Haltere morphology and campaniform sensilla arrangement across Diptera

One of the primary specializations of true flies (order Diptera) is the modification of the hind wings into club-shaped halteres. Halteres are complex mechanosensory structures that provide sensory feedback essential for stable flight control via an array of campaniform sensilla at the haltere base. The morphology of these sensilla has previously been described in a small number of dipteran species, but little is known about how they vary across fly taxa. Using a synoptic set of specimens representing 42 families from all of the major infraorders of Diptera, we used scanning electron microscopy to map the gross and fine structures of halteres, including sensillum shape and arrangement. We found that several features of haltere morphology correspond with dipteran phylogeny: Schizophora generally have smaller halteres with stereotyped and highly organized sensilla compared to nematoceran flies. We also found a previously undocumented high variation of haltere sensillum shape in nematoceran dipterans, as well as the absence of a dorsal sensillum field in multiple families. Overall, variation in haltere sensillar morphology across the dipteran phylogeny provides insight into the evolution of a highly specialized proprioceptive organ and a basis for future studies on haltere sensory function.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Digestive proteolysis in the Colorado potato beetle,Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.     

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.     

Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae)

The acrosome complex plays an indispensable role in the normal function of mature spermatozoa. However, the dynamic process of acrosome complex formation in insect remains poorly understood. Gampsocleis gratiosa Brunner von Wattenwyl possesses the typical characteristic of insect sperms, which is tractable in terms of size, and therefore was selected for the acrosome formation study in this report. The results show that acrosome formation can be divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase, based on the morphological dynamics of acrosome complex and nucleus. In addition, the cytoskeleton plays a critical role in the process of acrosome formation. The results from this study indicate that: (1) glycoprotein is the major component of the acrosome proper; (2) the microfilament is one element of the acrosome complex, and may mediate the morphologic change of the acrosome complex; (3) the microtubules might also shape the nucleus and acrosome complex during the acrosome formation.     

Complete mitochondrial genome of Pycnonotus xanthorrhous (Passeriformes,Pycnonotidae) and phylogenetic consideration

The complete mitochondrial genome (mitogenome) of Pycnonotus xanthorrhous was sequenced via next generation sequencing. The full length of the circular genome is 16,952 bp. It consists of 37 typical animal mitochondrial genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) and 2 ribosomal RNA (rRNA) genes. P. xanthorrhous also contains one control region (CR) and one pseudo-control region, and shares the identical gene arrangements with sequenced Pycnonotus spp. which differs from the typical vertebrates gene order. Phylogenetic analyses indicates that Passerida sensu stricto contains three major clades and the core Sylvioidea form a monophyletic group. Furthermore, we investigated the evolution of control region within this lineage and revealed the multiple independent origins of duplicate control region.     

Fat accumulation in differentiated brown adipocytes is linked with expression of Hox genes

Homeobox (Hox) genes are involved in body plan of embryo along the anterior–posterior axis. Presence of several Hox genes in white adipose tissue (WAT) and brown adipose tissue (BAT) is indicative of involvement of Hox genes in adipogenesis. We propose that differentiation inducing agents viz. isobutyl-methyl-xanthine (IBMX), indomethacin, dexamethasone (DEX), triiodothyronine (T3) and insulin may regulate differentiation in brown adipose tissue through Hox genes. In vitro culture of brown fat stromalvascular fraction (SVF) in presence or absence of differentiation inducing agents was used for establishing relationship between fat accumulation in differentiated adipocytes and expression of Hox genes. Relative expression of Pref1, UCP1 and Hox genes was determined in different stages of adipogenesis. Presence or absence of IBMX, indomethacin and DEX during differentiation of proliferated pre-adipocytes resulted in marked differences in expression of Hox genes and lipid accumulation. In presence of these inducing agents, lipid accumulation as well as expression of HoxA1, HoxA5, HoxC4 & HoxC8 markedly enhanced. Irrespective of presence or absence of T3, insulin down regulates HoxA10. T3 results in over expression of HoxA5, HoxC4 and HoxC8 genes, whereas insulin up regulates expression of only HoxC8. Findings suggest that accumulation of fat in differentiated adipocytes is linked with expression of Hox genes.     

Structural,phenotypic and functional maturation of bone marrow dendritic cells (BMDCs) induced by Chitosan (CTS)

The objective of the present work was to explore the effect of CTS on structural, phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDCs). The maturity of BMDCs post treatment with CTS was evaluated using transmission electron microscopy (TEM) for structure changes, flow cytometry (FCM) for changes of key surface molecules, FITC-dextran bio-assay for phagocytosis, test of acid phosphatase activity (ACP) for biochemical changes and enzyme linked immunosorbent assay (ELISA) for cytokine level. We found that CTS downregulated the numbers of phagosomes inside the BMDCs, up-regulated the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs, decreased activity of ACP and phagocytosis by BMDCs, and induced production of higher levels of IL-12 and TNF-α. It was therefore confirmed that CTS could effectively promote the maturation of BMDCs. Our study provided more detailed evidence and rationale to support the application of CTS as an immune stimulator for enhancing host immunity and as an adjuvant in the design of DC-based vaccines.     

