text:
- Research article
- Open Access
Antimicrobial peptide-like genes in Nasonia vitripennis: a genomic perspective
© Tian et al; licensee BioMed Central Ltd. 2010
- Received: 25 August 2009
- Accepted: 19 March 2010
- Published: 19 March 2010
Antimicrobial peptides (AMPs) are an essential component of innate immunity which can rapidly respond to diverse microbial pathogens. Insects, as a rich source of AMPs, attract great attention of scientists in both understanding of the basic biology of the immune system and searching molecular templates for anti-infective drug design. Despite a large number of AMPs have been identified from different insect species, little information in terms of these peptides is available from parasitic insects.
By using integrated computational approaches to systemically mining the Hymenopteran parasitic wasp Nasonia vitripennis genome, we establish the first AMP repertoire whose members exhibit extensive sequence and structural diversity and can be distinguished into multiple molecular types, including insect and fungal defensin-like peptides (DLPs) with the cysteine-stabilized α-helical and β-sheet (CSαβ) fold; Pro- or Gly-rich abaecins and hymenoptaecins; horseshoe crab tachystatin-type AMPs with the inhibitor cystine knot (ICK) fold; and a linear α-helical peptide. Inducible expression pattern of seven N. vitripennis AMP genes were verified, and two representative peptides were synthesized and functionally identified to be antibacterial. In comparison with Apis mellifera (Hymenoptera) and several non-Hymenopteran model insects, N. vitripennis has evolved a complex antimicrobial immune system with more genes and larger protein precursors. Three classical strategies that are likely responsible for the complexity increase have been recognized: 1) Gene duplication; 2) Exon duplication; and 3) Exon-shuffling.
The present study established the N. vitripennis peptidome associated with antimicrobial immunity by using a combined computational and experimental strategy. As the first AMP repertoire of a parasitic wasp, our results offer a basic platform for further studying the immunological and evolutionary significances of these newly discovered AMP-like genes in this class of insects.
- Disulfide Bridge
- Mature Peptide
- Parasitic Wasp
- Intron Loss
- Insect Defensin
AMPs constitute essential components of innate immunity to rapidly respond to diverse microbial pathogens [1, 2]. As key effectors, AMPs directly kill invaders by acting either as pore-formers or metabolic inhibitors [3]. Most AMPs are cationic polypeptides, usually smaller than 100 amino acids in length, with enormous sequence diversity. Based on structural characteristics, these molecules are roughly divided into three different groups [4]: 1) Linear peptides free of cysteines, often forming an α-helical conformation with amphiphilic surface, such as insect cecropins and mellitins, amphibian magainins and arachnidian meucins [5, 6]; 2) Cysteine-rich peptides. The most representative of this group are insect-derived defensins, which belong to the CSαβ polypeptide superfamily and are conserved across the kingdoms of eukaryotes [7, 8], even in bacteria [9, 10]; 3) Peptides with unusual bias in certain amino acids, such as glycine-rich tenecin3 [11], proline-rich apidaecin [12], histidine-rich histatin [13] and tryptophan-rich indolicidin [14]. AMPs in this group are unstructured and often enter into bacterial cells to inhibit different metabolic targets [3, 15].
Insects, as a rich source of AMPs, attract great attention of scientists in both understanding the basic biology of immune system and searching molecular templates for anti-infective drug design. So far, about 200 such peptides have been identified from insects. In Drosophila melanogaster, there are 20 AMPs characterized [16]. In the past ten years, a number of genome sequencing projects have promoted application of computational approaches for the discovery of components involved in innate immunity of a given species. Some examples include D. melanogaster, Anopheles gambiae, A. mellifera, Tribolium castaneum and Bombyx mori[17–21]. This will undoubtedly extend our knowledge in basic biology of insect innate immunity system. Furthermore, these data also provide new clues for elucidation of immunological adaptation of insects to environmental changes. For example, analysis of the complete immune system of A. gambiae results in the discovery of a marked deficit of orthologues due to gene loss and excessive expansion of some specific genes, which is significantly different from that of D. melanogaster. This possibly reflects differential selective pressures from different pathogens between these two species. These results also facilitate further in-depth analysis of the mosquito immune system's impact on the malaria parasite [17, 22].
Parasitoids (Hymenoptera, Insecta) are a group of parasitic insects. Some of them attach significant vectors of human disease, such as house flies, roaches and ticks, and some of them are extremely important regulators of agricultural pests [23]. To study molecular mechanism of the innate immunity of parasitic wasps, the basic biology of their AMPs is needed to be established. As the first parasitic hymenopteran insect to have its genome sequenced [24], N. vitripennis provides a new resource for the identification of AMP genes in this class of insects in a genomic scale [25, 26]. Here, we report systemic characterization of the AMP repertoire from the N. vitripennis genome, which provide a perspective for the understanding of a possible relationship between immunity and parasitism. In comparison with A. mellifera and several non-Hymenopteran insects, N. vitripennis has developed a more complex antimicrobial immune system through genetic duplication and exon-shuffling.
Computational identification of AMPs in the N. Vitripennis genome
To identify N. vitripennis AMP repertoire, we employed three complementary approaches to database search of the N. vitripennis genome (see additional file 1). Firstly, we chose A. mellifera AMPs (e.g. mellitin, apidaecin, apisimin, abaecin, hymenoptaecin, defensins) as queries to recognize their orthologues in N. vitripennis by BLASTP and TBLASTN. Secondly, we performed pattern search by the Scanprosite program based on the cysteine arrangement pattern of CSαβ-type defensins [8]. This strategy allowed identifying a large family of DLPs with a typical CXXXC and CXC motif and an amino-terminal signal peptide, which can be divided into three distinct subfamilies, including the known navidefensins [25] and two new subfamilies named nasonins and navitricins, respectively. Thirdly, we screened putative secreted peptides of < 150 amino acids from the N. vitripennis proteome by recognizing an amino-terminal signal sequence, from which AMP-like peptides rich in specific amino acids were identified. All AMPs found by the above approaches were again used as query to carry out BLASTP and TBLASTN iteratively until no new hits appeared. As a result, we identified a total of 44 AMPs in the genome of N. vitripennis, as shown in additional file 2, in which only nahymenoptaecin-1 and five defensins are recently reported [25, 26].
All these newly discovered peptides display remarkable AMP characteristics, as reflected by their small size and net positive charges at pH 7.0. Most peptides described here are smaller than 150 amino acids in length, except for nahymenoptaecin-1, nahymenoptaecin-2, nasonin-2 and nasonin-6, which all have undergone internal duplication (see additional file 2). N. vitripennis AMPs exhibit extensive sequence and structural diversity and can be distinguished into multiple molecular types including insect and fungal DLPs with the CSαβ fold; Pro- or Gly-rich abaecins and hymenoptaecins; horseshoe crab tachystatin-type AMPs with the ICK fold; and a linear α-helical peptide.
To confirm the reliability of our computational prediction, we undertook the N. vitripennis EST database search and found more than 60% peptides have corresponding transcripts, suggesting the genes encoding these peptides are expressed at the transcriptional level. For remaining 40% peptides whose cDNA sequences were not found in the EST database, one possibility is that their expression depends upon suitable microbial challenges [27] or is associated with different developmental stages of N. vitripennis, as observed in the D. melanogaster antifungal drosomycin [28], although pseudogene or wrong prediction resulted from computational methods cannot be completely excluded.
