 56%, 66% for males and 12%, 13%, 17% for abnormal sexes, respectively. X2-test revealed significant differences in sex ratios between the three TBECH groups and the solvent control group, and the sex ratios in the two higher concentration groups were male-biased. These observations show that TBECH has a masculinizing effect on gonadal differentiation and development in P. nigromaculatus, suggesting an androgenic activity of TBECH in vivo. To our knowledge, this is the first study demonstrating that TBECH could induce gonadal masculinization in an animal, which raises new concerns for reproductive risk of TBECH exposure.

Elbella luteizona (Mabille, 1877) (Lepidoptera, Hesperiidae: Pyrginae) in Brazilian Cerrado: larval morphology, diet, and shelter architecture

This study examined temporal variation in the abundance of immature stages of Elbella luteizona (Hesperiidae) and describes the morphology and behavior of the larvae on their host plants, Byrsonima coccolobifolia and Myrsine guianensis. Five hundred sixty-eight 10m diameter plots were searched for caterpillars in the Brazilian Cerrado over a period of one year. We inspected 5968 host plants, and found 31 eggs and 262 larvae on 244 plants. Similar numbers of immatures were found in both species of host plants. The abundance of immature stages varied monthly and was significantly higher in the dry season on both host plants, which may be due to the low density of natural enemies during that time. E. luteizona is univoltine, and larvae present relatively little morphological variation. However, during development, substantial changes occur in the architecture of leaf shelters that caterpillars construct. In addition, E. luteizona larvae develop very slowly, taking more than 300 days to complete metamorphosis.

Positive and relaxed selection associated with flight evolution and loss in insect transcriptomes.

The evolution of powered flight is a major innovation that has facilitated the success of insects. Previously, studies of birds, bats, and insects have detected molecular signatures of differing selection regimes in energy-related genes associated with flight evolution and/or loss. Here, using DNA sequences from more than 1000 nuclear and mitochondrial protein-coding genes obtained from insect transcriptomes, we conduct a broader exploration of which gene categories display positive and relaxed selection at the origin of flight as well as with multiple independent losses of flight. We detected a number of categories of nuclear genes more often under positive selection in the lineage leading to the winged insects (Pterygota), related to catabolic processes such as proteases, as well as splicing-related genes. Flight loss was associated with relaxed selection signatures in splicing genes, mirroring the results for flight evolution. Similar to previous studies of flight loss in various animal taxa, we observed consistently higher nonsynonymous-to-synonymous substitution ratios in mitochondrial genes of flightless lineages, indicative of relaxed selection in energy-related genes. While oxidative phosphorylation genes were not detected as being under selection with the origin of flight specifically, they were most often detected as being under positive selection in holometabolous (complete metamorphosis) insects as compared with other insect lineages. This study supports some convergence in gene-specific selection pressures associated with flight ability, and the exploratory analysis provided some new insights into gene categories potentially associated with the gain and loss of flight in insects.

Effects of 3-Nitro-1,2,4-triazol-5-one on Survival, Growth and Metamorphosis in the Northern Leopard Frog, Lithobates pipiens.

New explosive formulations are being developed to be less sensitive to impact and inadvertent explosion, increasing safety for the warfighter. Since testing and training make environmental releases imminent, the toxicity of 3-nitro-1,2,4-triazol-5-one (NTO), a component of Insensitive Munitions eXplosive (IMX) formulations, was assessed in a one-generation study to the northern leopard frog (Lithobates ( = Rana) pipiens). Because NTO in water creates acidic conditions, acute studies were conducted with non-pH-adjusted NTO, while a long-term (70-d) study was conducted with neutralized NTO. In the acute study, 48-h and 7-d LC50s were ~250 mg NTO/L. In the long-term study, tadpoles were dead by day 2 in 11,350 mg/L NTO, and by day 63 in 8382 mg/L. The 70-d LC50 was 3670 mg (neutralized) NTO/L. The number of organisms reaching complete metamorphosis was reduced by NTO; the lowest IC25 was 1999 mg NTO/L for the Number Completing Metamorphosis. The NOECs for Time to Front Limb Eruption or Time to Metamorphosis were the same at 1346 mg/L. Histopathology did not significantly distinguish between NTO-exposed and unexposed animals, although possible effects on the density of spermatogonia in NTO-exposed males was suggested. The test data indicate that acute toxicity to ambient NTO can be attributed primarily to its acidic nature; relatively low chronic toxicity of neutralized NTO is due to delays in metamorphosis. The consequence from this latter observation may be ecologically significant as delays of even a few days could increase mortality through predation and/or loss of the aquatic medium in temporary water bodies.

