text:
Tropical Fish Keeping - Aquarium fish care and resources (http://www.tropicalfishkeeping.com/forum.php)
- Introduce Yourself (http://www.tropicalfishkeeping.com/introduce-yourself/)
- - hi from michigan (http://www.tropicalfishkeeping.com/introduce-yourself/hi-michigan-57563/)
hi from michigan
Hi am am toxodon and I own 1 spotted leaf fish and my favorite t.v. shows are ncis and conan
Hi toxodon. I love NCIS too. Hope you like the forum.
Welcome to Tropical Fish Keeping!:-D
I love NCIS also! I have seen every episode from season 1 to 7 (thank you Netflix!), still waiting for season 8.
Hello and welcome to the forum :wave:
welcome aboard...leaf fish are awswome...do you have to feed yours live...and wich conan are we talking about...barbarian by crom...or o brien..anthow..glad you found us..
Hi, and welcome to Tropical Fish Keeping forum.
welcome to the forum
Welcome to TFK. Do you have any pics of your leaf fish that you can share? I love those fish. :-)
Hi toxodon. (sounds like a headache medicine..;-)) Welcome to TFK! Leaf fish are quite interesting. And I am in love with Conan (O'brien ..that is)
hello and welcome. :)
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Subway - La Roche sur Yon, FR
in Subway Restaurants
Restaurant Subway juste à côté du magasin Bébé 9
posted by: pilouille
location: Pays de la Loire, France
date approved: 1/15/2016
last visited: 10/3/2015
Subway Restaurant - Deer Creek Commerce Lane, Davenport, Florida
Subway Restaurant - Deer Creek Commerce Lane - Davenport, Florida
posted by: walkingwildly
location: Florida
date approved: 1/13/2016
last visited: 1/14/2016
Subway - S. La Brea Ave. - Los Angeles, CA
One of many Subways in the area.
posted by: bluesnote
location: California
last visited: never
Subway - Commonwealth Centre Pkwy. - Midlothian, VA
This is one of many Subways in the area.
location: Virginia
date approved: 1/5/2016
Subway - Main St. - Damascus, MD
A Subway near the center of town.
location: Maryland
last visited: 1/8/2016
Subway Store - Flying J Travel Center - Portland, OR
This Subway is located in a truck stop travel center.
posted by: thebeav69
location: Oregon
date approved: 1/4/2016
Subway Store - Washington Square Mall - Tigard, OR
This Subway is located on the second floor in a food court within Washington Square Mall.
Subway Store #30887 - Clackamas Town Center - Portland, OR
This Subway is located in a food court on the second floor of Clackamas Town Center.
Subway #11832 - Wysox, PA
This Subway is near the intersection of route 187 and route 6.
posted by: ripraff
location: Pennsylvania
date approved: 1/3/2016
Subway - Southpoint Pkwy. - Fredericksburg, VA
Subway - Wright City, MO
Located in strip mall and new to this town. Casual counter-serve chain for build-your-own sandwiches & salads, with health-conscious options.
posted by: YoSam.
location: Missouri
date approved: 1/1/2016
Subway Store #58083 - Westfield Palm Desert Mall - Palm Desert, CA
This Subway is located inside Westfield Palm Desert Mall food court on the 2nd floor.
date approved: 12/31/2015
last visited: 4/17/2016
Subway - Stonebridge Plaza Ave. - Richmond, VA
date approved: 12/26/2015
Subway - Greenville Commons - Greenville, TX
Subway #32259 is located in the Greenville Commons shopping center, 7215 I-30 #1 Lot 3B, Greenville, TX. Phone is 903-455-4700.
posted by: QuarrellaDeVil
location: Texas
date approved: 12/24/2015
Subway - Commons - Ithaca, NY
This Subway is on the Ithaca Commons.
location: New York
date approved: 12/20/2015
Subway - Ohio 108 - Wauseon OH
Located inside of Turnpike Shell on OH 108
posted by: nomadwillie
location: Ohio
last visited: 7/23/2016
Subway - Chisholm Trail - Jacksboro, TX
Subway #21917 is located at 112 Chisholm Trail, Jacksboro, TX, just north of the center of town. Phone is 940-567-5229. They also have a drive-thru.
date approved: 12/18/2015
Subway - Harlem Ave - Forest Park IL
Located on Harlem Ave
location: Illinois
date approved: 12/16/2015
Subway - Walton, NY
This store is on the main road into town. It is on route 206 and route 10.
Subway - Sepulveda Blvd. - El Segundo, CA
One of many Subway's in the area.
date approved: 12/13/2015
Subway #47080 at Legends - Sparks, NV
A Subway located at the Legends Outlet Stores Complex.
location: Nevada
date approved: 12/12/2015
Subway - Bally's - Las Vegas, NV
A Subway in the downstairs food court below the Casino.