Classification of N. Vitripennis AMPs
Defensin-like peptides
Classical insect-type defensins (CITDs)
Defensins with the CSαβ structure are crucial effectors of innate immunity [7] which have been found in many insect species, the majority of them coming from different orders of the subclass Neoptera (Diptera, Coleoptera, Lepidopera, Hemiptera and Hymenoptera). These AMPs are primarily active against Gram-positive bacteria likely by forming voltage-dependent channels in bacterial membrane leading to a loss of cytoplasmic potassium [29, 30]. Their protective roles have been well documented by in vivo targeted disruption of defensin gene which resulted in the death of A. gambiae after Gram-positive bacterial infection [31]. Insect defensins consist of 30-50 amino acids linked by three or four disulfide bridges. They share a common CSαβ structural motif with some functionally related scorpion neurotoxins targeting various ion channels, protease inhibitors, and even a plant sweet taste peptide [8, 32].
(2) Nasonins
In addition to the defensins mentioned above, we identified a new DLP subfamily without a propeptide, which contains fourteen members (named nasonin-1 to -14) (Figure 1). Sequence analysis revealed that nasonins are more closely related to two known non-classical defensins than CITDs in precursor organization, n-loop size and sequence similarity, one being antifungal termicin isolated from termites [39, 40]; another being defensin1 from the Mexican scorpion Centruroides limpidus limpidus[41]. Intriguingly, such similarity was also observed between nasonins and a scorpion K+ channel toxin cobatoxin [42, 43] (Figure 1). These observations suggest that nasonins could have diverse functional features. For nasonin-2 and nasonin-6, their defensin unit has undergone 4 to 5 internal repeats, as described in fungal DLPs [32]. What's the potential function of such repeats and how these repeats occur will be further discussed below.
We also predicted the structure of nasonin-1 by comparative modeling using scorpion neurotoxin cobatoxin (PDB: 1PJV) [43] as a template. Although having a shorter n-loop than CITDs, nasonins can also adopt a typical CSαβ architecture (Figure 2B). The reliability of this model was evaluated by Verify3D with a score of 0.248. Overall, nasonin-1 more resembles structurally termicin (PDB: 1MM0) [44] and cobatoxin (Figure 2B), but they differ in molecular surface charge distribution (data not shown), which could explain functional diversity of these peptides, as observed between human beta-defensin2 and the snake toxin crotamine [45].
Navidefensin3 is a unique non-classical defensin because it has identical precursor organization and gene structure to navidefensin1 and 2 but similar n-loop size and amino acid sequence to nasonins. In fact, several non-classical insect defensins with a short n-loop were also found in Lepidoptera insects (e.g. gallerimycin, spodoptericin and the B. mori defensins) [46–48]. In the tree presented in Figure 2A, navidefensin3 is clustered together with nasonins rather than navidefensins, suggesting that this DLP is an evolutionary link between these two multigene subfamilies of defensins. Its lack in A. mellifera is consistent with the absence of the nasonin subfamily in this species.
(3) Navitricins
As mentioned previously, navitricins have two additional cysteines which could form a fourth disulfide bridge. To confirm this, comparative modeling was again used to build the structure of navitricin-1 by choosing different templates, e.g. CITDs (sapecin and phormicin), the scorpion toxin BmTX1, and the plant sweet taste protein brazzein. In all these cases no acceptable models were generated and we thus tried to use the I-TASSER algorithm for structure simulation which produced a suitable model, as evaluated by Verify 3D (Figure 3B). Overall, this molecule is similar to the defensins described above including three identical disulfide bridges. Importantly, this model correctly predicts the fourth disulfide bridge linking the n- and c-loops together which makes the whole structure more compact. In fact, computational prediction was also applied to reveal an additional disulfide bridge in several insulin-like proteins [49].
(4) Biological activity
Subsequently, we evaluated antibacterial and antifungal activities of nasonin-1 using classical inhibition zone assays [52] and found that nasonin-1 only displayed activity against two Gram-negative bacteria at micromolar concentrations with a lethal concentrations (CL) of 1.52 μM for Stenotrophomonus sp. LZ-1 and of 15.4 μM for Escherichia coli ATCC 25922 (Figure 4C). No effect was observed on Gram-positive bacteria and fungi. This is the first example describing a DLP mainly active on Gram-negative bacteria. Recombinant navidefensin2-2 was recently reported to be active on Gram-positive bacteria [25]. Whether such remarkable difference in target selectivity is associated with the change of n-loop length in these two Nasonia defensins needs further investigation.
AMPs rich in specific amino acids
AMPs rich in specific amino acids (e.g. glycine, proline, histidine, tryptophan or arginine) represent an additional class of AMPs acting on diverse microorganisms (e.g. bacteria, fungi, and virus) [53, 54]. The majority of these molecules are unstructured and they often inhibit microbial growth by entering into cells and interacting with proteins involved in key metabolic processes [15]. Eighteen such AMPs have been identified here, including two known AMP families (abaecin and hymenoptaecin) [55, 56].
(1) Abaecin
(2) Hymenoptaecin
Nahymenoptaecin-2 presents a more interesting structural feature in the mature peptide region, whose amino- and carboxyl-termini are respectively a Pro-rich peptide (named pronavicin) and a hymenoptaecin-like peptide with four internal repeats. Overall, these repeats can be well aligned with hymenoptaecin except a deletion of 31 amino acids in each repeat. Similarly, a conserved phase 0 intron is also present in the amino-termini of bee and parasitoid hymenoptaecins, suggesting that these peptides diverged from a common ancestor after speciation.
Although hymenoptaecins are believed to be only present in Hymenoptera, our study reveals that similar peptides were also evolved by Dipteran Drosophila (Figure 6A). These fly-derived peptides display a chimeric characteristic because they have an acidic propeptides, as bee hymenoptaecins, but their mature peptides more resemble the repeat of nahymenoptaecin-2 in size. Moreover, Drosophila hymenoptaecins have lost their introns, consistent with the lineage-specific intron loss in these species [61]. All Drosophila hymenoptaecins clustering together in the tree (Figure 6B) indicates their monophyletic origin after separation of Diptera from Hymenoptera.
Antibacterial activity of pronavicin.
Lethal conc. (CL) (μM)
B. megaterium
3.11 (y = 1.0867x + 1.019, R2 = 0.998)
M. luteus
42.4 (y = 0.65x + 0.0175, R2 = 0.9956)
Bacillus sp.
19.5 (y = 0.7125x - 0.2313, R2 = 0.995)
S. typhimurium
15.8 (y = 0.3458x + 0.2525, R2 = 0.9993)
E. coli
61.8 (y = 0.2375x + 0.0712, R2 = 0.9774)
Stenotrophomonus sp. YC-1
78.0 (y = 0.3x + 0.0292, R2 = 0.9959)
Stenotrophomonus sp. LZ-1
12.0 (y = 0.3625x + 0.3, R2 = 0.9542)*
Tachystatin-type AMPs
Linear α-helical peptides
Linear peptides with an α-helical conformation are a large class of AMPs involved in immune response of arthropods, amphibians and vertebrates. Since cecropin, the first insect-derived linear AMP, was isolated from Cecropia moth [71], the number of such peptides dramatically increases in recent decades [72, 73]. Several linear α-helical AMPs have also been characterized in social wasps [74–76]. From the N. vitripennis genome, we identified one AMP-like peptide (called nahelixin) belonging to this class (Figure 7C). The precursor of nahelixin is 118 residues in length with an amino-terminal signal peptide and a putative carboxyl-terminal propeptide. The predicted mature peptide of 22 amino acids shares about 40% sequence identity with four known antibacterial peptides from frogs [77]. Secondary structure prediction indicated it could adopt an α-helical structure (data not shown), which was further confirmed by ab initio structural prediction. An amphiphilic surface, with hydrophilic and hydrophobic residues separately arranged at two directions of the helical axis, is present in the model, as seen at the helical wheel projection (Figure 7C). Such structural feature represents a prerequisite for antibacterial activity of linear AMPs [6].