Developmental studies of the enigmatic wormCaobangia billetiGiard, 1893 (Annelida; Sabellidae), a symbiont of freshwater snails

Caobangiids are an aberrant group of annelids with an unusual phoronid-like body plan. The most perceptible anatomical characteristic of caobangiids is the anal trunk, which is recurved outside the body and ends with an anal opening located near the head. So far neither the larval development nor the metamorphosis of these worms have been investigated thoroughly. This study describes the larval development and metamorphosis ofCaobangia billeti, and focuses mainly on the formation of the alimentary tract. The trochophore has eight chaetigers. Embryogenesis includes the development of segmentally arranged bands of cilia and ventral ciliary fields, the development of chaetae and the early formation of anterior radioles. Upon exiting the larval duct, larvae have eyes with lenses, two pairs of rudimentary radioles on the prostomium, a pair of nephridia on the peristomium, and two capillary chaetae in each parapodium of segments 3–9; parapodia of the 10th segment also bear a row of palmate hooks. Metamorphosis takes about 20 h and involves dorso-ventral folding of the body and enlargement of the pygidium along the dorsal side of segments 5–8. Thus, the anus develops on the dorsal side of the 5th segment, the anal trunk forms from the projection of pygidium whereas the prepygidial growth zone appears posterior to the 8th body segment. After the completion of metamorphosis, the prostomial and peristomial segments are fused and reduced, eight full thoracic chaetigers derive from the larva's body. Abdominal chaetigers grow posteriorly from the prepygidial growth zone.

Pituitary-thyroid axis development during the larval-juvenile transition in the pejerrey Odontesthes bonariensis.

The morphological development of the thyroid gland of pejerrey Odontesthes bonariensis during larval-juvenile transition was studied and related to whole-body concentrations of thyroxine (T4 ) and tri-iodothyronine (T3 ). A complementary (c)DNA fragment of the thyroid-stimulating hormone β-subunit (tshb) was sequenced and transcript levels quantified during this period. Follicles with eosinophilic and T4 -immunoreactive colloids were detected at hatching together with tshb transcript levels and whole-body concentrations of T4 and T3 hormones. Thyroid follicles were located in the subpharyngeal region associated with the ventral aorta below the hyoid bone. Follicle structure switched from the rounded form at hatching to oval in juveniles. Significant increase of follicle number per larva, mean colloidal area and total colloidal area was observed throughout development with maximum values at the end of the larval-juvenile transition. A significant decrease of tshb expression together with a significant increase in T4 and T3 whole-body concentrations was observed prior to achieving the juvenile phenotype. These results are in accordance with a negative feedback regulation of tshb expression by thyroid hormones and a possible association between thyroid hormone levels and the acceleration of metabolic processes necessary to complete metamorphosis.

Circadian regulation of caterpillar feeding and growth

Circadian clocks orchestrate many physiological processes in adult organisms. For example, rhythmic feeding behavior is regulated by the central clock in the nervous system in coordination with metabolic rhythms, which in turn depend mostly on peripheral clocks localized in many tissues. Disruption of the circadian clock leads to metabolic dysregulation both in mammals and in the model insect Drosophila melanogaster. Circadian coordination of feeding and metabolism has been studied mainly in adult insects and not in larval stages, which are dramatically different from adults in species with complete full metamorphosis. The goal of this study was to determine whether feeding and metabolism in lepidopteran larvae are subject to circadian regulation. We show that cotton leafworm caterpillars, Spodoptera littoralis, display rhythmic feeding behavior and that circadian clock genes are expressed in two peripheral tissues, the midgut and fat body. Even though both tissues display rhythmic circadian clock gene expression, the main component of the clock, per, is arrhythmic in the gut and rhythmic in the fat body. In both tissues, the presence of rhythmic physiological processes was observed, which suggested that metabolism is already driven by the circadian clock in the insect’s juvenile stages.

