LABORATORY OBSERVATION ON SPAWNING,FECUNDITY AND LARVAL DEVELOPMENT OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI TSINGTAUNESE)

Although amphioxus is widespread in temperate and tropical seas,its population is diminishing be-cause of environmental pollution.To keep the population of this evolutionarily important animal fromdiminishing,study on its reproduction and development is necessary.The main findings in this study onthe spawning and fecundity of the amphioxus reared in laboratory and its larval development are as fol-lows.1.Water temperature markedly affected the spawning.It spawned only when water temperature reached 21℃.2.Spawning of the amphioxus in laboratory was markedly extended.Initially,the amphioxus spawned at about 7:00 PM,but spawning time was postponed as spawning days went on.3.The number of eggs produced by a female ranged from 1400 to 12800,average of 5800.This al-so represents the fecundity of the amphioxus because it shedded all eggs within the ovary at a time.4.During the first few months of life of the amphioxus,its growth rate changed seasonally.Thegrowth rate in summer and fall was greater than that

A larval specific OBP able to bind the major female sex pheromone component in Spodoptera exigua(Hübner)

Odorant binding proteins （OBPs） in insects are postulated to solubilize and transport the hydrophobic odorants across the hydrophilic antennal lymph to the olfactory receptors （ORs） located on the dendrite membrane of the sensory neurons. OBPs in adult insects have been intensively reported, but those in larvae are rarely addressed. In our study, a full-length OBP cDNA, namely SexiOBP13, was cloned by RT-PCR and RACE strategy from the heads of Spodoptera exigua larvae. The quantitative real-time PCR （qPCR） measurement indicated that SexiOBP13 was highly expressed in larval head, but very low in other parts of larva and was not detected in any tissues of adult. The binding affinities of SexiOBP13 to plant volatiles and female sex pheromone components were measured by competitive binding assays. Interestingly, SexiOBP13 displayed a high binding affinity （Ki=3.82 IJmol L-1） to Z9,E12-14：Ac, the major sex pheromone component of S. exigua, while low affinities to the tested host plant volatiles （Ki〉27 μmol L-l）. The behavioral tests further confirmed that Z9,E12-14：Ac was indeed active to elicit the behavioral activity of the third instar larvae of S. exigua. Taken together, our results suggest that SexiOBP13 may play a role in reception of female sex pheromone in S. exigua larvae. The ecological significance of the larvae preference to the adult female sex pheromone was discussed.

2D-QSAR Using MLR and 3D-QSAR Using CoMSIA Studies on the Toxicity of Aromatic Hydrocarbons on Larval Sinonvaculina Constricta

Aromatic hydrocarbons,one of the persistent organic pollutants（POPs）,has been usually found in mussels,accumulated for their hard mobility and activities in harbours and estuaries.In this study,based on the 96 hr-LC50 of 12 aromatic hydrocarbons with larval sinonvaculina constricta,three-dimensional quantitative structure-activity relationship（3D-QSAR） technique：comparative molecular similarity indices analysis（CoMSIA） and 2D-QSAR technique：multiple linear regression（MLR） were described to obtain more detailed insight into the structure-activity relationships between the molecular structure and bio-activity.The results show the MLR model based on density functional theory（DFT） calculation carried out at the B3LYP/6-311＊＊ level with Gaussian 03 program yielded a very good correlation with a coefficient squared R2 of 0.716 and a cross-validated Q2 of 0.874.The dipole moment and enthalpy,as the thermodynamic parameters,were two important factors influencing pLC50.Correspondingly,CoMSIA based on the partial least-squares（PLS） methodology with steric,electrostatic,hydrophobic,H-bond donor and acceptor fields contributing simultaneously were employed and the values of R2 and the cross validation with leave-One-Out（LOO） Q2LOO were 0.585 and 0.990,respectively,which reveals the structure features,such as the electronegative substituent（nitro-group）,hydrophobic groups（the benzene ring） and H-bond（nitro-group）,related to the toxicity.The results of 2D-QSAR employing MLR model and 3D-QSAR employing CoMSIA model provide the useful information for predicting the toxicity of other aromatic hydrocarbons by comparing the molecular structures of similar compounds.

Characterization of larval instars of artemisia weevil,Adosomus sp.