Inducible expression profiles of N. Vitripennis AMP-like genes
AMPs in the parasitic wasp
With more and more genome sequences released, identifying new bioactive peptides/proteins by computational genomics approaches is becoming quite valuable. Some examples include hormones from nematode and mosquito, odorant binding-like proteins from honey bee, opossum immune genome, and reptile venom genes [78–82]. However, most strategies used are primarily based on sequence similarity that is useful in finding orthologues of known genes. An additional computational search strategy depends on the presence of conserved structural motifs within a peptide superfamily. Such examples include the identification of fungal DLPs and mammalian β-defensins [32, 83]. Here, we identified the N. vitripennis AMPs in a genomic scale by using an integrated strategy which combines similarity search, pattern recognition and AMP characteristics (see additional file 1).
In comparison with several non-Hymenopteran insects whose genome sequences have been released (e.g. D. melanogaster and A. gambiae from Diptera, B. mori from Lepidoptera, and T. castaneum from Coleoptera) [17–21], N. vitripennis still have evolved more AMP genes both in kinds and numbers (Figure 9A). Based on the phylogenetic relationship of holometabolous insects [85], we elucidate several evolutionary events (e.g. gene expansion, terminal extension and tandem repeat) which might have occurred along the parasitoid branch (Figure 9B) to result in the lineage-specific complexity increase in N. vitripennis AMPs for synergetic defense in parasitic condition [86].
Evolution of the N. Vitripennis AMPs
Three different strategies have been recognized to shape the complex antimicrobial immune system in N. vitripennis:
Gene duplication
Maximum likelihood estimates of parameters and sites inferred to be under positive selection in the amino-terminal abaecin unit.
Positive selected sites
ω = 0.15
p0 = 0.80, ω0 = 0.046
p1 = 0.20, ω1 = 1.00
p0 = 0.80, ω0 = 0.05
p1 = 0.00, ω1 = 0.05315
p2 = 0.20, ω 2 = 5.91067
p = 0.48 q = 1.62
p0 = 0.80, p = 1.06, q = 14.00
(p1 = 0.20), ω = 6.69
Exon duplication
By using a combined computational and experimental strategy, we established for the first time the N. vitripennis peptidome associated with innate immunity. Three basic evolutionary scenarios are recognized in the generation of a complex antimicrobial system in N. vitripennis. Our work presented here will offer a basic platform for further studying the immunological and evolutionary significances of these newly discovered AMPs in parasitic insects. Whether these AMP-like genes with a complicate structure or much more copies are involved in parasitism remains an open question which constitutes our next research direction. In particular, our efficient approaches for finding new AMPs in a genomic scale can easily be applied to other model organisms including humans, which will accelerate our understandings on AMP-mediated innate immunity at a defense network level. Further functional characterization of these parasitic wasp-derived AMPs will also help explore new-type of drug leads for anti-infective therapy.
Database searches
Strategies for gene discovery used here are provided in the supplementary information (see additional file 1). BLASTP and TBLASTN programs were used to characterize orthologues of known AMPs from A. mellifera against the database of N. vitripennis[93]. When searching for hymenoptaecin in flies, BLAST was carried out in Flybase [94]. The program WinGene [95] was employed to predict and translate a complete open reading frame from a selected nucleotide sequence. The N. vitripennis protein sequences downloaded from GenBank [96] and the FTP site [97] were applied to perform ScanProsite on the ExPaSy sever [98].
Characteristics identification
All sequences we identified as AMP-like peptides were submitted to SignalP 3.0 server [99] for signal peptide prediction. Peptide characteristics identified here were further analyzed on the ExPaSy server [100] for estimation of molecular weights and calculation of net charges at Protein Calculator [101]. Secondary structure prediction was done on the ExPaSy server [100]. To predict amphiphilic structure of peptides, helical wheels models were calculated on the server [102].
Sequence alignment and structural analysis
Amino acid sequences were aligned using Clustal X [103] with fine adjustment by hands and phylogenetic trees were constructed by MEGA 4.0 [104]. Structures were modeled from ab initio by I-TASSER on-line [105–107] or on the Structural Bioinformatics server by comparative modeling method [108]. Structure evaluation was carried out by Verify 3D [109, 110]. The MultiProt server was used to do structural superimposition and calculation of root mean square deviation (RMSD) [111]. All model structures predicted here have been despoited in Protein Model DataBase [112] under id number of PM0075686-PM0075709.
Maximum likelihood analysis
Codon-substitution models were selected to estimate the nonsynonymous-to-synonymous rate ratio (ω = dN/dS) using the CODEML program of the PAML software package [113]. Four models recommended by Yang make two likelihood ratio tests (LRTs) by M1a/M2a and M7/M8: M1a (nearly neutral model) constraints a proportion p0 of conserved sites with 0 < ω < 1, while a proportion p1 = 1-p0 of neutral sites with ω1 = 1; M2a (positive selection model) adds an extra class of sites with the proportion p2 = 1-p0 -p1 and with ω estimated from the data. M7 (β distribution model) does not allow for positively selected sites and M8(β and ω model) adds an extra class of sites to M7, allowing for ω > 1, which means the presence of positively selected sites. In addition, Model 0 (M0) assuming one ω for all sites that does not allow the existence of positive selection was chosen for negative control [114, 115]. Upon detection of the positively selected signals, the calculation of posterior probabilities was completed using the Bayes Empirical Bayes (BEB) method [115, 116].
Chemical synthesis and oxidative refolding
Pronavicin and reduced nasonin-1 were chemically synthesized by Xi'an Huachen Bio-Technology Co., Ltd. (Xi'an, China). The cyclization reaction to form disulfide bridges in the reduced nasonin-1 molecule was carried out in 0.1 M Tris-HCl (pH = 8.0) with 2 mM GSH and 0.2 mM GSSG. Refolded peptide was purified to be homogeneity by C18-RP-HPLC. Its MW was determined by MALDI-TOF MS on a Kratos PC Axima CFR plus (Shimazu Co. LTD, Kyoto).
Antimicrobial assays
Inhibition zone assays were carried out to evaluate antimicrobial activities of peptides [28, 52, 117]. Briefly, 50 μl bacteria or fungal spores with OD600 = 0.5 were inoculated to pre-heated 6 ml Luria-Bertaini's medium (1% bactotryptone, 0.5% bactoyeast extract and 0.5% NaCl) for bacteria or MEA medium (1% Malt Extract, 0.1% Peptone, 2% Glucose) for fungi containing 0.8% agar. The mixture was then spread on a 9-cm Petri dish, giving a depth of 1 mm. After settling, 2-mm wells were punched in the plate and then 2 μl of peptide under different concentrations was added to each well. Diameters of the inhibition zones were measured 16 hours after incubation at 30°C. C L was calculated by the Hultmark's method [52]. The microorganisms used in this assay were listed in additional file 3.
Establishment of the infection model
N. vitripennis was maintained as a laboratory culture on pupae of the housefly Musca domestica. They were reared under a 14:10 light-dark cycle at 25°C in glass containers fed on 20% (v/v) honey solution. A standard method was applied to establish an infection model [118] of N. vitripennis. Briefly, a bacterial mixture of E. coli ATCC 25922 and M. luteus which had been separately incubated overnight was injected into abdomen of N. vitripennis adults with a micro-injector. About 50 wasps were challenged in total. Non-injected wasps were used as a control.
Preparation of total RNA
For isolating N. vitripennis total RNA, 50 wasps challenged for 16 hours by bacteria were grounded into fine powder in liquid nitrogen. The Trizol reagent (SBS Genetech, Beijing) was used to prepare total RNA according to the supplier's instructions. This method was also applied to prepare non-challenged total RNA [25].
cDNA Cloning
Reverse transcription of total RNAs prepared from non-infected or infected N. vitripennis was performed using RT PreMix kit (TransGen, Beijing) and a universal oligo (dT) - containing adaptor primer dT3AP. The first-strand cDNA amplification in both non-infected and infected wasps was performed by using an AMP-like gene-specific primer and the universal primer 3AP according to the method described [119]. PCR product was cloned into pGEM-T (TIANGEN, Beijing), and transformed into E. coli DH5α competent cells (TransGen, Beijing). Their sequences were confirmed by DNA sequencing. Primers used in this study are all listed in additional file 4.