Growth characteristics and rates of the mash frog Pelophylax ridibundus Pall. introduced into water bodies of the Middle Urals

The characteristics and growth rates of mash frogs introduced upon the completion of metamorphosis into the cooling ponds of heating stations have been studied. The age composition of frog populations has been determined. It has been shown that an extra year must be added to the visible adhesion lines in the tissue of long bones. It has been found that the population of mash frogs inhabiting the Verkhnii Tagil Reservoir is characterized by not only a longer life span than in the Reftinskoe Reservoir, but also by a sufficiently high frequency of occurrence in the samples of older age groups.

Weather, hydroregime, and breeding effort influence juvenile recruitment of anurans: implications for climate change

AbstractAmphibians that primarily breed in ephemeral wetlands are especially vulnerable to climate change because they rely on rainfall or temperature to initiate breeding and create suitable hydroregimes (water duration, timing, frequency, depth) for reproductive success. Hydroregime effects on reproductive success are likely to differ among species because of differences in reproductive strategies: the length and timing of breeding period, rate of larval development, and timing of metamorphosis. We applied an information‐theoretic approach to 22 consecutive years of continuous amphibian trapping data at eight ephemeral wetlands to test hypotheses regarding environmental (hydroregime, weather) and biological (adult breeding effort) factors affecting juvenile recruitment (JR) by six focal species representing four reproductive strategies. We hypothesized that (1) JR by species with similar reproductive strategies would be influenced by similar variables; (2) JR would be higher for all species when models encompassed the maximum time span of potential tadpole occurrence and development; and (3) JR rates within individual wetlands and breeding cycles would correlate most closely between species with similar breeding strategies. The best model for all focal species (except Scaphiopus holbrookii) encompassed the maximum time span and indicated that ≥1 hydroregime variable, total precipitation, or both were important drivers of reproductive success; average air temperature was not. Continuous hydroperiod through peak juvenile emigration was an important predictor of JR for species with prolonged breeding periods, slow larval development, and a “fixed” late spring start date for juvenile emigration (regardless of when oviposition occurred, or cohort age; Lithobates capito, Lithobates sphenocephalus), but not for species with rapid larval development and continual emigration as cohorts complete metamorphosis (Anaxyrus terrestris, Anaxyrus quercicus, Gastrophryne carolinensis, S. holbrookii). Total rainfall was positively associated with recruitment for most species; depth characteristics affected species differently. Annual JR was positively correlated among species with similar reproductive strategies. Our results indicate that weather and hydroregime characteristics interact with reproductive strategies that differ among amphibian species and influence reproductive plasticity, opportunity, and success. Effects of altered weather patterns associated with climate change on amphibian reproductive success may correspond more closely among species having similar reproductive strategies, with critical implications for population trends and assemblages.

Bioaccumulation and Biodistribution of Selenium in Metamorphosing Tadpoles.

Selenium is an important macronutrient with a very narrow margin between essentiality and toxicity. Amphibians are hypothesized to be particularly sensitive due to the potential for metamorphosis-driven mobilization, which could transfer or concentrate contaminant burdens within specific organs. We explored the potential role of tissue degeneration and remodeling during anuran metamorphosis as a mechanism for altering tissue-specific Se burdens. Limnodynastes peronii tadpoles were exposed to dissolved 75Se (as selenite) for 7 days and depurated until completion of metamorphosis. Bioaccumulation and retention kinetics were assessed in whole tadpoles and excised tissues using gamma spectroscopy, and temporal changes in biodistribution were assessed using autoradiography. Tadpoles retained Se throughout metamorphosis, and partitioned the element predominantly within digestive and excretory tissues, including livers > mesonephros > guts > gallbladder. Importantly, our results demonstrate that Se biodistribution varies significantly throughout development. This is indicative of tissue transference, and particularly in tissues developing de novo after depuration. To the best of our knowledge, this is the first study demonstrating Se transference during metamorphic tissue remodelling. Further research is warranted to explore the fate and metabolism of Se (and other metal and metalloids) during anuran development and the implications of transference for influencing toxicity.