Subway - Szewska - Krakow, Poland
Located at Szewska 16 in Krakow.
posted by: Metro2
location: Małopolskie, Poland
date approved: 12/9/2015
last visited: 9/19/2015
Subway Restaurant - 88239 Wangen, Baden-Württemberg, Germany
Subway Restaurant im Argencenter - 88239 Wangen... -
Subway restaurant in Argen Center - 88239 Wangen
posted by: jotheonly
location: Baden-Württemberg, Germany
Subway - Slawkowska - Krakow, Poland
Located at Slawkowska 22, 31-014 Kraków,
date approved: 12/1/2015
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text:
The Jigoshop Smart Send shipping plugin integrates Australian shipping cost calculations for 'Smart Send' online shipping.
Major update - added: - Multiple price point / delivery quotes to choose from - Option for receipted delivery: Always, Never, and user choice - Option for transport assurance: Always, Never, and user choice - Option for tail-lift delivery based on item weight, both pickup and delivery - Checkout updates automatically on any option changes - Improvements to cart view shipping estimate - Fixes to locations caching
First submission to plugin repository
Requires: 3.4 or higher
Compatible up to: 3.5.2
Last Updated: 4 years ago
Active Installs: Less than 10
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A versatile piece from John Lewis thats perfect on its own or layered under a warm jacket. The two shades of blue add a pleasing contrast and soft cotton is kind to sensitive skin.
Thank you for visiting John Lewis Boy Raglan Long Sleeve T-Shirt, Navy/Blue on shoppingfind.co.uk
We bring the products from many websites to one website for you to search, save and compare.


text:
Financial Staffing Agencies Bismarck ND
Fargo, ND
Fargo, ND
temporary, long-term, temporary/part time, part time, payroll
Bismarck, ND
Fargo, ND
Information Technology
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Career Profile for Financial Managers, Branch or Department - Education & Training
Financial Managers, Branch or Department
Overview of Education & Training
Unfortunately, no educational attainment
data exists for this profession.
Most Significant Source of Postsecondary Education or Training
In-Depth Look at Education & Training
Most financial managers need a bachelor's degree, and many have a master's degree or professional certification. Bank managers often have experience as loan officers or in other sales positions. Financial managers also need strong interpersonal, math, and business skills.
Education and training. A bachelor's degree in finance, accounting, economics, or business administration is the minimum academic preparation for financial managers. However, many employers now seek graduates with a master's degree, preferably in business administration, finance, or economics. These academic programs develop analytical skills and teach financial analysis methods and technology.
Experience may be more important than formal education for some financial manager positions—most notably, branch managers in banks. Banks typically fill branch manager positions by promoting experienced loan officers and other professionals who excel at their jobs. Other financial managers may enter the profession through formal management training programs offered by the company.
Licensure. Many financial managers work in accounting departments. Accounting positions normally require workers to be certified public accountants (CPAs).
Other qualifications. Candidates for financial management positions need many different skills. Interpersonal skills are important because these jobs involve managing people and working as part of a team to solve problems. Financial managers must have excellent communication skills to explain complex financial data. Because financial managers work extensively with various departments in their firm, a broad understanding of business is essential.
Financial managers should be creative thinkers and problem-solvers, applying their analytical skills to business. Financial managers must have knowledge of international finance because financial operations are increasingly being affected by the global economy. In addition, a good knowledge of regulatory compliance procedures is essential.
Certification and advancement. Financial managers may broaden their skills and exhibit their competency by attaining professional certification. Many associations offer professional certification programs. For example, the CFA Institute confers the Chartered Financial Analyst designation on investment professionals who have at least a bachelor's degree, work experience, and pass three difficult exams. The Association for Financial Professionals confers the Certified Treasury Professional credentials to those who pass a computer-based exam and have a minimum of 2 years of rele...
Click here to read the rest of this article from College Toolkit


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Those who do not understand Unix are condemned to reinvent it, poorly. -- Henry Spencer


text:
Koi at the Trump Soho Hotel Suing Insurance Company Over Sandy Claim
The restaurant says it lost thousands of dollars after the power went out.By Hugh Merwin
The restaurant says it lost thousands of dollars after the power went out.By Hugh Merwin
Plus: 'Smash' cast members drown their sorrows at Catch, and more, in this week's roundup.By Lauren Duca
Meanwhile, Mel Gibson and Florence Henderson defined dichotomy while dining at Cafe de Rey.By Hadley Tomicki
The glitzy Japanese restaurant is offering a limited edition flight of crispy rice.By Hadley Tomicki
The book will detail the Kogi mastermind's wild youth in L.A. through the founding of his Korean taco truck.