Semi-quantitative RT-PCR
For semi-quantitative RT-PCR, an AMP-like gene were amplified from non-infected or infected cDNAs by using a gene-specific primer and 3AP and PCR products obtained from different cycles (i.e. 25, 30, 35, 40, 45) were taken for comparison of the amounts of these products by electrophoresis on 1.5% agarose gels. N. vitripennis ribosome protein RP49 was chosen as an internal control which was amplified by using primers NvRp49-F2/3AP and the same cDNA templates.
We thank Human Genome Sequencing Center, Baylor College of Medicine http://www.hgsc.bcm.tmc.edu/ for providing N. vitripennis sequence data for public use and Prof. Chuanling Qiao (Institute of Zoology, Chinese Academy of Sciences, China), Prof. Fengyan Bai (Institute of Microbiology, Chinese Academy of Sciences, China) and Dr. Jianguo Zhou (Oak Ridge National Laboratory, USA) for providing microbial strains used in this study. We also thank Prof. Fengqin He (Institute of Zoology, Chinese Academy of Sciences, China) for providing M. domestica. This work was supported by grants from the National Natural Science Foundation of China (30730015, 30621003 and 90608009) and the 973 Program from the Ministry of Science and Technology of China (2010CB945304).
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Stilwell, Oklahoma Passport Office Information
Residents of Stilwell in Adair County, Oklahoma have only a single location from which they can get a passport application sealed at. The passport agent in Stilwell can provide you with passport forms or witness your signature and seal your passport documents (if you don't need your passport application sealed then you do not need to visit an acceptance agent). Besides the 1 location there are 91 other locations in the state of Oklahoma from which you can get a passport application sealed at. If you are not needing a passport in Stilwell for a least (3) weeks, you can visit the sole passport acceptance agent listed below the map on this web page. If you need it faster than (3) weeks we have provided the closest regional passport offices to Stilwell, Oklahoma and Expedited Passport service options for Stilwell.
Regional Passport Offices Close to Stilwell:These are passport issuing facilities. You will need to make an appointment and present proof of upcoming travel but in most cases, you will be able to get passport renewal, new passports, child passports, or a lost passport replacement Same Day or Next Day.
Arkansas Passport Center - 108.9 Miles, Dallas Passport Agency - 210.7 Miles, Houston Passport Agency - 364.4 Miles, New Orleans Passport Agency - 418.8 Miles, Chicago Passport Agency - 489.6 Miles
Fast Rush Passport Expediting Service in Stilwell, Oklahoma3rd Party Passport Expediting in Stilwell OK: Expedited Passports & Visas are the experts for expediting passports in Stilwell, if you're in a hurry, this is probably going to be your best option. Passport servces offered for Stilwell: Expedited Passport Renewal, Expedited New Passports, Fast Passport Name Changes, Expedited Child Passports, Expedited Lost/Stolen Passport Replacement
How to Get a Passport in Stilwell, OklahomaGetting a passport in Stilwell OK depends on what type of passport application you are needing. The process in these guides assumes that you are applying at a local Stilwell Passport Office in which case it will take you 4 to 6 weeks to get a passport in Stilwell.
Please select the appropriate guide from the list below to get started:
Passport Office in Stilwell, OK
The single passport acceptance agent (local passport office) located in Stilwell is listed below:
- Stilwell Post Office
16 S 2nd St
Stilwell, OK 74960
Other Passport Offices Near Stilwell, Oklahoma:Listed below are additional passport offices that are located within 50 miles of Stilwell Oklahoma:
Cherokee County Court Clerk - Tahlequah - 18.6 Miles
Sequoyah County District Court Clerk - Sallisaw - 21.7 Miles
Van Buren Main Post Office - Van Buren - 22.9 Miles
Farmington Main Post Office - Farmington - 23.3 Miles
Siloam Springs Post Office - Siloam Springs - 23.3 Miles
Passport Service for Cities Near StilwellResidents of the following closeby cities looking to get a passport should consider using the passport office(s) located in Stilwell for document sealing and 3-week passport expediting. Be sure to check other nearby passport offices listed above, as there may be a closer passport office to your city.
Zion Oklahoma, Salem Oklahoma, Westville Oklahoma, Lincoln Arkansas, Prairie Grove Arkansas, West Fork Arkansas, Chester Arkansas, Farmington Arkansas, Winslow Arkansas, Siloam Springs Arkansas, Notiechtown Oklahoma, Fort Smith Arkansas, Alma Arkansas, Gentry Arkansas, Johnson Arkansas, Fort Gibson Oklahoma, Barling Arkansas, Fort Chaffee Arkansas, Mulberry Arkansas, Decatur Arkansas

subset: commoncrawl


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In recent years my work has focused on immigration, women's rights and education, taking me from Afghanistan to Zimbabwe, from India to criss-crossing the Western Balkan migration route. I shared the story of that journey at TEDx Lausanne and at the Power of Storytelling in Romania.
Previously, I worked as a multimedia editor and photographer at the St. Louis Post-Dispatch and as a freelance photojournalist and filmmaker based in Lima, Peru, where I cofounded Everyday Latin America.
While in St. Louis, I also taught courses in multimedia production and photojournalism for Webster University and led National Geographic Student Expeditions to London, Bar Harbor, and Ecuador.
I regularly collaborate with humanitarian organizations to tell character-driven stories, dissecting how war, climate change and poverty affect lives across the globe. I am a proud member of the Community Team at The Everyday Projects where I edit and write regularly for Re-Picture. We document everyday life around the world, using photos to challenge stereotypes and as a tool to drive awareness and connection.
My work has been published by the New York Times, PRI's The World, Al Jazeera, National Geographic Traveler and Der Spiegel, among others. I've also collaborated with the Fuller Project for International Reporting, Chemonics, Pro Mujer, Oxfam and Catholic Relief Services. I graduated with honors from the University of Missouri School of Journalism.


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Nail art enthusiast Dorota Pankowska is behind this clever LEGO-themed manicure. In order to make this manicure extra fun, Pankowska actually cut the tips off of real LEGO pieces and glued them to her nails. This enabled her to stick various LEGO accessories to her freshly manicured nails, making them far more entertaining than the average nail art design.
Nail art allows women to express their moods and personalities through vibrant colors and interesting patterns. This toy-infused nail design tells viewers that Pankowska is extremely imaginative and playful.
This fantastic LEGO-themed manicure allows women to entertain themselves using LEGO attachments, which is guaranteed to attract attention. This manicure is perfect for girls who haven't been able to outgrow their love for building blocks.
Building Block Manicures
More Stats +/-
Bizarre Feminine Ritual Art
24 Carat Gold Manicures
Musician Marketed Manicure Art
Love-Inspired Nail Decals
Prickly Manicure Designs
Dorota Pankowska Created This LEGO-Themed Manicure
- By: Lacey-Jane WalshJan 24, 2014
References: dorithegiant & sweet-station
References: dorithegiant & sweet-station


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The Houston City Council could vote as early as next week to force truckers to pay extra fees to run inside city limits but off the interstate network if their trucks are oversize or overweight.
Officials with the Owner-Operator Independent Drivers Association say the fees are excessive and could work against interstate commerce.
The City Council was scheduled to vote Wednesday, July 18, on whether to adopt the ordinance, but several council members wanted more time to gather information, a city staff member told Land Line.
Council members moved to change the voting day to Wednesday, July 25. The deadline for public comment – Friday, July 20 – had not been changed as of Wednesday afternoon, though city staff said that it could be extended.