Effects of CFT Legumine (5% Rotenone) on Tadpole Survival and Metamorphosis of Chiricahua Leopard Frogs Lithobates chiricahuensis, Northern Leopard Frogs L. pipiens, and American Bullfrogs L. catesbeianus

AbstractAmphibians may experience collateral effects if exposed to CFT Legumine (5% rotenone), a piscicide that is used to remove invasive fish. A series of 48‐h static toxicity tests assessed the acute effects of CFT Legumine on multi‐aged tadpoles of the federally listed Chiricahua leopard frog Lithobates chiricahuensis, the widespread northern leopard frog L. pipiens, and the increasingly invasive American bullfrog L. catesbeianus. At the earliest Gosner stages (GS 21–25), Chiricahua leopard frogs were more sensitive to CFT Legumine (median lethal concentration [LC50] = 0.41–0.58 mg/L) than American bullfrogs (LC50 = 0.63–0.69 mg/L) and northern leopard frogs (LC50 = 0.91 and 1.17 mg/L). As tadpoles developed (i.e., increase in GS), their sensitivity to rotenone decreased. In a separate series of 48‐h static nonrenewal toxicity tests, tadpoles (GS 21–25 and GS 31–36) of all three species were exposed to piscicidal concentrations of CFT Legumine (0.5, 1.0, and 2.0 mg/L) to assess postexposure effects on metamorphosis. In survivors of all three species at both life stages, the time to tail resorption was nearly doubled in comparison with that of controls. For example, mid‐age (GS 31–36) Chiricahua leopard frog tadpoles required 210.7 h to complete tail resorption, whereas controls required 108.5 h. However, because tail resorption is a relatively short period in metamorphosis, the total duration of development (days from posthatch to complete metamorphosis) and the final weight did not differ in either age‐group surviving nominal concentrations of 0.5‐, 1.0‐, and 2.0‐mg/L CFT Legumine relative to controls. This research demonstrates that the CFT Legumine concentrations commonly used in field applications to remove unwanted fish could result in considerable mortality of the earliest stages of Lithobates species. In addition to acute lethality, piscicide treatments may result in delayed tail resorption, which places the tadpoles at risk by increasing their vulnerability to predation and pathogens.Received October 1, 2016; accepted January 17, 2017 Published online March 30, 2017

Effects of the herbicide triclopyr on metamorphic northern red-legged frogs.

Aquatic herbicides are used to manage invasive emergent plants in and around wetlands. Metamorphic frogs that emerge during the aquatic weed management season may be at risk of herbicide exposure. Metamorphic northern red-legged frogs (Rana aurora) were exposed to a triclopyr tank mix labeled for control of broadleaf emergent aquatic weeds such as invasive purple loosestrife (Lythrum salicaria). The tank mix consisted of Renovate® 3 (triclopyr triethylamine salt 44.4%), the modified vegetable oil surfactant Competitor®, and the marker dye Hi-Light® . Metamorphs were exposed to the tank mix and a clean-water control for 96 h, and then reared in clean water for 60 d. Exposure to the tank mix resulted in no treatment-related mortalities, no effects on behavior immediately post exposure, and no effects on body or liver condition indices. Exposure to the tank mix resulted in lethargy during exposure and a 1-d delay in completion of metamorphosis. Deformities present in the rearing population confounded results for some endpoints. Observed effects were minimal, especially compared with the potential for ecological impacts from unmanaged invasive plants. Environ Toxicol Chem 2017;36:2316-2326. © 2017 SETAC.

Transgenic tomato expressing dsRNA of juvenile hormone acid O-methyl transferase gene of Helicoverpa armigera (Lepidoptera: Noctuidae) affects larval growth and its development

Helicoverpa armigera is an important pest infesting a number of crops of high commercial value leading to huge economic losses globally. RNA interference (RNAi) is a potent tool for the control of insect pests and towards this objective, transgenic tomato expressing dsRNA of Juvenile hormone acid methyl transferase (JHAMT) gene of H. armigera was developed. The H. armigera larvae fed on HaJHAMT tomato leaves led to a severe (90%) down regulation of the cognate gene expression thus adversely affected the feeding and metamorphosis. Reduction in larval and pupal weight and inability to undergo complete metamorphosis were observed that led to larval-pupal intermediates and subsequently affected the eclosion. Thus, JH down regulation is an attractive target due to specificity to insects and its important role in insect growth and development to engineer insect resistance in crops using RNAi.

Thyroid Hormone Receptor α Controls Developmental Timing and Regulates the Rate and Coordination of Tissue-Specific Metamorphosis in Xenopus tropicalis.

