Maternal Thermal Effects on Female Reproduction and Hatchling Phenotype in the Chinese Skink(Plestiodon chinensis)

We maintained gravid Chinese skinks(Plestiodon chinensis) at three constant temperatures(25, 28 and 31 °C) during gestation, and randomly assigned eggs from each female to one of the same three temperatures for incubation to determine maternal thermal effects on female reproduction and hatchling phenotype. Maternal temperature affected egg-laying date, hatching success and hatchling linear size(snout-vent length, SVL) but not clutch size, egg size, egg component, and embryonic stage at laying. More specifically, females at higher temperatures laid eggs earlier than did those at low temperatures, eggs laid at 31 °C were less likely to hatch than those laid at 25 °C or 28 °C, and hatchlings from eggs laid at 31 °C were smaller in SVL. Our finding that maternal temperature(pre-ovipositional thermal condition) rather than incubation temperature(post-ovipositional thermal condition) affected hatching success indicated that embryos at early stages were more vulnerable to temperature than those at late stages. Our data provide an inference that moderate maternal temperatures enhance reproductive fitness in P. chinensis.

Advertisement Call Variability in the Black-spined Toad Bufo melanostictus (Anura: Bufonidae) during the Breeding Season in Lishui, Zhejiang, China

Advertisement calls play an important role in influencing female mate choice and are regarded as one of the key characters responsible for reproductive isolation and speciation events in frogs and toads. The black-spined toad, Bufo melanostictus, is one of the most common amphibians in Southeast Asia and is frequently observed in human habitations throughout its distributional range. The advertisement calls of the toad have not been well described. Here, we report the advertisement calls of this species from Lishui, Zhejiang, China during its breeding season. The advertisement calls of B. melanostictus are composed of single notes with a single harmonic frequency emitted sporadically. The call parameters recorded in this study were highly variable across recording times and with different body sizes. Dominant frequency was negatively correlated with body size. Comparing the calls of the B. melanostictus population from Lishui with the populations from five other localities(Bangkok, Thailand; Coorg, India; Burma, Myanmar; Bali, Indonesia; Halimun-Salak National Park, Indonesia), we found that call structure was similar among five different populations with no harmonics, while the Halimun-Salak National Park population contained two clear harmonics. Dominant frequency was similar between Bali(1.56 kHz) and Coorg(1.6 kHz) populations, while it was highly variable among other populations. Besides call duration, call interval and pulse/call rate may also be significantly different among the comparing populations. Pooling the available data, we observed some similarities and differences in call parameters among the six populations.

Toxic Effects of Three Heavy Metallic Ions on Rana zhenhaiensis Tadpoles

Heavy metal pollution is widespread in some areas of China and results in contamination of land, water, and air with which all living organisms interact. In this study, we used three heavy metallic ions （Cu^2＋, Pb^2＋ and Zn^2＋） to assess their toxicity effects on mortality, blood biomarker and growth traits （body length and body mass） of Rana zhenhaiensis tadpoles. The results showed that the toxicity levels of the three metallic ions were different when conducted with different experiment designs. For acute toxicity tests, Cu^2＋ was the most toxic with the highest tadpole mortality. The mortalities of tadpoles showed significant differences among the treatments at the same exposure time endpoints （24, 48, 72 and 96h）. Results from repeated measures ANOVA indicated that metallic ion concentration, exposure time and their interactions significantly affected the mortalities of R. zhenhaiensis tadpoles. Also, the toxicity effects of all binary combinations of the three metallic ion treatments showed synergism. The half lethal concentrations （LCs0） decreased with increasing exposure time during the experimental period, and the safe concentration （SC）values of Cu^2＋, Pb^2＋ and Zn^2＋ were different from each other. Combined and compared LC50 values with previous data reported, it is suggestes that the toxicity levels of metal pollution to anuran tadpoles should be species-and age-related. For blood biomarker tests, Zn^2＋ was the most toxic with the highest total frequencies of abnormal erythrocytic nucleus. All three metallic ions caused higher abnormal erythrocytic nucleus compared with control groups. In a chronic toxicity test, Pb^2＋ was the most toxic with lowest growth traits. Survival rate （except for 18 days）, total body length and body mass showed significant differences among the treatments. These findings indicated that tadpoles of R. zhenhaiensis should be as a bioindicator of heavy metals pollution.