Proposed fees start at $145 for a single trip off the network but within the city limits of Houston and go up from there. A one-year package for running oversize is a proposed $2,035 per truck.
By restricting oversize and overweight trucks, Houston city officials in favor of the fees hope to gain revenue to offset road-repair costs.
A draft of the ordinance obtained by Land Line does not provide a timeframe for when the ordinance would take effect other than "when signs are erected."
Sizes, dimensions and weights outlined in the ordinance closely resemble dimensions outlined for interstate transportation in federal regulations.
– By David Tanner, staff writer


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Trump administration denies imposing Russia sanctions against organizations and overseas companies executing business ostracized Russian defense and intelligence set up. The administration was needed by law to shortlist the companies and persons and probably permit them under a 2016 law denoting punishment to Russia for its intrusion in 2016 US election and human right infringement takeover of Crimea and continuing military functions in eastern Ukraine.
A State Department representative said that the administration has manifested to place foreign governments and private sector establishments on notice that the noteworthy undertakings with tabulated Russian entities will result in sanctions. The representative said that punishment on certain entities or individuals will required to be levied because the legislation is performing as a damper.
The declaration was made on the day when the continuous FBI investigation into President Donald Trump's prospective crusade links to Moscow during the 2016 election over again influenced the news and over again pitched questions about policy commitment his administration is making on Russia.
FBI Deputy Director Andrew McCabe, long been a thorn in Trump's eye towards the FBI about its consideration into potential collusion between his campaign and Russia, relinquished in a stupefied move. Trump's confederate have lately strengthen their campaign against the investigation, alleging FBI abuses of a surveillance law.
Rep. Eliot Engel, the ranking Democrat on the House Foreign Affairs Committee, said that his anxiety reaches no bounds. However, they won't function well as seen from past experience.


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About us
The COGRI (Children of God Relief Insititute) Nyumbani Charity currently provides a primary and secondary school for children in the area as well as a state-of-the-art HIV screening laboratory which helps to keep the children free of AIDS.
Club Member Alison Stedman has been involved with Nyumbani for many years and has successfully brought £000's to this very deserving Project.
You can find out more about the Nyumbani charity and it's activities by clicking on this link;
So now to business ....Welcome to the website of the Rotary Club of Arbroath, based in the historic Scottish east coast fishing port 17 miles north of Dundee.
Our fify one members are always delighted to welcome visiting Rotarians and we hope that if you're travelling to or in Scotland, you'll join the Club's Members in fellowship at one of our weekly meetings.
The town is home to the world famous smokie (smoked haddock) and was the setting for the signing of the Declaration of Scottish Independence in the presence of King Robert the Bruce in 1320. The ruins of Arbroath Abbey, where the signing took place, still dominate the town's skyline. The club meets every Thursday in the Meadowbank Inn, just outside Arbroath, at 6.00 for 6.15pm - see Meeting Information to the right of this text!
If you need to make contact with our club, or are interested in joining Rotary and would like to know more about what Arbroath Rotary Club get up to then an email to our secretary should work just fine; email@example.com
Our Chartered Rotaract Club is on Facebook at;
Please check out what our young Rotaractors have been up to!!!
Thank you for visiting our Club's website, I hope you find it interesting.
David Miller
Webmaster, Rotary Club of Arbroath, December 2017
The Rotary Club of Arbroath is a Registered Charity, number SCO37945
Happening soon...
Meetings and events happening soon
Thursday 20th June 18.00
Weekly Meeting
Thursday 27th June 18.00
Weekly Meeting
Ian Ballantyne Outgoing President
Thursday 4th July 18.00
Weekly Meeting
Dale Hatton Incoming President
Thursday 11th July 18.00
Weekly Meeting
Speaker: Ian Hunter
Thursday 18th July 18.00
Weekly Meeting
Speaker- Hannah Robb
Rotary News
Be part of a global network of volunteers, trying to make the world a better place. Please also see the 'links & news' page for information from the local district and live feeds from Rotary International and Rotary International in GB&I.
Family Rotary club use skills to kick-start local charities
Rotary members unite to tackle world hunger
Meetings & Venue
Where and when:
The Meadowbank Inn,
Montrose Road,
Arbroath, DD11 5RA 01241 875755


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8 perfect autumn pub walks in Hertfordshire
PUBLISHED: 18:38 17 October 2018 | UPDATED: 10:49 25 October 2018
Make the most out of autumn with these eight walking routes that take in a variety of Hertfordshire's terrains and - most importantly - have a cosy pub along the way
1. The Lea Valley Circle
This walk starts in the pretty village of Wheathampstead before heading down Rose Lane, an ancient road surrounded by foliage that will be displaying its hues of orange and brown during the autumn months. Continue north and the trail takes a Roman road past lush fields before heading towards Marshalls Heath which is rich in wildlife including butterflies and moths. Cross over the Lea River by Leasey Bridge, the final stretch back towards Wheathampstead takes walkers through patchwork fields parallel to the river.
The pub: upon arrival back in the village, pay a visit to The Swan on the High Street to satiate you after your walk. Start with a pint of real ale while you peruse the menu and daily changing specials board and when you're ready, tuck into freshly prepared food.
For a map of the trail, click here.
Related content:
Absorb the beautiful scenery of the Ver Valley on The Three Burys walk. Referring to the pretty hamlets of Childwickbury, Redbournbury and Gorhambury, the walk, between St Albans and Redbourn, offers picturesque views over fields and farmland. At a moderate eight miles, this walk starts and finishes at the Verulamium Museum in St Albans before travelling north past the pretty Batch Wood – on fire with colour in the autumn – and up through Childwick Green before turning and heading west at Bamville Wood. Travel down Beesonend Lane where the trail takes walkers back towards St Albans at Redbournbury Mill, along the River Ver.
The pub: there are plenty of eateries in St Albans to enjoy after your ramble. Why not try The Six Bells on St Michael's Street which is located very close by the Verulamium Museum? With a roaring open fire and real ale on tap, you can enjoy the cosy atmosphere that is well deserved after your hike.
Click here for a map of the walk with detailed instructions.
3. Three villages walk: Ashwell, Caldecote, Hinxworth
Take this pretty walking route through a trio of villages starting at The Three Tuns in Ashwell. Pass Newnham Hill – which has mesmerising views over the countryside – and head towards the second village on the trail, Caldecote. The trail takes walkers past the stunning, historic Caldecote Manor before leading towards the third village, Hinxworth. You will pass through fields and kissing gates and the medieval manor, Hinxworth Place, on your way before arriving in Hinxworth. From the village you will walk back to Ashwell through more pretty countryside and over the River Rhee.
The pub: stomachs should be rumbling when you arrive back in Ashwell so head inside the lovely Bushel and Strike, one of our 2016 Food & Drink Awards finalists, for something from the BBQ menu which includes beef brisket with a chilli jam. For a quirky post-meal adventure, discover the history of the bubonic plague that can be explored in the village.
For a map of this walk, click here.
4. Sarratt and the Chess Valley walk
The green in Sarratt is the start of this walk through the Chess Valley. Heading northwest will take walkers through pretty farmland and woodland before arriving in Flaunden. Walk through the village and into the woods at the other side heading towards Latimer. Take in the autumn flora and fauna of Baldwin's Wood before reaching Latimer and heading east back towards Church End and Sarratt, enjoying the eclectic terrains and wildlife that surround you including woodland, water meadows and cosy cottages.
The pub: The Boot in Sarratt is the perfect post-walk pub to enjoy a satisfying meal and a drink. This 18th century inn serves elevated pub classics such as beer battered haddock and chips, sausage and mash or a generously portioned beef burger.
Click here for a map with walk instructions and landmarks.