Thyroid hormone (T3) receptors (TRs) mediate the effects of T3 on organ metabolism and animal development. There are two TR genes, TRα and TRβ, in all vertebrates. During animal development, TRα expression is activated earlier than zygotic T3 synthesis and secretion into the plasma, implicating a developmental role of TRα both in the presence and absence of T3. Using T3-dependent amphibian metamorphosis as a model, we previously proposed a dual-function model for TRs, in particular TRα, during development. That is, unliganded TR represses the expression of T3-inducible genes during premetamorphosis to ensure proper animal growth and prevent premature metamorphosis, whereas during metamorphosis, liganded TR activates target gene transcription to promote the transformation of the tadpole into a frog. To determine if TRα has such a dual function, we generated homozygous TRα-knockout animal lines. We show that, indeed, TRα knockout affects both premetamorphic animal development and metamorphosis. Surprisingly, we observed that TRα is not essential for amphibian metamorphosis, given that homozygous knockout animals complete metamorphosis within a similar time period after fertilization as their wild-type siblings. On the other hand, the timing of metamorphosis for different organs is altered by the knockout; limb metamorphosis occurs earlier, whereas intestinal metamorphosis is completed later than in wild-type siblings. Thus, our studies have demonstrated a critical role of endogenous TRα, not only in regulating both the timing and rate of metamorphosis, but also in coordinating temporal metamorphosis of different organs.

The copepod Acartia tonsa as live feed for fat snook (Centropomus parallelus) larvae from notochord flexion to advanced metamorphosis

From early development until the completion of metamorphosis, fat snook (Centropomus parallelus) larvae are commonly fed the rotifers Brachionus spp. and Artemia spp. nauplii. In this study, cultivated copepods Acartia tonsa were evaluated as feed for 15-to 45-day-old larvae. Two experiments were performed using twelve 30-L tanks stocked with 3.3 fat snook larvae L-1. Their initial mean weight and length were 1.35 ± 0.01 mg (mean ± standard deviation) and 3.83 ± 0.33 mm for 15-day-old larvae and 2.79 ± 1.2 mg and 6.99 ± 0.88 mm for 31-day-old larvae. Three dietary treatments were carried out in four replicates, including Rotifer (R), Artemia (A) and/or Copepod (C). Experiment 1 included Diet RA (control), Diet RC and Diet RCA; while experiment 2 included Diet A (control), Diet C and Diet AC. The survival and growth of larvae fed the Diet RCA in experiment 1 were significantly higher than the others. In experiment 2, the inclusion of copepods in the diet did not improve survival and growth, however, larvae fed Diet C had the highest DHA/EPA ratio. We conclude that the copepod Acartia tonsa provides an important nutritional benefit to fat snook larvae undergoing metamorphosis.

Growth, Development, and Intestinal Remodeling Occurs in the Absence of Thyroid Hormone Receptor α in Tadpoles of Xenopus tropicalis

During development in all vertebrates, thyroid hormone receptors (TRs) are expressed before as well as during and after the peak in plasma thyroid hormone (TH) levels. Previously, we established a role for unliganded TRα in gene repression and developmental timing using tadpoles of TRα knockout (TRαKO) frogs. Here, we examined the role of liganded TRα on growth, development, and intestinal remodeling during natural and TH-induced metamorphosis. Disrupted TRα had little effect on growth during the larval period, but after metamorphosis, TRαKO juveniles grew more slowly than wild-type (WT) juveniles. TRαKO tadpoles developed faster throughout premetamorphosis when TH was low or absent, and despite their decreased responsivity to exogenous TH, TRαKO tadpoles not only were able to complete TH-dependent metamorphosis but also did so earlier than WT tadpoles. In contrast to external morphology, larval epithelial cell apoptosis and adult cell proliferation of intestinal remodeling were delayed in TRαKO tadpoles. Also, TRαKO intestines did not shrink in length to the full extent, and fewer intestinal folds into the lumen were present in TRαKO compared with WT juveniles. Such delayed remodeling occurred despite higher premetamorphic expression levels of TH target genes important for metamorphic progression-namely, TRβ, Klf9, and ST3. Furthermore, the decreased TH-dependent intestinal shrinkage was consistent with reduced TH response gene expression during natural and TH-induced metamorphosis. As in the TRα null mouse model, TRαKO frogs had statistically significant but surprisingly mild growth and development phenotypes with normal survival and fertility.

Sublethal effects on wood frogs chronically exposed to environmentally relevant concentrations of two neonicotinoid insecticides.