Effects of Predation by Invasive Western Mosquitofish(Gambusia affinis) on Survival of Eggs,Embryos and Tadpoles of Pelophylax nigromaculatus and Duttaphrynus melanostictus in South China

Alien species are one of the most serious threats to the decline and extinction of native amphibian populations. In this study, we examined the predation of invasive Western Mosquitofish Gambusia affinis on the eggs, embryos, and tadpoles of Duttaphrynus melanostictus and Pelophylax nigromaculatus in south China. Our results suggested that the survival of eggs and embryos remaining in the egg capsules of P. nigromaculatus and D. melanostictus was significantly higher than those removed from the egg capsule at 12-h intervals within 72 h in the presence of G. affinis. The survival of P. nigromaculatus eggs and embryos without egg capsules was significantly lower than those of D. melanostictus without egg capsules. The survival of P. nigromaculatus eggs and embryos with egg capsules was significantly higher than those of D. melanostictus with egg capsules from 24 h to 72 h except for 12 h. The survival of D. melanostictus tadpoles was significantly higher than that of P. nigromaculatus tadpoles in the presence of G. affinis. The survival of Gosner stage 26 tadpoles of P. nigromaculatus was significantly higher than that of Gosner stage 30 tadpoles from 12 h to 60 h, but there were no significant differences at 72 h. In contrast, the survival of Gosner stage 26 tadpoles of D. melanostictus was significantly lower than that of Gosner stage 30 tadpoles within 72 h, recording every 12 h. The increasing temperature caused a significant increase in predation by G. affinis on P. nigromaculatus eggs and embryos. The outer jelly capsule surrounding anurans eggs might serve as a mechanical defense against predation by G. affinis due to its large diameter, relatively stationary state and unpalatability. The differences in the vulnerability of P. nigromaculatus and D. melanostictus embryos and tadpoles to G. affinis probably due to differences in the unpalatability, black skin and activity. Based on the magnitude of predation by G. affinis on the eggs, embryos and tadpoles of these two species and the combined impact of temperature, we might speculate that invasive G. affinis and global warming would have more detrimental impacts on population viability of P. nigromaculatus than D. melanostictus in China.

Density But Not Kinship Regulates the Growth and Developmental Traits of Chinese Tiger Frog (Hoplobatrachus chinensis) Tadpoles

Kinship and density are believed to affect important ecological processes such as intraspecific competition, predation, growth, development, cannibalism, habitat selection and mate choice, In this work, we used Chinese tiger frog Hoplobatrachus chinensis tadpoles as an experimental model to investigate the effects of kinship and density on growth and development of this species over a 73 day period. The results showed that density can affect the growth and developmental traits （survival rate, larval period, size at the limb bud protrusion/metamorphic climax and body mass at different life stages） of H. chinensis tadpoles, while kinship does not. Tadpoles took longer to develop and potential metamorphosis was greater in high density groups of both sibling and non-siblings. The interaction of kinship and density did not significantly influenced growth traits of H. chinensis tadpoles during the experimental period. For coefficient variations of each growth trait, no differences were detected between sibling and non-sibling groups. These findings provide valuable information on the basic ecology of H. chinensis which will be helpful in future studies of other anuran species.