5. Shaw's Corner walk
Enter the world of George Bernard Shaw, influential Irish playwright, who lived in the village of Ayot St Lawrence. This 7.5-mile walk will take you around the landscape that Shaw himself used to tread and even stops off at his eponymous home, Shaw's Corner. Start in the village of Wheathampstead at the East Lane car park and head north. Cross over the River Lea before heading east a little way next to the water. Walking north through fields and woodland will take you past Lamer House in the pretty Lamer Park, and then up towards Harepark Spring and into Ayot St Lawrence. Here you will find Shaw's Corner, long time home of George Bernard Shaw, now owned by the National Trust and open for visitors to explore. Once you have your fill at Shaw's Corner, head south past stunning woodland until reaching the River Lea again which, when followed west, will take you back to Wheathampstead.
The pub: continue your day of tranquillity with a visit to The Cross Keys, a charming pub based in the secluded woods of Hertfordshire, a short drive from Wheathampstead.
Click here for step by step walk instructions.
6. Lilley, Chilterns AONB (long route 5 miles, short route 2 miles)
Starting on East Street in the village of Lilley outside of Hitchin, this walk takes in some hills in the Chilterns Area of Outstanding Natural Beauty. Along East Street, take the Baulk bridleway on your right heading out of the village before going downhill towards the Kingshill Plantation which will be flourishing with autumnal colour. You could take a detour to visit Telegraph Hill for arresting views over Bedfordshire or continue along the Icknield Way. Walkers will pass Galley Hill with ancient excavated graves, before traversing back along Wardswood Lane to the village of Lilley.
The pub: a kilometre from Lilley, in the neighbouring village of Great Offley, is The Red Lion pub, a freehouse surrounded by countryside that serves tasty hot food. Featuring unpretentious pub classics such as steak and ale pie or lamb shank in mint gravy, it's the perfect spot for a hearty post-walk feed.
Find full walk details here.
7. River Stort walk
The beautiful River Stort, navigated by canal boaters and bursting with wildlife throughout the year, runs through Bishop's Stortford to Sawbridgeworth, providing a pretty, five-mile ramble that's perfect to witness the turning of the seasons. Start at the Bishop's Stortford train station and head in the direction of the town centre but take the footpath along the river just before the Riverside Bridge. As you leave the market town, the riverside route becomes rural, plunging the walker into a kaleidoscope of autumn colour.
The pub: cosy up next to the flickering fire either before or after your walk in The Coach & Horses, one of our favourite real ale pubs in the county. Quirky decorative features paired with open brick fireplaces create an eclectic and cosy place to refuel with a few glasses of real ale and some homemade food.
Full walking instructions here.
8. Ashridge Estate woodland walk
Nestled in an Area of Outstanding Natural Beauty, the Ashridge Estate is comprised of lush meadowland, cascading countryside and historical wooded patches. Start from the visitor centre and take the woodland path which provides stunning views of the pretty Aldbury village. Along this circular trail, you will pass country cottages, dramatic vistas and Dockey Wood, sprinkled with bluebells in the spring but carpeted with fallen leaves at this time of year. Walkers can choose their own routes that range from 1.5 to 4.5 miles.
The pub: when you've finished and worked up an appetite, head to The Greyhound Inn in Aldbury, a perfect country pub boasting high accolades from locals and professional bodies alike. The lunch menu features four sharing platters from the ploughman's style board with cheese, ham, scotch egg, pickle and crusty bread to the seafood platter of crab paté, king prawns and smoked salmon or maybe a garlic and rosemary infused baked camembert with bread and fig jam. Enjoy a well-earned feast!
For the full walk and other routes on the estate, click here.
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And we go live! Introduction.
2018 promised to be year full of excitement and new beginnings - and it hasn't disappointed, already 4 months later!
If you have been following me, you know that every 1 or 2 years I re-boot my website and decide to build it from scratch again... so here we are! I have finally found the way to separate my two branches of work. Curly Sketches will now be solely for my illustration work and personal projects.
Aafke Mertens is where I will promote my visualisation and live drawing services. I think this helps to give me the space I need for my creativity without sacrificing the more serious look I would like to keep for my commercial clients.
My plan for this blog will be to record my new ideas and projects, sharing them with you. I want to get over the fear of not having the perfect picture, and really just sharing with you my sketchbook and art table.
I really hope you enjoy it!


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NOTE: I thought Thesaurus Rex would be a cute idea for a children's book. So I googled it. Turns out it already is. Thesaurus Rex by Laya Steinberg. Not sure if I've seen the book before and it subconsciously came to mind while I was on thesaurus.com or if Mrs. Steinberg and I just share a love of dinosaurs and words. So if you like the idea go buy her book here.
It might keep me from getting sued.


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Introduction to Microsoft Word 2013
Computer Skills Series
This workshop is designed to help customers learn the fundamentals of the Microsoft Word 2013 program. Topics include:
- Understanding how tools and buttons are organized
- Opening and saving documents
- Creating and formatting a document
- Using the Copy and Paste feature
Requirements or Prerequisites:
Basic computer skills are needed for this workshop.
Workshop Materials & Resources:
Introduction to Microsoft Word 2013
- Bullets Practice
- Copy Cut and Paste Practice
- Formatting a Research Paper Practice
- Formatting Practice
- Formatting Paragraphs Practice
- Margins Practice


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TOWSON, Md. — Last Saturday against Sacred Heart, the Fordham football team's potent offense was stuck in neutral. The Rams could muster only two field goals in the second half of their 37-27 victory, with the opportunistic defense bailing out the struggling offense by intercepting the Pioneers twice in the fourth quarter.
"[That's] the beauty of playing a team game," Rams coach Joe Moorhead said this week. "This team truly doesn't care of who gets the credit, regardless of how many points we win by, or who scores points or how many we hold them to. There's one stat that matters, and that's the one in the left-hand column — did we win or lose the game? In terms of the flow of the game in the second half, the defense absolutely helped turn the [tide] and made a couple of momentum-changing plays [that] really helped seal the game."
That defense will have to keep creating good opportunities for the offense on Saturday, when Fordham will take on Towson at Johnny Unitas Stadium in the second round of the FCS playoffs.
The Tigers — who are seeded seventh in the playoffs, but ranked fourth in the latest Sports Network FCS poll — are going to be a tough test for the unseeded Rams, and Moorhead knows it.
"The big message for this week, relative to a very, very talented Towson team is that our margin of error is going to be much smaller," Moorhead said. "Some of the things that maybe we were able to get away with in the Sacred Heart game and still pull away with a victory, I'm not so sure that we'll be able to do that against a team of Towson's caliber."
In order for the Rams to give themselves a chance, they will have to fine-tune the same offense that sputtered in the second half against Sacred Heart, and Moorhead is confident his offense will get back to its high-scoring ways.
"You look at two plays in the second half — Tebucky [Jones had] a touchdown in his hands in the end zone and he bobbled it and dropped it, and that took seven points off the board," he said. "We didn't get the field goal there. We had another touchdown where Brian [Wetzel] scored and we had an unnecessary hold that really didn't have any factor in the play. You take 14 points off the board right there, and we're in line for another 50-point game."
The biggest challenge Fordham will face is Towson running back Terrance West, who is second in the FCS in rushing yards (1,865) and has already tallied 30 touchdowns on the ground. The Rams have had trouble stopping the run this season, allowin 5.3 yards per rush.
Moorhead said the 5-foot-11, 223-pound West possesses both breakaway speed and brute physicality, and detailed what it will take to slow him down.
"It's really about gap fits — making sure the defensive linemen hold a point in the front four," he said. "Our linebackers have their assigned gap, and particularly on the back end, that our safeties are fitting in to force them to run back to the linebackers."
Sacred Heart running back Keshaudas Spence recorded runs of 65 and 57 yards against the Rams last week, which Moorhead attributed to errors in execution.