Trump and Koi, together at last. What took them so long?By Hugh Merwin
Plus: George Lucas met with Francis Ford Coppola, Ivanka Trump celebrated her husband's birthday, and more, in our weekly roundup of celebrity dining.By Eric Sundermann
Sorry kids, it's 20% every time. Yes, EVERY time.By Hadley Tomicki
The L.A. Times thinks Sotto is the first serious contender to the dominance of Northern Italian restaurants in L.A.By Hadley Tomicki
Meanwhile, Taylor Swift gives thanks to Ismail Mohan at RockSugar.By Hadley Tomicki
Only 1,000 calories? What if we're still hungry?By Jenny Miller
Plus: Capital Grille gets artsy again, Sunset Park getting a "jungle-themed club," and more, in our daily neighborhood watch.
And in other not-so-surprising news, underage Miley Cyrus appears to have gotten plastered again.By Hadley Tomicki
Grammy's week brought Justin TImberlake, Cee-Lo Green, and Miranda Lambert out to eat in L.A.By Hadley Tomicki
A star-packed week for restaurants after The Golden Globes.By Hadley Tomicki
"I would have hoped that the head legal officer of this State would not jump to conclusions on a basis of guilt by association," says a representative of the restaurant.By Hadley Tomicki
Attorney General Jerry Brown rails against the restaurant's flashy image and yellowtail tartare.By Hadley Tomicki
"I don't like watery coffee. Skim milk ruins it. If I'm really treating myself, I'll take heavy cream."By Helen Rosner
Plus: Russell Brand's star power transcends his scruffy attire, Ramona Singer is dining and of course wining, and more in our weekly roundup of celebrities' meals out.By Alex Rees
Was there a conspiracy to make Alvin Gentry puke during the playoffs and could Bam Bam beat The Hulkster?By Hadley Tomicki
This Obscure News Story, Which Should Be Huge, Shows How Trump Gets Away With Corruption
15 Stars Reminisce About the Actors Who Used to Beat Them Out for Roles
Trump Staffers Plant Alternative Facts to Stop Him From Tweeting Them
Zach Galifianakis Overstays His Welcome on The Late Late Show With James Corden
In Get Out, Allison Williams Makes the 'Good White Person' Terrifying
Why Was Times Theater Critic Charles Isherwood Fired?
Report: Manafort Faced Blackmail Over Russia Connections During Trump Campaign
George Clooney Wants You to Remember That Donald Trump and Steve Bannon Are Hypocritical 'Hollywood Elitists'
Trump: We're Submitting a Health-Care Plan! Tom Price: No, We're Not!
Republicans Accuse Protesters of Organizing for Political Change


text:
The Case Mini Copperhead is a knife you will never run out of uses for. Its 2-blade design makes it highly versatile and capable of handling nearly any task set before it. The blades are all honed to a razor-sharp edge, so take care not to cut yourself as you admire the beauty and elegance of this classic knife.
Technical Information
- Blade Steel: Surgical Stainless Steel
- Handle Material: Genuine Stag
- Blade Length: 2.25" Clip, 1.6" Pen
- Closed Length: 3.125"
- Weight: 1.4 oz
Additional Information


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Hostelworld rekommenderar
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Pension Aduar
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Albergue Inturjoven Marbella is situated in the historic centre of Marbella.
This is a beautiful building with andalusian typical style, garden and sunny terraces with beautiful mediterranean view... Mer...
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Hostal Tak has a total of 7 bedrooms offering individual beds, bunk beds a double bed and a... Mer...
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Definitions for forgot
This page provides all possible meanings and translations of the word forgot
Webster Dictionary
of Forget
of Forget
imp. & p. p. of Forget
Chambers 20th Century Dictionary
Forgotten. See Forget.
British National Corpus
Written Corpus Frequency
Rank popularity for the word 'forgot' in Written Corpus Frequency: #1542
Chaldean Numerology
The numerical value of forgot in Chaldean Numerology is: 4
Pythagorean Numerology
The numerical value of forgot in Pythagorean Numerology is: 9
Sample Sentences & Example Usage
Honey, I forgot to duck.
Most of what I know I forgot.
Hey Pilgrim, you forgot your pop-gun
We traveled long and forgot why poetry was invented.
I once had a memory pillow, but I forgot where I put it!
Images & Illustrations of forgot
Translations for forgot
From our Multilingual Translation Dictionary
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Use the citation below to add this definition to your bibliography:
"forgot." Definitions.net. STANDS4 LLC, 2017. Web. 20 Feb. 2017. <http://www.definitions.net/definition/forgot>.


text:
Travel and Hotel
Many destinations very welcome to greet whoever across the world. If you are the one who love traveling, do not miss all these charming places where will fulfill your magnificent holiday. Our websites with more than hundreds of the most famous tourist attractions will provide you the information of any paradises on earth no matter where you want to visit or just drop in. Have no idea how or when to get start? We have all information for any gadabouts to guide you in traveling including accommodations, transportation, diet, and so forth. We provide you with the best accommodations ranged from the most luxury hotels to cheap hotels. Feel bored about humdrum traveling? Various kinds of holiday trip packages are waiting for you to choose like one-day trip to a famous city, interesting and exciting activity, etc. We present interesting content with updated travel guide around the planet.