Developmental and Liver Metabolite Changes Induced by TPhP Exposure in Brown Frog(Rana zhenhaiensis)Tadpoles

As an organophosphorus compound that frequently detected in water samples,triphenyl phosphate(TPhP)has been showed to have multiple toxicological effects on aquatic species.However,no attention has been paid to its potential impact on non-model amphibian species.Here,tadpoles of the Zhenhai brown frog(Rana zhenhaiensis)were exposed to different concentrations of TPh P(0,0.02 and 0.1 mg/L)throughout the developmental period to assess physiological and meta bolic impacts of TPh P exposure on amphibian larvae.After 30-day TPh P exposure,the developmental stage of tadpoles from the high-concentration treatment appeared to be more advanced than that from the other two treatments,but other measured traits(including body size,tail length and liver weight)did not differ among treatments.Metabolite profiles in tadpole livers based on liquid chromatographymass spectrometry(LC-MS)revealed a distinct metabolic disorder in exposed animals.Specifically,significant changes in various hepatic amino acids(such as glutamine,glutamate,valine and leucine)were observed.Overall,our results indicated that chronic TPhP exposure potentially caused developmental and hepatic physiological changes in R.zhenhaiensis tadpoles,although its impact on tadpole growth appeared to be minor.

Comparison on Purification Effect of Two Macroalgae Species on Prawn Tailwater

[Objectives]This study was conducted to investigate the purification effects of two common large seaweeds on the tail water of prawn farming in greenhouses,and to determine the best culture density of seaweeds.[Methods]Two large seaweed species,Gracilaria lichevoides and Ulva lactuca,were selected to set four culture densities of 0.5,2,4 g/L and a blank control group,respectively.The seaweeds were cultured in 100 L white polyethylene buckets,each of which contained 50 L of tail water from prawn culture.[Results]After 5 d,the nutrient removal rates of the two seaweeds were directly proportional to the density.There was no significant difference in NH_(4)-N removal rate between G.lichevoides and U.lactuca(P>0.05)by two-way analysis of variance,and the NH_(4)-N removal rate of the latter was higher.The removal rates of NO_(3)-N,TN and TP by G.lichevoides were significantly higher than those by U.lactuca(P<0.05).The specific growth rates of seaweeds were negatively correlated with their culture densities.The specific growth rates of G.lichevoides were 5.73%,1.654%and 0.48%,respectively,and those of U.lactuca were 2.01%,1.187%and 0.138%,respectively,when the culture densities were 0.5,2.0 and 4.0 g/L.Two-factor analysis of variance showed that the former was significantly higher than the latter,when the culture density of the two species of seaweeds was 0.5 g/L(P<0.05).The two-way analysis of variance showed that when the culture density of the two kinds of seaweeds was 0.5 g/L,the specific growth rate of G.lichevoides was significantly higher than that of U.lactuca(P<0.05).Based on the above research,the two macroalgae could reduce the nutrients in the wastewater to a large extent,but the culture density determined the scale and economic benefits of seaweed cultivation and further affected the normal growth,metabolism and quality of the seaweeds.[Conclusions]This study provides some theoretical basis for large-scale seaweed farming and biological selection of in-situ ecological restoration of eutrophic seawater.

Treatment of Shrimp Effluent by Integrated Culture of Bivalves and Macroalgae

[Objectives]To provide a theoretical basis for constructing the best species suitable for local shrimp-bivalves-algae IMTA through the screening of different bivalves and the determination of seaweed density.[Methods]The preliminary studies of different kind of bivalves and macroalgae(Gracilaria lichevoides)used for the purification of shrimp effluent were described.Through the screening of benthic bivalves,the best ratio of integrated culture of bivalves and algae was determined.[Results]Both bivalves and macroalgae had certain purification effects on aquaculture wastewater,but the effects significantly differed from species and breeding density.The removal rate of nutrient declined from Potamocorbula laevis,Sinonovacula constricta,Tegillarca granosa.The mixotrophic culture of 8 ind/L P.laevis and 120 g G.lichevoides had the highest efficiency of purification and removal rates of nutrient:NH4-N:90.67%,TP:86.18%,TN:72.66%,NO3-N:51.85%,respectively.There was a significant difference between the blank control group and the other three groups(P<0.01).The 8 ind/L+120 g group was significantly higher than the 4 ind/L+120 g group(P<0.05)in TP removal rate.Additionally,the difference between four groups was significant in the removal rate of NH4-N and TN(P<0.05),but 2 ind/L+120 g group and 8 ind/L+120 g group in the removal rate of NO3-N had no significant difference(P>0.05).[Conclusions]This research provides a reference for the use of filterable shellfish and large seaweed to treat aquaculture wastewater,and also provides the theoretical basis for constructing the local multi-level breeding structure.