"Where the runs got out, we didn't fit the runs properly and had guys out of position," he said. "When you're committing that many men to the box and one person is out of position, it's going to get out."
Despite the challenge in front of them, the Rams are confident, as they already have recorded two victories over Top-10 teams this season — Villanova, which was ranked No. 8 in the Sports Network FCS poll at the time, and Lehigh, then ranked No. 10 in the FCS Coaches' poll.
Fordham quarterback Michael Nebrich said after last Saturday's game the offense won't fear any defense it faces. This Saturday, we'll see if the Rams can shake off their second-half offensive slump from a week ago and get back to their prolific ways.


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Colorado is in the spotlight once again as the Denver Broncos recruit legendary quarterback Peyton Manning. Just last season, we were all glued to our televisions over the rising star Tim Tebow, not only because of his athletic skills but also because of his character and image.
But what do these two men mean to Colorado outside of football field? The answer is that both Manning and Tebow are extremely charitable men who give their time, their public platform and a great deal of money to causes that are dear to them.
The Tim Tebow Foundation was started when Tebow was still an undergraduate at the University of Florida. Its inspiring mission is to bring faith, hope and love to those needing a brighter day in their darkest hour of need. It has raised money for Shands Hospital in Florida, Uncle Dick's Orphanage in the Philippines, funding for playrooms in Children's Hopitals around the world and has provided Disney trips for children. In addition to his foundation work, Tebow also invites special needs children to his games and gives them quality time and attention.
The Peyback Foundation, founded by Peyton Manning and his wife Ashley in 1999, focuses on the development of underprivileged youth. The foundation supports programs in the areas of leadership development, mentoring, after school programming and healthy living. The geographic focus is currently Indiana, Louisiana and Tennessee, but it makes sense that Colorado would eventually be added if (or should we say WHEN?) Manning moves to the Broncos.
Colorado nonprofits could benefit greatly from Peyton Manning's move to the Broncos. His Peyback Foundation is a grantmaking foundation, while the Tim Tebow Foundation utilizes a targeted approach.
Professional athletes are extremely important to the nonprofit community when they utilize their public platform to bring issues to light and to raise money to support the missions closest to their hearts. Colorado is fortunate to have many great, charitable athletes and we welcome Peyton Manning to our community!


text: Search Heraldmag.org For a more thorough search of the Herald site please go to http://www.google.com/ and type; heraldmag.org:{your search subject}. For example, if you wanted to search all Herald articles about Jeremiah, after going to Google, you would type: "heraldmag.org:Jeremiah" in the search box. This will give you the best results from our extensive archives, which date back to 1918. Share this:TwitterFacebookMorePrintEmail


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Hayzl Gets Over Excited III
Hi, As you can see we got a little over excited as we were taking this set and just had to get it on. Hope you enjoy. Luv Hayzl xXx
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You loved our Top This Hats, now we have put together and exclusive to Whimsical Yarn the Top This Blanket kit. This kit contains all yarn needed to create a fun, whimsical blanket. This blanket kit contains all the yarn to create a blanket with a mix of textures and colors and a coordinating character to top a corner of the knitted blanket.
Package includes one skein of Bernat Giggles Solid, one skein of Bernat Li'l Tots, one Top This yarn kit with coordinating character, and printed directions.
Size 10.5 US 24" circular knitting needles are not included.
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Benguela Area Part 1
Near Benguela, there are extensive outcrops of red beds of silt, mud, and sandstones that are mapped as Albian in age. These red rocks show cross-bedding, bioturbation, and tree trunks that indicate that are terrestrial deposits, and therefore a good candidate for yielding dinosaur bones. Moreover, we collected a few dinosaur bone fragments from equivalent beds in the Namibe region.
But reality is not always as we wish: a lot of walking... we found no vertebrate fossils. The most significant finding were some beautiful gymnosperm tree trunks.
The Upper Cretaceous marine beds were more productive, and we found many invertebrates, shark teeth, and some marine reptiles pieces, but mostly fragments. These finds are good for building the record, but we keep looking for new localities and fossils that can tell much more about evolution of vertebrates in Angola.
On July 16th, Louis Jacobs, Ana Marques, and John Graf flew up to Cabinda. Cabinda is a province to the north separated from the main country by the Republic of Congo.
From July 17th to July 20th, we prospected four beaches which had outcroppings that were of more recent ages. For the last four years, members of Project PaleoAngola have collected samples and measured sections at these localities for the purposes of determining the faunal assemblage of these outcrops and determining the ages of the rocks (something that had not previously been accomplished).
During these four days, we found two new localities that were fossil rich. From these localities, we were able to recover the same types of fossils found at the other localities along with a crocodile genus not described from this area before and an Arsinoitherium tooth.
Arsinoitheres are hippo-like mammals with two large horns forming a V at the end of their snouts that lived during the Late Eocene and Early Oligocene.
Image from http://farm6.static.flickr.com/5257/5404145675_50b1457eea_z.jpg
These new fossils will add to the already rich fossil faunal assemblage found in Cabinda and will help narrow down the age of the rocks that they were discovered in.
On July 21st, we flew back to Luanda. On July 22nd, we flew down to Benguela to rejoin Michael and Octavio for the rest of the field season.
Malanje Trip
Octávio Mateus and Michael Polcyn drove to the Malange area while Louis Jacobs, John Graf, and Ana Marques went to Cabinda. In Malange (also spelled Malanje) and Lunda Norte provinces are in the north central part of the country and previous reports in Baixa de Cassange have shown a rich fauna of Triassic fossil fishes. These areas haven't been visited by paleontologists since the 1960's so we decided to give it a try.
We arrived to Malange on the evening of the 16th and met with our friend Pedro Vaz Pinto, a biologist that studies the giant sable, the Palanca-Negra-Gigante, the national symbol of Angola. He has been capturing sables to fit them with radio tracking collars and told us that while they were darting a sable from a helicopter that day, a large male lion tried to take the sable. Fortunately the helicopter scared the lion away and the sable was safely collared. This sighting is extremely significant, being the first large predator in the area since the war ended.
Yesterday (the 17th) we drove to Xá-Muteba (Lunda Norte), the closest town to the Triassic fossil localities. The local authorities alerted us about the possible presence of landmines in the area we wanted to visit, so we met the de-mining team who informed us that the area surrounding the fossiliferous sites has not been completely cleared, so we headed back to Malanje to regroup and plan for the next day.
Today (the 18th) we decide to see the continental Cretaceous formations near Kiwapa Mzoji (former Brito Godins). There are good rock exposures along the road cuts as the road expansion works progresses. The rocks are a varved mudstone and sandstone with some ripple-marks.... and no fossils. Although we were not able to find any fossils on this leg of the expedition, we did learn a lot about the area, the people and the rocks. In a few years, we expect the mines will be cleared and we will be able to go tot he Triassic localites. Tomorrow, we head back to the coast and plan to base in the port town of Benguela.
The team met in Luanda on the 12th and after gathering the proper permits, and getting the vehicles and supplies organized, we set out for the localities in the north around the village of Iembe. This locality has thus far yielded 4 holotypes; Angolasaurus bocagei, Tylosaurus iembeensis, Angolachelys mbaxi, and Angolatitan adamastor.
Our goals this year for the Iembe locality were threefold. First, we needed to collect fossil molluscs for stable carbon isotope analysis to build a chemostratigraphy of the area which we will compare with the global record to provided better age control for the fossils we have recovered from the site. Second, we needed to revisit previous dig sites to see if any additional material had weathered out of the outcrops, and third, we wanted to prospect for new localities in the area.
We accomplished all of our goals and were especially excited to find a new productive locality to the north of the outcrops that had yielded our previous finds in the area. We found fragmentary material including a disarticulated turtle carpace, plesiosaur limb material, and a disarticulated mosasaur that appears to be Angolasaurus. We collected the plesiosaur limb and Angolasaurus material. Judging from the associated fish and molluscs, we believe this new locality is about the same horizon as the type locality of Angolasaurus and Angolatitan.