Ottawa, Canada
The city is located along the southern banks of the Ottawa River, the river that forms the local boundary of Ontario and Quebec provinces and is the main water way of provinces here. Ottawa is the capital city of Canada. Diversity of cultures of Ottawa is established with the mixture of local origins - Odawa - and modern civilizations. Here in the nice heart of Ottawa you can touch the atmosphere that seems like a young attractive guy's personal advertisement including vibrant, clean, bilingual, naughty, and the like. Due to these all factors, the charming Ottawa is considered and described one of the world's most beautiful capital cities. Although Ottawa is a small city located on the province of Ontario, the city attracts a large number of tourists every year as well as the other big city attractions. Furthermore, Chinatown and Little Italy temporary stay here.
Ottawa, Canada
The city is located along the Ottawa River on the southern banks, the river that forms the local boundary of Ontario and Quebec provinces and is the main water way of provinces here. Ottawa is the capital city of Canada. Diversity of cultures of Ottawa is established with the mixture of local origins—Odawa—and modern civilizations. Furthermore, Chinatown and Little Italy temporary stay here.
Ottawa, Canada
Why do many tourists visit here every year? Because of this green city combined with the parked and opens space where is filled with the numerous of national attraction, interesting museum, sightseeing, and so on. For example the Museum of Civilization or the Canadian Museum is the place where you can learn the history of 1,000 years Canadian life with it fascinating and precious convention. Another is the ByWard Market or a traditional farmers' market which is a two-story building where visitors can find a restaurant or boutique shop for relax sitting or talking with friends in a warmth atmosphere and taking a grab of snack or a glass of beer or wine. After a tired day wandering around the city's interesting attractions, find a nice hotel for a great relaxation at Ottawa's various hotels where you can spend your days and nights resting among the warmest atmosphere.


text:
1001 Great Ideas for Teaching and Raising Children with Autism Spectrum Disorders
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- Brief Synopsis:
- This is the greatest book for teaching kids with autism spectrum disorders as well as children in general. It can be motivating for both parents and children alike.
- Long Synopsis:
- This book is a cool guide for parents and teachers. It even has suggested sites to check out with your kids. You'll find it very helpful.
- Book Quality:
- Excellent
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- 222 Pages
- 9781932565195
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- Date of Addition:
- 10/15/08
- Copyright Date:
- 2004
- Copyrighted By:
- Ellen Notbohm and Veronica Zysk
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- Self-Help, Disability-Related, Health, Mind and Body, Nonfiction, Parenting and Family, Reference
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Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases.
Methodology/Principal Findings
An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans.
This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.
Author Summary
Leishmaniasis is a vector-borne disease with a complex ecology and epidemiology. It has three main clinical forms of which visceral leishmaniasis (VL) is the most severe, as it is fatal if untreated. It is caused by a protist parasite, Leishmania spp., and is transmitted to humans by phlebotomine sandflies. The best method to interrupt any vector-borne disease is to reduce man-vector contact. Vector-targeted strategies are particularly attractive because the vectorial capacity to transmit infectious diseases to humans is proportional to vector density and, in an exponential way, to vector survival. Biological control is an effective means of reducing or mitigating pests through the use of natural enemies and is more environmentally friendly than traditional insecticide treatments. Nevertheless, there is very scanty information on the biological control of sandflies and their potential control agents. In this context, a detailed knowledge of the microorganisms that are associated with these vectors would aid in the development of novel strategies for controlling them. This is the first study to survey the taxa associated with leishmaniasis vectors and, more importantly, with any infectious disease vector, using an unbiased and high-throughput approach.
Citation: McCarthy CB, Diambra LA, Rivera Pomar RV (2011) Metagenomic Analysis of Taxa Associated with Lutzomyia longipalpis, Vector of Visceral Leishmaniasis, Using an Unbiased High-Throughput Approach. PLoS Negl Trop Dis 5(9): e1304. doi:10.1371/journal.pntd.0001304
Editor: Philippe Solano, IRD/CIRDES, Burkina Faso
Received: March 28, 2011; Accepted: July 22, 2011; Published: September 6, 2011
Copyright: © 2011 McCarthy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was principally supported by a Fundación Bunge y Born grant (FBBEI5/08) to CBM; it was also supported by a Max Planck Gesellschaft Partner laboratory grant and Buenos Aires Provincial Government grant to RVRP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Leishmaniasis is a vector-borne neglected infectious disease of worldwide incidence and its most severe clinical form is visceral leishmaniasis (VL). Each year VL causes an estimated 500,000 new cases and more than 59,000 deaths [1], a death toll that is only surpassed by malaria among the parasitic diseases [2]. Furthermore, both figures are approximations since VL is frequently not recognized or not reported [3]–[4]. Leishmaniasis is transmitted through the bite of two phlebotomine sandfly genera, Phlebotomus in the Old World and Lutzomyia in the New World. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis [5]. This phlebotomine sandfly is only found in the New World, with a wide distribution from Mexico to Argentina [6]. The geographical distribution of leishmaniasis has undoubtedly expanded and is now being reported in areas that were previously non-endemic. The worldwide phenomenon of urbanisation, closely related to the sharp increase in migration, is one of the major risk factors that is making leishmaniasis a growing public health concern for many countries around the world [7] and Argentina is not an exception. Between 1925 and 1989 only 14 leishmaniasis human cases were reported in Argentina and none was attributed to Le. chagasi. Moreover, there were only two isolated reports of Lu. longipalpis (in 1953 and 2000) which were not associated with VL [8]. Nevertheless, this situation has changed dramatically, mostly due to an indiscriminate advance of urbanisation, and the first Argentine VL outbreak was recently reported [9]. From 2006 to date the morbidity and mortality toll of this disease have amounted to 83 human cases (35% corresponding to children under ten years of age), 7 deaths and more than 7,000 infected dogs (National Health Surveillance System, Epidemiology Bureau, National Ministry of Health, Argentina).