Path analysis of desirable traits and evaluation of reproductive performance of Crassostrea sikamea in different ages

Kumamoto oysters(Crassostrea sikamea)are important marine aquaculture shellfish because of their taste and round shell shape.Due to its small size,selective breeding is required to improve its market size,but there are no reports on traits suitable for direction selection and reproductive productivity of Kumamoto oysters of different ages.In this study,path analysis and one-way ANOVA were used to explore the influential indices of oyster body weight and fecundity.The results showed that shell height was the main factor affecting the body weight of Kumamoto oysters at one-year-old and two-year-old,followed by shell length.Body weight was the main factor influencing fecundity,and shell height showed a significant correlation with fecundity.Therefore,shell height should be considered the first parameter for selection when selecting Kumamoto oysters for body weight as the direct selection target.When one-year-old and two-year-old Kumamoto oysters were selected as parents from Xiangshan Bay,hatching parameters and growth parameters of F1 generation larvae were measured.The fecundity and egg diameters were 1.72×10^(6)and 46.60±3.62μm,respectively,which were significantly greater than one-aged oysters(5.86×10^(5),40.26±3.52μm).The hatching parameters(fertilized egg size,fertilization rate,and hatching rate)and larval growth performance(D-stage veliger and veliger)were similar and did not differ significantly(P>0.05),and after metamorphosis,there was also no significant difference in shell height size at 40 days of age(P>0.05).Therefore,there was no significant effect of age on reproduction,larval development,and the early growth of Kumamoto oysters.These results could provide a basis for parental sources and proper traits for Kumamoto oyster selection,which is beneficial to developing and utilizing this excellent germplasm resource.

Leukocyte Membrane-Coated Liquid Metal Nanoswimmers for Actively Targeted Delivery and Synergistic Chemophotothermal Therapy

We report a leukocyte membrane-coated gallium nanoswimmer(LMGNS)capable of ultrasound-propelled motion,antibiofouling,and cancer cell recognition and targeting.The LMGNS consists of a needle-shaped gallium core encapsulating an anticancer drug and a natural leukocyte membrane shell.Under the propulsion of an ultrasound field,LMGNSs could autonomously move in biological media with a speed up to 108.7μms^(-1).The velocity and motion direction of the LMGNSs can be modulated by regulating the frequency and voltage of the applied ultrasound field.Owing to the leukocyte membrane coating,LMGNSs can not only avoid biofouling during the motion in blood but also possess cancer cell recognition capability.These LMGNSs could actively seek,penetrate,and internalize into the cancer cells and achieve enhanced anticancer efficiency by combined photothermal and chemical therapy.Such biofunctionalized liquid metal nanoswimmer presents a new type of multifunctional platform for biomedical applications.

Acoustophoretic Motion of Erythrocyte-mimicking Hemoglobin Micromotors

of main observation and conclusion Biocompatible micromotors have received increasing attention,due to their powerful motion,diverse functionalities and autonomous navigation in the field of biomedical applications.Herein,we report an acoustically-propelled,magnetic-guided,erythrocyte-mimicking hemoglobin micromotor with oxygen-carrying capability.These hemoglobin micromotors were fabricated by encapsulating magnetic nanoparticles in hybrid hemoglobin/calcium carbonate microparticles,followed by covalently layer-by-layer assembly of hemoglobin multilayers.The asymmetric distribution of magnetic nanoparticles enables these micromotors to move rapidly under the propulsion of an acoustic field.The velocity of acoustophoretic motion of hemoglobin micromotors increases with the increase of input voltage of acoustic field,up to 58.2 pirn s^-1 at 10 V.The hemoglobin micromotors are directionally navigated by applying an external magnetic field,and also could pass through microchannels toward the targeted region.Moreover,such hemoglobin micromotors show effective oxygen loading and releasing capability,holding considerable promise as a new platform for oxygen delivery applications.