Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

Development and application of 15 novel polymorphic microsatellite markers for sect. Paeonia (Paeonia L.)

Herbaceous peony, which belongs to sect. Paeonia, Paeonia L., Paeoniaceae, is not only a famous traditional flower in China; it is also highly regarded as an ornamental in Europe and the USA. On account of the abundance in germplasm resources and wide distribution ranges, herbaceous peony can be divided into three cultivar groups, the Chinese Peony Cultivar Group, the European Peony Cultivar Group and the Hybrid Peony Cultivar Group. However, most studies on genetic relationships in Paeonia are limited to the first group. This study used 15 polymorphic microsatellite markers that were developed by magnetic bead enrichment to explore the genetic diversity of 89 genotypes collected from Beijing, the USA and Canada and introduced to China. From 145 allelic loci that were detected from 15 primers, 140 (96.6%) were polymorphic. The number of allelic loci ranged from 4 to 16 with an average of 9.3, and the polymorphic index content ranged from 0.362 to 0.825 with an average of 0.678. Both cluster analysis and principal component analysis based on the 15 polymorphic primer pairs could distinguish the species from cultivars although the cluster analysis was better able to reflect the genetic relationships between all individuals. Some new insights about Paeonia L. are suggested. Furthermore, the 15 SSR markers have great significance as they will allow for furthering the studies on genetic diversity, genetic relationships and even the molecular marker-assisted breeding of Paeonia L.     

Morphometric,meristic and ISSR marker systems for species identification and evolutionary analysis in five Indian Channids

Snakehead species belonging to Channidae are primary group of freshwater air breathing fishes having their confined distribution in African and Asian continents. ISSR – PCR was used to investigate the phylogenetic relationship among five Channidae species viz. Channa striatus, Channa marulius, Channa punctatus, Channa diplogramme and Channa gachua. In addition, morphometric and meristic characters were subjected to principal component analysis (PCA) and the bootstrap values within the species were also calculated. The genetic identity between the species ranged from 0.5526 to 0.7632 and the genetic distance ranged from 0.2703 to 0.5931. The Nei's gene diversity (H) was calculated as 0.2653 and the Shannon's information index (I) was 0.3842. UPGMA dendrogram arrived by the morphological and molecular markers revealed the closeness between C. striatus and C. marulius among the five species.     

When physical oceanography meets population genetics: The case study of the genetic/evolutionary discontinuity in the endangered goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species

Epinephelus itajara is one of the marine fish species most threatened for extinction and it is considered to be “critically endangered” by the IUCN. The present study evaluated the genetic diversity of the species and the genetic/evolutionary relationships of its populations along the Atlantic coast of South America. The results indicate relatively reduced genetic variation, re-emphasizing the low adaptive potential of the species. One of the populations presented relatively high degrees of genetic diversity and it is evolutionary isolated from the all other populations. The evidences indicate the existence of two Evolutionarily Significant Units comprising E. itajara in the Atlantic coast of South America and the conservation prospects for the species must take these evidences into account.     

Genetic diversity of wild population of Pyropia haitanensis based on SSR analysis

Genetic diversity analysis was conducted on 80 individuals of 4 populations of non-bred Pyropia haitanensis by simple sequence repeat (SSR) method. Using 15 pairs of microsatellite primers, 37 polymorphic loci were amplified, representing 94.9% of all loci. At the population level, the percentage of polymorphic bands (P) and polymorphism information content (PIC) were 66.67–84.62% and 0.481–0.488, with average value at 73.72% and 0.483, respectively. Expected heterozygosity (H_e) and Shannon's information index (I) were 0.279 and 0.434, respectively, at the species level, and 0.233 and 0.356 at population level. According to the coefficient of gene differentiation (G_ST), a large proportion of genetic variance (83.6%) of P. haitanensis was among individuals within populations, only 16.4% genetic variance was among populations, which was identified with the moderate gene flow value (N_m = 2.542). UPGMA clustered the 4 populations into 3 groups, and no significant correlation was found between the genetic distance and the corresponding geographic distance among the populations.     

The genetic diversity and population structure of wild soybean evaluated by chloroplast and nuclear gene sequences

Glycine soja, also called wild soybean, is the wild ancestor of domesticated soybean (Glycine max), and one of the world's major cultivated crops. Wild soybean is a valuable resource for the breeding of cultivated soybean and harbors useful genes or agronomic traits. To use and conserve this valuable resource, we conducted a study to evaluate the genetic diversity and population structure of wild soybean using the sequencing data of two nuclear loci (AF105221 and PhyB) and one chloroplast locus (trnQ-rps16) of more than 600 individuals representing 53 populations throughout the natural distribution range. The results showed that most of the variation was found within the populations and groups, but significant genetic differentiation was also detected among different eco-geographical groups. Correlations between genetic and geographical distance at all the loci were consistent with the isolation by distance gene flow model. G. soja exhibited the highest genetic diversity in middle and downstream of Yangzi River (MDYR) region, followed by North East China (NEC), and was the lowest in North West China (NWC). We concluded that both in situ and ex situ conservation strategies required for wild soybean populations, especially which are native to MDYR and NEC regions.     

Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

Molecular characterization of Lathyrus species using chloroplast DNA trnH-psbA

Lathyrus L. is an important genus contributing in human food, animal feed and fodder. The genetic variation is studied among and within six species sampled over a large geographical area: Lathyrus cicera, Lathyrus sativus, Lathyrus sylvestris, Lathyrus tuberosus, Lathyrus ochrus and Lathyrus aphaca. The phylogenetic relationship among these species was assessed using sequences of chloroplast DNA trnH-psbA (intergenic spacer). The highly polymorphic spacer' length was 330 bp. The phylogenetic analyses using Maximum Parsimony and Genetic Distances, agreed with the universal taxonomy of Kupicha. L. sativus and L. cicera could be considered as sister species, sharing a common ancestor.     

Evolutionary and demographic history among Maghrebian Medicago species (Fabaceae) based on the nucleotide sequences of the chloroplast DNA barcode trnH-psbA

Data from trnH-psbA intergenic spacer (cpDNA) were analyzed to elucidate molecular evolution within and among Maghrebian species of Medicago. The spacer highlighted a high interspecific variation and a low intraspecific diversity among species. Haplotype and nucleotide diversities revealed high level of variation. Parsimony and median-joining Network methods revealed (1) the segregation into 17 haplotypes; (2) the ancestral behaviour of the annual Medicago minima and (3) the clusters are independent of the geographic origin. The neutral evolution of Wright and Fisher is rejected since the Tajima's D values deviated from 0. Besides, the statistical analyses are in agreement with an evolution into stable populations' size.     

The silkworm glutathione S-transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development

Insect molting and metamorphosis are tightly controlled by ecdysteroids, which are important steroid hormones that are synthesized from dietary sterols in the prothoracic gland. One of the ecdysteroidogenic genes in the fruit fly Drosophila melanogaster is noppera-bo (nobo), also known as GSTe14, which encodes a member of the epsilon class of glutathione S-transferases. In D. melanogaster, nobo plays a crucial role in utilizing cholesterol via regulating its transport and/or metabolism in the prothoracic gland. However, it is still not known whether the orthologs of nobo from other insects are also involved in ecdysteroid biosynthesis via cholesterol transport and/or metabolism in the prothoracic gland. Here we report genetic evidence showing that the silkworm Bombyx mori ortholog of nobo (nobo-Bm; GSTe7) is essential for silkworm development. nobo-Bm is predominantly expressed in the prothoracic gland. To assess the functional importance of nobo-Bm, we generated a B. mori genetic mutant of nobo-Bm using TALEN-mediated genome editing. We show that loss of nobo-Bm function causes larval arrest and a glossy cuticle phenotype, which are rescued by the application of 20-hydroxyecdysone. Moreover, the prothoracic gland cells isolated from the nobo-Bm mutant exhibit an abnormal accumulation of 7-dehydrocholesterol, a cholesterol metabolite. These results suggest that the nobo family of glutathione S-transferases is essential for development and for the regulation of sterol utilization in the prothoracic gland in not only the Diptera but also the Lepidoptera. On the other hand, loss of nobo function mutants of D. melanogaster and B. mori abnormally accumulates different sterols, implying that the sterol utilization in the PG is somewhat different between these two insect species.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect's genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the “Transposon Model” of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the “Life Cycle Model” that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.     

The role of miR-2∼13∼71 cluster in resistance to deltamethrin in Culex pipiens pallens

Excessive and continuous application of deltamethrin has resulted in the development of deltamethrin resistance among mosquitoes, which becomes a major obstacle for mosquito control. In a previous study, differentially expressed miRNAs between deltamethrin-susceptible (DS) strain and deltamethrin-resistant (DR) strain using illumina sequencing in Culex pipiens pallens were identified. In this study, we applied RNAi and the Centers for Disease Control and Prevention (CDC) bottle bioassay to investigate the relationship between miR-2∼13∼71 cluster (miR-2, miR-13 and miR-71) and deltamethrin resistance. We used quantitative real-time PCR (qRT-PCR) to measure expression levels of miR-2∼13∼71 clusters. MiR-2∼13∼71 cluster was down regulated in adult female mosquitoes from the DR strain and played important roles in deltamethrin resistance through regulating target genes, CYP9J35 and CYP325BG3. Knocking down CYP9J35 and CYP325BG3 resulted in decreased mortality of DR mosquitoes. This study provides the first evidence that miRNA clusters are associated with deltamethrin resistance in mosquitoes. Moreover, we investigated the regulatory networks formed between miR-2∼13∼71 cluster and its target genes, which provide a better understanding of the mechanism involved in deltamethrin resistance.