The artemisia weevil, Adosomus sp., has become an important wormwood （Artemisa ordosoca and A. sphaerocephala） pest in recent years in Ningxia Province, China. The number of larval instars should be determined before further studies on its life history, biology and integrate management of pest （IMP）. In this study, we measured the length and width of head capsule, based on frequency histograms and Dyar＇s law, from which we concluded that the larvae ofAdosomus sp. have four instars. Of the two variables measured, the width of head capsule provided the best division of instars. The average width of head capsule of the 1st instar larvae was 1.348±0.152 mm, the 2nd 2.109±0.199 mm, the 3rd 3.563±0.492 mm and the 4th 5.974±0.581 mm.

COMPARATIVE STUDIES ON THE LARVAL DEVELOPMENT OF THE PENAEID SHRIMPS,PENAEUS CHINENSIS,P.MERGUIENSIS AND P.PENICILLATUS

The morphology of larval and 1st postlarval stages of Penaeus penicillatus are described.Results from comparative studies on larval development of P.penicillatus,P.merguiensis and P.chinensis are as fol-lows: These three species could not be identified during their naupliar stages. In the 1st protozoea,the antennule L1/L2 value is 1.7—2.0 in P.merguiensis,but lessthan 1.7 in P.chinensis and P.penicillatus;in the 2nd protozoea,the supra-orbital spine in P.chinensis isnot bifurcated,while those of P.merguiensis and P.penicillatus are bifurcated;in the 3rd protozoea,thereis a minute(or no)dorso-median spine on the posterior margin of the 1st and 2nd abdominal somite in P.chinensis,but they are prominent in P.merguiensis and P.penicillatus.In the mysis and 1st postlarvalstages,P.chinensis differs from P.merguiensis and P.penicillatus in having 9(8 in the other 2 species)longsetae on the exopod of pereopods 1—3;additionally,one dorsal tooth appears on the rostrum of P.chinensis in the 2nd mysis and that

Dynamic metabolite alterations of Portunus trituberculatus during larval development

A mass mortality often occurs from molting to the megalopa stage during the larval development of the swimming crab Portunus trituberculatus. Larvae with insuffcient nutrient accumulation during the zoeal stages are probably unable to develop into juvenile swimming crabs. However, the nutritional information such as the primary metabolites is scarce for P. trituberculatus larvae. The aim of this work is to obtain an insight into the metabolite traits of P. trituberculatus at early developmental stages. 1 H nuclear magnetic resonance spectroscopy coupled with multivariate data analysis was used to determine how the metabolite pro?les shift during larval development in P. trituberculatus. Our results show that the trend of total metabolites exhibited a rise from zoea 1 to zoea 3, followed by a drop from zoea 4 to megalopa and recovery during the ?rst juvenile stage. A large-scale depletion of total metabolites in the zoea 4 and megalopa stages suggests a deep depression of metabolic activity, which may be linked to the mass mortality from molting to the megalopa stage. These ?ndings provided essential metabolic information about the larval development of P. trituberculatus and important clues for understanding the nutritional requirements of P. trituberculatus in early developmental stages.

Larval habitats of mosquito fauna in Osogbo metropolis,Southwestern Nigeria

Objective:To determine the larval habitats of mosquito fauna and possible impact of land use/land cover changes on the epidemiology of mosquito-bome diseases in Osogbo metropolis,Southwestern,Nigeria.Methods:All accessible larval habitats were surveyed between May and September,2011in Osogbo metropolis while Land Use/Land cover of the city was analyzed using 2 Lansat Multispectral Scanner satellite imagery of SPOT 1986 and T.ANDSAT TM 2009.Results:A total of six species namely,Aedes aegypti,Aedes albopictus,Aedes vittatus,Anopheles gambiae complex,Culex quinquefascialus and Erelmapodite chrysogaster were encountered during the study.The occurrence and contribution of disused tyres was significantly higher(P<0.05)than the other habitats encountered,while there were no significant differences in the contribution of gutters/run-offs.septic tanks/drums,ground pools/open drains and discarded containers lo the breeding of mosquitoes(P>0.05).The accessible land use/land covered of the study area between 1986 and 2009 showed that the wet land coverage and settlement area increased from 0.19 lo 9.09 hectare and 1.00 to 2.01 hectare respectively while the forest area decreased from 60.18 to 50.14 hectare.Conclusion:The contribution of the habitats coupled with the increasing rale of flooded environment which could provide ample breeding sites for mosquitoes call for sustained environmental sanitation and management in Osogbo metropolis.