We returned to Luanda on the 15th to begin the next leg of the expedition.
Lack of internet access in the field and available time the last week delayed the blog updates. I am not sure if this entry really counts as a "field blog" update, as I am sitting in a nice comfortable chair in my air conditioned family room at home, but I did want to provide a final update for this years field work.
June 11th through the 14th. The morning of the 11th I wrapped up work on "Big Mo", the large Prognathodon I discovered last year. We had determined that collecting the specimen was just too ambitious for this year given our available time and crew size, so we have decided to rebury the specimen for collection in 2013. In the afternoon I shifted my focus to collecting other specimens found in previous years and some from this season. I checked out the articulated vertebrae that Octavio had found the previous day. We had a student from the Methodist University in Luanda working with us this year, Ana Marques, and together we began to excavate the specimen. It turned out to be the portion of the tail preserved in articulation was the section that bears a dorsal fleshy fluke and ventral bend. There
were also a number of isolated vertebrae from the anterior part of the tail surrounding it, but it appeared that this was the only portion of the animal preserved; However, the next morning we decided to move more of the overburden and witihin five minutes of arriving on site we found what appears to be a relatively complete skull and artculated neck vertebrae and some isolated limb and girdle elements. Over the next two days and with the help of Ana, Ricardo, and Louis we recovered the entire specimen in two large jackets and a number of smaller ones.
Articulated portions of tail of russellosaurian mosasaur.
Skeleton of the new specimen.
Ricardo and Ana excavating tail block.
Ricardo has been largely focused on collecting plesiosaur specimens to augment the collection made in previous years, the material on which his Phd work is based. This year he was able to find additional specimens that allow us to definitively link a number of partial skeletons to specific taxa, giving us a better composite view of their anatomy and thus providing better data to determine their relationships.
Ricardo excavating plesiosaur specimens.
Octavio has spent most of his time prospecting for dinosaurs in the terrestrial sediments near Bentiaba, finding a large number of bone fragments, some identifiable as sauropod limb elements. He also found yet another partial skull of the turtle Euclastes
Octavio and Ana excavating a partial Euclastes skull.
Partial Euclastes skull
On the 15th I drove Louis, Ricardo, Tyrone, and Ana to Lubango. Tyrone and Ana are finished with their field work for this year. Louis and Ricardo will meet another member of our crew, Scott Myers, in Luanda on the 17th. Those three will then travel to the northern province of Cabinda to collect fossil and isotope samples and collect data to work out the stratigraphy and paleotemperature of these fossil bearing sediments. During the trip to Cabinda the crew collected numerous fish teeth, a complete skull of a turtle, a crocodile humerous, and a single vertebra of a small snake. A major focus of the Cabinda leg was to sample and examine the rocks and fossils of Cabinda that are relevant to human evolution and to the climatic and environmental history of Africa and the Tropics. One of the most important topics in African paleontology is human evolution, documented mainly in East Africa. There has been no Tropical lowland West African record of fossil primates until our discovery of a single tooth in Cabinda. The importance of this find cannot be over estimated because it opens a new chapter that cannot be found elsewhere. Additioanally, they sampled the rocks for magnetic and chemical analysis that will be used to understand climatic changes in the African Tropics associated with such global events as the onset of Antarctic glaciation, which provide an environmental context for human evolution. Because the age of the rocks is imprecisely known, our work will have a major impact on this facet of science.
Cabinda localities. Jungle covers just about everything except the cliffs along the shore. These beach localities are the focus on our work here.
Ricardo and Scott on the largely vertical outcrops.
Louis and Scott collecting samples.
Turtle skull collected this year at Landana
Johanna Salminen (University of Helsinki, Finland) joined the team in Lubango on the 15th, returning with me to Bentiaba. She is a specialist in paleomagnetism. Throughout its history, the magentic polarity of the Earth has changed occassionally, and in some cases, as rocks are formed , they record that polarity. The dates of these polarity changes are now well known and the reversals can be used to correlate rocks over long distances. She collected rock samples from the Cretaceous sediments of Bentiaba and the K-Pg boundary section at Namibe to assist us with more precise dating of the rocks. Her colleague David Evans (Yale University) joined her in Lubango on the 27th to sample some of the oldest rocks in Angola to understand how the continent of Africa was formed. Certain kinds of rock found within granite in Namibe and Huíla can show through their magnetic properties how the continent of Africa was originally assembled. This is a fundamental scientific investigation and a direct outgrowth of discussions centered on our original work.
Johanna Salminen (University of Helsinki, Finland) with coring equipment.
Johanna sampling for paleomag at the K-Pg boundary in Namibe province.
Mike, Johanna, and Octavio at K-Pg boundary locality.
I spent the last few days at Bentiaba collecting the large Prognathodon specimen I found this year. Octavio collected a portion of a pterosaur wing. Although most of the specimen was left covered, it is the most complete found so far, consisting of at least three bones in articulation. He also began to uncover one of the turtle specimens I found this year. When I found it, only a few peripheral plates were showing, weathering out of the hill. As Octavio began to uncover the specimen,
the costal ribs were found in place and it became apparent that we had a semi-articulated (and given previous experience here), a relatively complete skeleton. However, this was the last day in the field and we were running out of time, so this fossil was also reburied for excavation next year.
Pterosaur wing block. Top left is the defined block prior to plastering, top right is a detail of the bone that was weathering on the surace, and the bottom image is teh block just prior to removal.
Octavio digging an articulated turtle. This one will need to wait till next year due to time constraints.
During the last week in Angola, Octavio and I began managing the crating and shipping of the collection. Joined by student Claudio Raposo (Agostinho Neto University, Angola), we also took one day to go to the Turonian locality of Iembe to collect the remains of a ninety million-year–old specimen of the turtle
previously named by Projecto PaleoAngola Angolachelys mbaxi. This specimen was discovered by Johan Lindgren who joined us during our 2009 field season, and is the best preserved and most complete specimen found thus far. Louis, Octavio and I had collected the skull block and part of the postcrania in 2011, but the large block collected by Octavio and I this year contains much of the postcrania. On our return drive from Iembe, we stopped to prospect sediments north of Barra do Dande and discovered another locality. It is interesting because it appears to contain a Campanian fauna and we found a couple of teeth of a rare durophageous mosasaur Globidens, that appears to be a different taxon from those reported elsewhere. The rest of the crew joined us in Luanda mid-week to finish the shipping prepartation that includes the collections made in 2011 and 2012 field seasons.
Removing the final block of the 2009 Angolachelys specimen.
Octavio and I talking to the students about fossils at the school in Iembe, the type locality of Angolatitan, Angolasaurus, and Angolachelys.
Repacking crates in Luanda.
Crates being prepared for shipment.
The crew left Luanda on the 30th of June.
The field team for the 2012 Projecto PaleoAngola Field Expedition included the following participants:
Louis L. Jacobs (Southern Methodist University, USA)
Octávio Mateus (FCT, UNL, Portugal)
Michael J. Polcyn (Southern Methodist University, USA)
Scott Myers (Southern Methodist University, USA)
Ricardo Araújo (Southern Methodist University, USA)
Ana Soraya Marques (Angola Methodist University, Angola)
Claudio Raposo (Agostinho Neto University, Angola)
Tyrone Rooney (Michigan State University, USA)
Johanna Salminen (University of Helsinki, Finland)
David Evans (Yale University, USA)
Funding for the field season was provided by Vida Foundation, Esso Angola through the Liga Nacional dos Amigos de 4 de Abril, the Institute for the Study of Earth and Man at Southern Methodist University, The Saurus Institute, and Yale University.
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