In the natural environment, phlebotomine larvae feed on organic matter from soil [10], while adults from both sexes feed on sugars from plant sources [11]–[12]. Only female adults need blood to obtain necessary proteins for the development of their eggs [5]. It is widely accepted that many insects derive their microbiota from the surrounding environment, such as the phylloplane of food plants or the skin of the animal host, and although the degree of persistence of strains of the ingested species is unknown, these microorganisms can influence the insect life cycle [13]. To comprehensively understand the biology of insects, microorganisms must be considered as a very important component of the ecological system [14]. Moreover, an inventory of the associated microbiota of phlebotomine sandflies, especially of wild specimens, would aid in understanding the annual and regional variations recorded for this disease [15] and in the development of novel strategies for controlling these vectors, among others [13]. One serious obstacle for the biological control of VL sandfly vectors is that their precise breeding sites are poorly known. Furthermore, its practical application seems to be limited to the adult VL vector stage [16] because, as Lu. longipalpis larvae appear to be thinly dispersed [17], this complicates the employment of biolarvicides in the field.
There is scanty information on the microbial colonisation of Lu. longipalpis and it is not yet clear if they possess an indigenous community. Previously, midgut bacteria were examined from wild and laboratory reared Lu. longipalpis populations [18]–[20] which showed a predominance of Gram negative bacteria. Various genera found ubiquitously in the environment (water, soil and debris) were identified in these studies, including Acinetobacter, Serratia, Pseudomonas, Stenotrophomonas, Flavimonas and Enterobacter. These bacteria have also been found associated with the gut of several other insects [21]–[24], suggesting they are a part of the natural or transient microbiota. Prior studies on guts and malpighian tubes from wild P. papatasi and P. tobbi showed a high incidence of mycoses which were similar to Aspergillus sclerotiorum and Saccharomyces cerevisiae [25]. Various types of virus have also been found infecting phlebotomine sandflies [26], including Vesiculovirus [27]–[28] and Cytoplasmic Polyhedrosis Virus [29]. Furthermore, in addition to Leishmania, Trypanosoma, Endotrypanum and possibly other trypanosomatids [30], neotropical sandflies may harbour other parasites including microsporidians [31]–[32], gregarines [33]–[36], some Plasmodium spp. that parasitise lizards [37] and nematodes [38]–[41]. Nevertheless, there is little information on the pathological effects these parasites may produce in their sandfly hosts.
Metagenomics facilitates the culture-independent analysis of microbial communities [42], an approach which does not require prior assumptions about the composition of the target community. Metagenomic sequencing of communities containing eukaryotes, in particular protists, is mostly cost-prohibitive because of their enormous genome sizes and low gene coding densities [43]. Nevertheless, from an ecological perspective, excluding eukaryotes from a metagenomic analysis compromises the ability to assess the microbial community in its entirety. A possible approach to bypass the problem of large amounts of non-coding eukaryotic sequence data consists in obtaining molecular data at the RNA level. Given the recent VL outbreak in Argentina and with the ultimate goal of identifying possible biological control agents, this study used unbiased high-throughput pyrosequencing technology [44] to compare the diversity of the taxonomic groups associated with wild male and female adult Lu. longipalpis from endemic (Posadas, Misiones) and non-endemic (Lapinha Cave, Minas Gerais) VL locations in Argentina and Brazil, respectively. As in this study phlebotomine sandflies were considered environmental samples, the term "associated with" was used here in its broadest sense, referring to a wide variety of possible interactions ranging from casual associations due to random environmental contact (e.g., plant pathogenic fungi spores adhering to the hairy surface of the sandflies when sugar-feeding on plants) to closer pathogenic or symbiotic interactions (e.g., protists that parasitise phlebotomines or permanent gut microbiota, respectively). This analysis revealed the presence of sequences from bacteria, fungi, protists, plants and metazoans.