Larval stages of digenetic trematodes in Melanopsis praemorsa snails from freshwater bodies in Palestine

Objective:To detect the species of larval trematodes(cercariae) in Melanopsis praemorsa snails from 5 different fresh water bodies in Palestine.Methods:A total of 1 880 Melanopsis praemona snails were collected from different fresh water bodies in Palestine from October,2008 to November,2010.Cercariae in Melanopsis praemona snails were obtained by lighting and crushing methods.The behavior of cercariae was observed using a dissecting microscope.Results:Three different species of larval trematodes were identified from Melanopsis praemona snails collected only from Al-Bathan fresh water body,while snails from other water bodies were not infected. These species were microcercous cercaria,xiphidiocercaria and brevifurcate lophocercous cercaria.These cercariae called Cercaria melanopsi palestinia 1,Cercaria melanopsi palestinia H and Cercaria melanopsi palestinia III have not been described before from this snail in Palestine. The infection rate of Melanopsis praemona collected from Al-Bathan fresh water body was 5.7%, while the overall infection rate of snails collected from all fresh water bodies was 4.3%.Details are presented on the morphology and behavior of the cercariae as well as their development within the snail.Conclusions:These results have been recorded for the first time and these cercariae may be of medical and veterinary importance.

Environmental characteristics of anopheline mosquito larval habitats in a malaria endemic area in Iran

Objective:To determine the effects of environmental parameters of lanal habitats on distribution and abundance of anopheline mosquitoes in Rudan county of Iran.Methods:This cross-sectional study was conducted during the mosquito breeding season from February 2010 to October 2011. The anopheline larvae were collected using the standard dipping method,the specimens were identified using a morphological-based key.Simultaneously with larvay collection,environmental parameters of the larval habitats including water current and turbidity,sunlight situation,and substrate type of habitats were recorded.Water samples were taken from breeding sites during lanal collection.Before collection of samples,the water temperature was measured.The water samples were analysed for turbidity,conductivity,total alkalinity,total dissolved solid.pH and ions including chloride,sulphate,calcium,and magnesium.Statistical correlation analysis and ANOVA test were used to analyze the association between environmental parameters and larval mosquito abundance.Results:In total 2 973 lanae of the genus Anopheles were collected from 25 larval habitats and identified using morphological characters.They comprised of six species: An.dthali(53.21%),An.stephensi(24.22%),An.culicifacies(14.06%),An.superpictus(4.07%),An. turkhudi(3.30%),and An.apoci(1.14%).The most abundant species was An.dthali which were collected from all of the study areas.Larvae of two malaria vectors.An.dthali and An.stephensi, co-existed and collected in a wide range of habitats with different physico-chemical parameters. The most common larval habitats were man-made sites such as sand mining pools with clean and still water.The anopheline mosquitoes also preferred permanent habitats in sunlight with sandy substrates.The results indicated that there was a significant relationship between mean physicochemical parameters such as water temperature,conductivity,total alkalinity,sulphate,chloride, and mosquito distribution and abundance.Conclusions:The results of this study showed a correlation between certain environmental parameters and mosquito lanae abundance,and these parameters should be considered in planning and implementing larval control programs.

Quantitative Evaluation of the Larval Density of Sea Potato Acaudina spp.with Mitochondrial Molecular Marker

The sea potato Acaudina spp.is a species of sea cucumber,belonging to the class Holothuroidea under Phylum Echino-dermata.It becomes blooming recently in the East Sea of China,causing serious ecological problems.However,until now there are no molecular data for its larval identification and population genetic analysis.In this study,we firstly screened a mtDNA fragment and demonstrated that it was the species-specific molecular marker for the identification of Acaudina spp.We then developed a quantitative polymerase chain reaction(PCR)method to evaluate the larval density of Acaudina spp.based on this molecular probe.Utilizing this method,we examined 116 plankton samples collected in four seasons from 13 stations along the coastal region in Fu-jian province,China.The results showed that the high larval density was presented at stations 1,2,and 3,which were near a quay in the coast.The larval density increased from April and reached the highest value in June and July,suggesting temperature might be the main environmental factor on the effects of its population distribution and density.Our work provides an important molecular tool for species identification and risk evaluation of a potentially invasive species.