Neonicotinoids are prophylactically used globally on a variety of crops, and there is concern for the potential impacts of neonicotinoids on aquatic ecosystems. The intensive use of pesticides on crops has been identified as a contributor to population declines of amphibians, but currently little is known regarding the sublethal effects of chronic neonicotinoid exposure on amphibians. The objective of the present study was to characterize the sublethal effect(s) of exposure to 3 environmentally relevant concentrations (1 μg/L, 10 μg/L, and 100 μg/L) of 2 neonicotinoids on larval wood frogs (Lithobates sylvaticus) using outdoor mesocosms. We exposed tadpoles to solutions of 2 commercial formulations containing imidacloprid and thiamethoxam, and assessed survival, growth, and development. Exposure to imidacloprid at 10 μg/L and 100 μg/L increased survival and delayed completion of metamorphosis compared with controls. Exposure to thiamethoxam did not influence amphibian responses. There was no significant effect of any treatment on body mass or size of the metamorphs. The results suggest that current usage of imidacloprid and thiamethoxam does not pose a threat to wood frogs. However, further assessment of both direct and indirect effects on subtle sublethal endpoints, and the influence of multiple interacting stressors at various life stages, is needed to fully understand the effects of neonicotinoids on amphibians. Environ Toxicol Chem 2017;36:1101-1109. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Semicarbazide-induced thyroid disruption in Japanese flounder (Paralichthys olivaceus) and its potential mechanisms

Semicarbazide (SMC) is a carcinogenic and genotoxic substance that has been found in aquatic systems. SMC may also cause thyroid follicular epithelial cell injury in rats, but the thyroid-disrupting properties of SMC and its potential mechanisms remain unclear. In this study, we exposed fertilized eggs of Japanese flounder (Paralichthys olivaceus) to 1, 10, 100, and 1000μg/L SMC for 55 d to assess the impact of SMC exposure on the thyroid system. The number of larvae in each metamorphic stage was counted, the concentrations of whole-body thyroid hormones (THs) 3,5,3′-triiodothyronine (T3) and thyroxine (T4) were measured, and the transcription levels of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis and gamma-aminobutyric acid (GABA) synthesis were quantified. The results showed that 10μg/L SMC significantly increased whole-body T3 levels, and 100 and 1000μg/L SMC markedly enhanced whole-body T4 and T3 levels. Furthermore, 100μg/L SMC exposure shortened the time it took for flounder larvae to complete metamorphosis by 2 d as compared to the control group. Thus, this study demonstrated that SMC exerted thyroid-disrupting effects on Japanese flounder. SMC-mediated stimulation of TH levels was primarily related to transcriptional alterations of pituitary-derived thyroid stimulating hormone β-subunit (tshβ) and hepatic deiodinase (id). In the 10 and 100μg/L SMC exposure groups, the increased TH levels may have resulted from inhibition of TH metabolism caused by down-regulation of id3 mRNA expression, while at 1000μg/L SMC-exposed group, up-regulation of tshβ and id1 transcripts was expected to enhance the synthesis of T4 and the conversion of T4 to T3 and, consequently, result in higher T4 and T3 levels. In addition, 1000μg/L SMC-induced down-regulation in glutamic acid decarboxylase gad65 and gad67 transcription may have also contributed to the increased TH levels. The thyroid-disrupting effects of 10 and 100μg/L SMC indicated that environmentally relevant concentrations of SMC posed potential environmental risks to aquatic organisms. Overall, our results demonstrated for the first time that SMC exhibited thyroid-disrupting properties by affecting the HPT axis and GABA synthesis, providing theoretical support for environmental risk assessment.

Chronic Effects of Fluoride Exposure on Growth, Metamorphosis, and Skeleton Development in Bufo gargarizans Larvae.

Bufo gargarizans tadpoles were chronically exposed to waterborne fluoride at measured concentrations ranging from 0.4 to 61.2mg F-/L for 70 days from Gosner stage 26 to completion of metamorphosis. The chronic exposure caused a concentration-dependent mortality in all tested fluoride concentrations. Total length, snout-to-vent length (SVL), body mass, and developmental stage of tadpoles were significantly inhibited at 42.6mg F-/L. In addition, significant metamorphic delay and increase in size at completion of metamorphosis occurred after exposure to 19.8mg F-/L. Moreover, 19.8mg F-/L suppressed the bone mineralization of larvae at completion of metamorphosis. However, the bone mineralization could be enhanced by 4.1mg F-/L. In conclusion, our results suggested that the presence of high concentrations of fluoride could increase mortality risk, delay metamorphosis, and suppress skeletal ossification in B. gargarizans larvae.