Metabolic reorganization in winter: Regulation of pyruvate dehydrogenase (PDH) during long-term freezing and anoxia

Wood frogs, Rana sylvatica, can undergo prolonged periods of whole body freezing during winter, locking as much as 65–70% of total body water into extracellular ice and imposing both anoxia and dehydration on their cells. Metabolic rate depression (MRD) is an adaptation used by R. sylvatica to survive these environmental stresses, where a finite amount of ATP generated through anaerobic metabolism is directed towards maintaining pro-survival functions, while most ATP-expensive cellular processes are temporarily reduced in function. Pyruvate dehydrogenase (PDH) is a vital metabolic enzyme that links anaerobic glycolysis to the aerobic TCA cycle and is an important regulatory site in MRD. PDH enzymatic activity is regulated via reversible protein phosphorylation in response to energetic demands of cells. This study explored the posttranslational regulation of PDH at three serine sites (S232, S293, S300) on the catalytic E1α subunit along with protein expression of four pyruvate dehydrogenase kinases (PDHK1-4) in response to 24 h Freezing, 8 h Thaw, 24 h Anoxia, and 4 h Recovery in the liver and skeletal muscle of R. sylvatica using Luminex multiplex technology and western immunoblotting. Overall, inhibitory regulation of PDH was evident during 24 h Freezing and 24 h Anoxia, which could indicate a notable reduction in glycoytic flux and carbon entry into the tricarboxylic acid cycle as part of MRD. Furthermore, the expression of PDHK1-4 and phosphorylation of PDH at S232, S293, and S300 were highly tissue and stress-specific, indicative of how different tissues respond differently to stress within the same organism.     

Herbivory-induced glucose transporter gene expression in the brown planthopper,Nilaparvata lugens

Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.     

Subcellular localization of the fatty acyl reductase involved in pheromone biosynthesis in the tobacco budworm,Heliothis virescens (Noctuidae: Lepidoptera)

Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.     

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.     

Insulin-like growth factor (IGF)-like peptide and 20-hydroxyecdysone regulate the growth and development of the male genital disk through different mechanisms in the silkmoth,Bombyx mori

It is well established that ecdysteroids play pivotal roles in the regulation of insect molting and metamorphosis. However, the mechanisms by which ecdysteroids regulate the growth and development of adult organs after pupation are poorly understood. Recently, we have identified insulin-like growth factor (IGF)-like peptides (IGFLPs), which are secreted after pupation under the control of 20-hydroxyecdysone (20E). In the silkmoth, Bombyx mori, massive amounts of Bombyx-IGFLP (BIGFLP) are present in the hemolymph during pupal-adult development, suggesting its importance in the regulation of adult tissue growth. Thus, we hypothesized that the growth and development of adult tissues including imaginal disks are regulated by the combined effects of BIGFLP and 20E. In this study, we investigated the growth-promoting effects of BIGFLP and 20E using the male genital disks of B. mori cultured ex vivo, and further analyzed the cell signaling pathways mediating hormone actions. We demonstrate that 20E induces the elongation of genital disks, that both hormones stimulate protein synthesis in an additive manner, and that BIGFLP and 20E exert their effects through the insulin/IGF signaling pathway and mitogen-activated protein kinase pathway, respectively. These results show that the growth and development of the genital disk are coordinately regulated by both BIGFLP and 20E.     

Ontogenetic trajectory and allometry of Diplonychus rusticus (Fabricius), an Oriental aquatic bug (Hemiptera: Belostomatidae) from the Western Ghats of India

Despite being one of the dominant groups in freshwater ecosystems, morphological and ontogenetic studies on aquatic Hemiptera have received little attention in the Oriental region. We present the ontogenetic trajectory and allometry of the widespread Oriental belostomatid species, Diplonychus rusticus (Fabricius) for the first time. We have measured nine different morphological variables throughout the growth of the bug using both field captured and laboratory reared specimens. Our results suggest that the developmental instars can be distinguished by the size variables, as seen in the Principal Component Analysis. On the basis of a CHAID (Chi-squared Automatic Interaction Detection) based regression tree, we also show that the characters – total length without head and maximum width – prove to be adequate for effective instar identification. The multivariate allometric growth pattern shows that different body parts exhibit different types of allometry. This is apparent in the allometry exhibited by forelegs and mid and hind legs, which show allometry of opposite polarities. This may be due to the different functions attributed to these body parts. Our results show that the growth pattern in D. rusticus is comparable with the New World genus Belostoma, suggesting a conserved growth pattern in the family Belostomatidae.     

Comparison of photosynthesis and leaf ultrastructure on two black locust (Robinia pseudoacacia L.)

Polyploidy is common in many plant species. Up to date, few studies were reported on photosynthesis and leaf anatomy of tetraploid black locust (Robinia pseudoacacia L.), which has considerable value for agriculture and forest. This study compared photosynthesis and leaf ultrastructure on two black locusts. The values of P_n and C_i in tetraploid were significantly higher than those in the corresponding diploids. Significantly lower stomata dimensions (6.0 μm in length and 2.4 μm in width) and tomatal density were observed in 4×. Leaf trichome density was statistically different between 2× and 4×. However, no substantial difference in the ultrastructure of chloroplasts and mitochondria between diploid and tetraploid was observed under any stress. These ultrastructural characteristics may contribute to tetraploid a better protection mechanism than diploid.     

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d₃₁-hexadecanoic acid (16:0), d₂₇-octadecadienoic acid (18:2), produced from d₃₁-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d₂₇-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.     

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation.Fish were fed 4 g kg^?1 MOS (Bio-Mos^® Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents.Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet.The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.