Effects of heavy metal ions on larval Penaeus chinensis and comparative study on their elimination methods

In order to establish a new method to eliminate toxicities of heavy metal ions to larval penaeid shrimp, the effects of copper, zinc, lead and cadmium ions on larval metamorphosis of Penaeus chinensis at each stage were determined in detail, and two methods eliminating toxicities were compared. The results indicate that sensitivity of larvae to heavy metal ions decreases in the order of nauplii, protozoea, mysis and postlarvae. The phytoplankton food can reduce, by chetating, the toxicities of heavy metal ions. Both EDTA chelation and PHMA absorption can be used to eliminate the toxicities effectively and there is no significant difference between the two methods. EDTA is harmful to larval P. chinensis at high concentration but affects neither survival nor metamorphosis at below 10 mg/dm3. It is preliminarily observed that copper and zinc ions at low concentration are beneficial to the development of larvae after protozoea stage. It is suggested that it is unnecessary to e-liminate the effects of heavy metal ions in the culture of penaeid shrimp, under normal conditions after protozoea stage.

Construction of Larval cDNA Expression Library and Immunological Identification of Positive Clones in Tick Haemaphysalis qinghaiensis

A primary cDNA expression library with a titer of 5.0 × 105 PFU mL-1 was constructed from mRNA extracted from larval Haemaphysalis qinghaiensis ticks in order to identify certain genes,which would then be used as candidate molecules for development of effective vaccines to control this parasite.Totally 11 positive clones,which designated as HqL01-11,were obtained by immunoscreening of the library using a polyclonal antibody generated in rabbit with larval tick protein extract.Results of sequence analysis from BLASTN searching revealed that 6 of them had no significant homology with the adult H.qinghaiensis ticks’ known genes,4 of them had no significant homology with all genes deposited in GenBank database.HqL07,HqL08,HqL09,and HqL11 were deposited to GenBank database,and accession numbers were EF605263,EF605264,EF605265,and EF605266,respectively.Subsequently,HqL07 and HqL09 were expressed in vitro and the molecular weights of the corresponding expressed products were 60 and 70 kDa,respectively.Western blot analyses showed that HqL07 and HqL09 had immunogenicity.This study laid the foundation for future production of genetically engineered vaccines for the immunological control of H.qinghaiensis.

Isozyme changes during larval development of Penaeus chinensis

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Using iTRAQ-Based Quantitative Proteomics Analysis to Identify Differentially Expressed Proteins Related to Larval Development of Portunus trituberculatus

The swimming crab Portunus trituberculatus is an economically important marine crustacean species.Here isobaric tags for relative and absolute quantitation(iTRAQ)analysis were used to identify proteins that are differentially expressed during larval development of P.trituberculatus to elucidate the underlying mechanisms.In comparison with the first zoea stage(Z1),3980 proteins were identified from 32789 peptides,which were matched with 115497 spectrums.A total of 241 proteins were screened with significantly differential expressions in all development stages.These 241 proteins are involved in various biological processes,such as cytoskeleton organization,protein synthesis,energy production and substance metabolism,physiological activities,and transport.Cluster analyses of the 241 differentially expressed proteins led to the generation of four protein clusters based on the overall similarity in protein expression patterns.Exactly 54,70,36,and 45 proteins clustered in profiles 10(0,0,0,−1,0,0),15(0,1,0,1,0,1),18(0,1,2,2,1,0),and 19(0,1,2,3,4,5),respectively.Muscle development and exoskeleton renewal were important processes throughout the development stages.In addition,protein synthesis,degradation,and digestion actively occurred,especially at the Z4 stage.These results provide novel insights into the mechanisms underlying larval development of swimming crab and can assist in larval rearing.

Chromatin accessibility profiling provides insights into larval cuticle color and adult longevity in butterflies

Butterflies are diverse in virtually all aspects of their ontogeny,including morphology,life history,and behavior.However,the developmental regulatory mechanisms underlying the important phenotypic traits of butterflies at different developmental stages remain unknown.Here,we investigated the developmental regulatory profiles of butterflies based on transposase accessible chromatin sequencing(ATAC-seq)at three developmental stages in two representative species(Papilio xuthus and Kallima inachus).

Larval Instars and Division Features of Apriona germari

Apriona germari larvae were obtained via sample collection in woodland and indoor rearing. In this study, larval instars were divided by Dyar＇s law, and the results showed that the larval stage of A. germari comprised six instars, while the width of head capsule was the best instar-dividing structure. On the basis of instar division, tubercle numbers on the transverse furrow downside of first abdominal dorsal ambulatory ampullae, seta numbers of abdominal telson and lateral seta numbers of mandible were observed and statistically calculated. The accuracy of instar-dividing was verified by comparing with the Dyar＇s law. The coincidence rate of two instar-dividing results was 90. 47%. It is approved that the above morphological features could be used in instar division ofA. germari larvae.