Ethics statement
This study was carried out in strict accordance with the recommendations in the Manual for the Use of Animals/FIOCRUZ (Manual de Utilização de Animais/FIOCRUZ) of Fundação Oswaldo Cruz, FIOCRUZ, Ministry of Health of Brazil (National decree Nr 3,179). The protocol was approved by the Ethics Committee for the Use of Animals of the Fundação Oswaldo Cruz - FIOCRUZ, Ministry of Health of Brazil (Nr 242/99).
Field sampling and specimen preparation
Lu. longipalpis specimens from the non-endemic VL location, Lapinha Cave (Minas Gerais, Brazil), situated in the Sumidouro National Public Park, were kindly provided by Dr. Paulo Pimenta (Laboratory of Medical Entomology, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, FIOCRUZ). Sandflies from this location were chosen as reference because they have been extensively studied. Lu. longipalpis specimens from the endemic VL location, Posadas (Misiones, Argentina), where they occur in high density, were kindly provided by Dr. María Soledad Santini and Mr. Enrique Adolfo Sandoval (Research Network for Leishmaniasis in Argentina, REDILA, and Posadas Municipality Quality of Life Department).
Captures were made using CDC light traps [45] on the 15th and 26th of May 2009 in the Lapinha Cave and in Posadas, respectively. The Lapinha Cave (S19 33 42.42 W43 57 34.96) is a network of interconnected caves located in a vast tropical savanna ecoregion called cerrado, characterised by great plant and animal biodiversity. The trap was left 50–80 cm above ground level in an external small annex cave (2 mt long) where a chicken was kept to attract the sandflies and as a source of food (see Table 1 for a detailed description of the site). Posadas, the densely populated capital city of the province of Misiones, is located in the subtropical fields and grasslands ecoregion. In the Posadas area this ecoregion contacts the Paranaense forest and has a savanna-type landscape. The trap was installed in the peridomicile of a worst-case scenario homestead (domestic animals, dense vegetation, nearby spring) (S27 23.266 W55 53.403) (see Table 1 for a detailed description of the site).
Sandflies were transported alive in a nylon cage to the corresponding laboratories in Belo Horizonte (Minas Gerais) and Posadas (Misiones) and no mortality was registered on arrival. Other insect species were captured together with the sandflies including hymenopterans, lepidopterans and mosquitoes. Sandflies were killed at low temperature, identified and separated according to sex, and stored alternatively in Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) or RNAlater® (Qiagen). A total of four groups of 100 sandflies each, two per location, were separated and named according to: SS1, females from the Endemic VL location (EVL females); SS2, EVL males; PP1, females from the Non-Endemic VL location (NEVL females); and PP2, NEVL males.
Sample preparation
Individual samples were ground in Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) with a Teflon pestle and total RNA was immediately extracted, according to the manufacturer's instructions. Total RNA was amplified using a modified sequence-independent amplification protocol [46]. Briefly, M-MuLV Reverse Transcriptase (Fermentas, Vilnius, Lithuania) was used for a first-strand reverse transcription which was initiated with a random octamer linked to a specific primer sequence (5′-GTT TCC CAG TAG GTC TCN NNN NNN N-3′) [47]. cDNA was then amplified with the Expand Long Template PCR System (Roche) using a 1∶9 mixture of the above primer and a primer targeting the specific primer sequence (5′-CGC CGT TTC CCA GTA GGT CTC-3′) [48]. The following profile was used: initial denaturation cycle at 94°C for 2 minutes; five low stringency cycles with denaturation at 94°C for 30 seconds, 25°C for 30 seconds and 68°C for 6 minutes, were followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds and 68°C for 6 minutes and a final extension cycle at 68°C for 5 minutes. Pooled samples were submitted for high-throughput pyrosequencing (Macrogen Inc., Korea).
Sequence accession numbers
Reads were submitted to the NCBI Sequence Read Archive (SRA) (submission SRA026595) under accessions SRR089611 (adult EVL female Lu. longipalpis; Posadas, Misiones, Argentina; SS1), SRR089612 (adult EVL male Lu. longipalpis; Posadas, Misiones, Argentina; SS2), SRR089613 (adult NEVL female Lu. longipalpis; Lapinha Cave, Minas Gerais, Brazil; PP1) and SRR089614 (adult NEVL male Lu. longipalpis; Lapinha Cave, Minas Gerais, Brazil; PP2).