Larval habitat of <i>Ochlerotatus albifasciatus</i>(Diptera: Culicidae) in the southern edge of the Americas, Tierra del Fuego Island

The information about ecological topics of mosquitoes at the southernmost tip of South America is fragmentary and scarce. The present study evaluates lentic freshwater habitat located in the surroundings of main roads of the Argentine sector of Tierra del Fuego as larval habitat of Ochlerotatus albifasciatus, also analyzes the relationships between their presence and several environmental variables: water turbidity, percentage of gramineans, percentage of macrophytes, presence of crustaceous cyanobacteria, and filamentous chlorophyceans. Mosquito inmatures were collected with dip nets. A generalized linear model (GLM) with negative binomial error distribution was used to determine the effects of different variables of the water bodies on abundance of Oc. albifasciatus in the larval habitats. Collections were made in 45 lentic freshwater bodies. Preimaginal stages were found in 17.70% of the studied habitats. Oc. albifasciatus was the only culicid registered. The GLM explained 93.17% of the variability, and showed a negative relationship between the abundances of Oc. albifasciatus and water turbidity, and a positive relationship with percent-age of gramineans. The gramineans would improve food supply, because the plants are providing suitable substrate for different types of microbiota, a layer of leaves would protect eggs from extreme temperatures, and could help the larvae to hide from potential predators. The negative association between abundance of this species with water turbidity could be related to the presence of vegetation that favors retaining the substrate, reducing water turbidity.

Altitudinal Distribution of Papilionidae Butterflies along with Their Larval Food Plants in the East Himalayan Landscape of West Bengal, India

The altitudinal distribution of Papilionidae butterflies across the East Himalayan Landscape of West Bengal, India is presented here. 26 butterfly species are known to occur across 11 altitudinal belts. Species Richness (R) and Species Diversity (H′) are said to be highest between 1200 - 1400 masl (meters above sea level). In contrast, lowest values of Species Richness and Species Diversity occur at the highest altitude of 3000 masl and above. Maximum number of individuals occurs between 900 - 1100 masl while the minimum number of individuals was present at the highest altitude of 3000 masl or above. 35 species of plants belonging to 6 families served as the larval food plant of these butterflies. Thus the presence of suitable larval host plants probably governs the altitudinal distribution of these papilionid species of butterflies. 30.77% of butterfly species are strictly monophagous in nature.

Expression Characteristics and Potential Function of Neuropeptide MIP in Larval Settlement of the Echiuran Worm Urechis unicinctus

Larval settlement and metamorphosis are important developmental events in marine invertebrates.Some neuropeptides may play a key role in these processes.In this study,we revealed the expression characteristics of myoinhibitory peptide(MIP)in the embryos and larvae of the echiuran worm Urechis unicinctus using RT-qPCR,in situ hybridization and immunofluorescence techniques,and found its location in the top cells of the upper hemisphere was conservative in larvae.Furthermore,we used the active MIP peptide to verify its role in inducing the larval settlement of U.unicinctus,and confirmed that MIP can trigger an obvious larval settlement when 10μmol L^(−1) or 15μmol L^(−1) MIP was added in the seawater where early-segmentation larvae were cultured.Our finding indicated that neuropeptide MIP participates in the larval settlement in U.unicinctus.

Cloning, Characterization and Bioinformatic Analysis of the Gene Encoding the Larval Serum Protein 2 in Diapause of the Onion Maggot, Delia Antiqua

The full-length cDNA encoding Larval serum protein 2 (LSp-2) in the onion maggot,Delia antiqua, was cloned and sequenced by rapid ampli?cation of cDNA ends methods. The result showed that the cDNA was 2203 bp long and the open reading frame (ORF) of 2106 bp encoded 701 amino acid with a calculated molecular weight of 80.5 kDa and an isoelectric point of 5.87. The onion maggot LSp-2 shows highest homology (83%) to that ofCalliphora vicinaat amino acid level. Its signal peptides, domains and structures were predicted and analyzed by using bioinformatic methods. The amino acid sequence of LSP-2 suggests that it would be a typical hexamerin.