Sequence analysis
Reads ranged in size from approximately 100 to 1200 base pairs (bp) (350 bp average). Raw sequence reads were trimmed to remove sequences derived from the amplification primer. With the purpose of reducing database search efforts and improving the homology detection sensitivity [49], Cd-hit [50] was used to generate non-redundant nucleotide datasets but these represented less than 1% in every case (data not shown). For this reason, singlet sequences were used for the nucleotide database search. Non-redundant (nt) and non-human, non-mouse ESTs (est-others) NCBI databases last modified on 23/04/10 and 25/04/10, respectively, were downloaded locally (ftp://ftp.ncbi.nlm.nih.gov/blast/db/). After trimming, singlet sequences were compared to these databases using BLASTN (nucleotide homology) [51], with a 1e-50 cutoff E-value. The resulting BLAST alignments were analysed and classified according to their taxonomical hierarchies using custom applications written in Mathematica (Wolfram Mathematica 7; available upon request). 16S sequences were confirmed by alignment to type-species 16S rRNA sequences from the Ribosomal Database Project (http://rdp.cme.msu.edu/) [52]–[53]. Hits for every taxon were individually revised and confirmed and only those which showed unequivocal results were included in the final analysis. Fisher's Exact Test [54] (p<0.05) was used to establish the significance of sequences in the different samples using a custom application written in Mathematica (Wolfram Mathematica 7; available upon request).
The vast majority of reads obtained for each sample corresponded to Lu. longipalpis sequences (∼85%) and an important fraction showed no significant hits in the homology searches against the different databases (∼14%). Hits which corresponded to taxa other than Lu. longipalpis represented less than 0.2% of the total reads.
Results for each taxon were organised separately in this section. Figure 1 emphasises the treatment of these vectors as environmental samples. It shows an overview of the workflow used in this study, summarising and associating information on the sampling sites and on the taxa identified by sequence homology in each adult Lu. longipalpis sample. A detailed ecological description of both sampling sites is given in Table 1. Figure 2 integrates and summarises results for all the samples, indicating the taxa that were identified in each case, the species that were found for each taxon and the number of sequences for each species. Table S1 lists all the reads that showed significant hits and a brief description of each hit.
This figure summarises and associates sampling site characteristics with taxa identified in male and female adult Lu. longipalpis from both locations. In all cases figures are only schematic and not an exact representation of either the sampling sites, phlebotomine sandflies or identified taxa. A) Shows the most significant ecological characteristics of both phlebotomine capture site locations in Argentina and Brazil: Posadas and Lapinha Cave, respectively. Only those animal species confirmed at the time of sampling were represented schematically (Table 1 includes a detailed list of animal and plant species in both locations). (B) Total RNA was extracted from male and female adult Lu. longipalpis specimens and amplified using sequence independent amplification (see Methods section). (C) Shows the different taxa identified by sequence homology in all four samples (bacteria, fungi, protists, metazoans and plants). Taxa are represented schematically and the particular species identified for each taxonomical group are not shown, except in the case of metazoans (see Figure 2 for a detailed list). Grey rectangular boxes group taxa found in female adult Lu. longipalpis. White rectangular boxes group taxa found in male adult Lu. longipalpis. VL: Visceral Leishmaniasis.
Results for all the samples were integrated and summarised in this figure, which indicates all the taxonomical groups that were identified, the species that were found for each taxon and the number of sequences for each species. Solid black lines group the different taxa (plants, protists, metazoans, bacteria and fungi). For bacteria, the dotted black lines separate and differentiate Gram+ and Gram- rods. The different species that were found are named beneath each column. The number of sequences for each species is indicated on the top part of each cylinder. SS1: adult females from the Endemic VL location (EVL); SS2: adult EVL males; PP1: adult females from the Non-Endemic VL location (NEVL); PP2: adult NEVL males.
BLASTN analysis of the high-throughput sequencing data identified bacteria in females from both locations (SS1 and PP1) and in NEVL males (PP2) (Figures 1 and 2). Bacteria were identified mostly by homology to completely sequenced bacterial genomes (7 reads), followed by rRNA genes (4 reads) and lastly to plasmid sequences (2 reads) (Table S1). Ten different bacterial types were identified, six of which showed homology at the species level (five to genomic sequences and one to plasmid sequences) and four to diverse uncultured environmental samples (three to 16S rRNA genes and one to genomic sequences). The bacterial composition was different and unique in every case and included sequences from Ralstonia pickettii, Anoxybacillus flavithermus, Geobacillus kaustophilus, Streptomyces coelicolor, Propionibacterium acnes, Acinetobacter baumannii, uncultured Veillonella sp. and uncultured bacterium clones isolated from environmental samples (cow faeces, wetland soil and water) (Figure 2; Table S1).
The totality of identified bacteria showed a predominance of Gram negative rods (53.8%, 7 reads) and a significant proportion of Gram positive bacteria (38.5%, 5 reads), in accordance with previous studies [18]–[20], [55]. Of all the bacterial sequences that were identified in this study, only A. baumannii, which was found in NEVL males (PP2), had been previously identified in adult female Lu. longipalpis. This species had been isolated from female laboratory reared specimens from the same non-endemic VL location (Lapinha Cave) [20] and from wild female specimens from endemic VL locations in Brazil (Jacobina, Bahia, and São Luís, Maranhão) [18].
Fungi were only found in NEVL males and females (PP1 and PP2) (Figure 1). A total of four different species was identified by homology to rRNA genes (Figure 2; Table S1). These differed between males and females and have not been found to date associated with phlebotomines. The identified species included Peronospora conglomerata, Cunninghamella bertholletiae, Mortierella verticillata and Toxicocladosporium irritans (Figure 2; Table S1).
Protist sequences were only identified in EVL female and male specimens (SS1 and SS2) (Figure 1), of which the vast majority (99.8%) were found in males (Figure 2; Table S1). Protists were identified by homology to sequenced rRNA genes (360 reads, 61.4%), cDNA (208 reads, 35.5%) and chromosomal sequences (18 reads, 3.1%) (Table S1). Ten species and one genus of apicomplexan parasites were identified that parasitise Diptera (Ascogregarina taiwanensis, Psychodiella chagasi), birds (Eimeria tenella, Sarcocystis falcatula, Sarcocystis cornixi), mammals (Cryptosporidium muris, Sarcocystis arieticanis, Besnoitia besnoiti, Plasmodium falciparum, Plasmodium berghei) and reptiles, birds and mammals (Sarcocystis sp.) (Figure 2; Table S1). Most of the apicomplexan sequences (64.8%, 379 reads) were homologous to the mammalian parasites C.muris, S. arieticanis, B. besnoiti, P. falciparum and P. berghei. Of these, more than half (53.6%, 203 reads) were homologous to published cDNA libraries and the rest to rRNA genes (41.7%, 158 reads) and chromosomal DNA (4.7%, 18 reads). The second most numerous group of apicomplexan sequences was homologous to the avian parasites E. tenella, S. cornixi and S. falcatula rRNA genes (28.2%, 165 reads) and the rest were homologous to the dipteran parasites A. taiwanensis and P. chagasi rRNA genes (7%, 41 reads).
Metazoan sequences (mammals, birds and reptiles) were also found in all the samples and included Homo sapiens, Gallus gallus and Anolis carolinensis (Figures 1 and 2). Homo sapiens was identified by homology to genomic chromosomal sequences (16 reads). Gallus gallus was identified by homology to genomic chromosomal sequences (46 reads), cDNA (18 reads) and rRNA genes (1 read). Anolis carolinensis was identified by homology to cDNA (1 read) (Table S1). Human sequences were found in males and females from both locations, whereas chicken sequences were found in NEVL females (PP1) and EVL males (SS2) and lizard sequences were only found in NEVL females (PP1) (Figures 1 and 2).
A total of ten different plant species were identified in males and females from both locations, namely Elaeis guineensis, Capsicum annuum, Juglans hindsii, Artemisia annua, Brassica napus, Vitis vinifera, Solanum tuberosum, Nicotiana tabacum, Oryza sativa and Rhapidophyllum hystrix (Figures 1 and 2). All plant sequences were identified by homology to cDNA libraries (56 reads), except for R. hystrix which was identified by homology to rRNA genes (1 read) (Table S1). EVL males and females showed a greater number of species (5 and 6 species, respectively), followed by NEVL males (4 species) and lastly NEVL females (3 species) (Figure 2). However, these differences were significant (p<0.05) only between females from both locations (Table 2). EVL females showed the highest number of plant sequences (23 reads), followed by EVL and NEVL males (13 reads) and lastly NEVL females (8 reads) (Figure 2). The only case in which these differences were not significant (p<0.05) was between EVL females and NEVL males (Table 3).
C. annuum (bell pepper) was found in EVL males and females and in NEVL males. E. guineensis (African oil palm) was found in EVL females and NEVL males and females. S. tuberosum (potato) was found in EVL males and females and in NEVL females. J. hindsii (Northern California walnut) was found in EVL males and females and A. annua (sweet wormwood) was found in males from both locations. R. hystrix (needle palm) and B. napus (rapeseed) were only found in EVL females and V. vinifera (grapevine) was only found in EVL males. O. sativa (rice) was only found in NEVL females and N. tabacum (tobacco) was only found in NEVL males (Figure 2).
This is the first study to survey taxa associated with an infectious disease vector applying an unbiased and comprehensive metagenomic approach. To ensure an unbiased description of the microbial community, the rationale chosen for this study included the extraction of total RNA and sequence-independent amplification. Total RNA was extracted from wild adult male and female Lu. longipalpis from an endemic (Posadas, Misiones) and a non-endemic (Lapinha Cave, Minas Gerais) VL location in Argentina and Brazil, respectively, and submitted to high-throughput pyrosequencing [44]. Given the high background level of vector sequences (∼85%), this approach proved to be very sensitive since it enabled the identification of taxa present in percentages up to 0.00036%. Moreover, as the different taxa were identified by homology to both rRNA and mRNA, the chosen approach was adequate for the objectives of this study.
The bacterial community identified in females from both locations and in NEVL males was distinct in every case. The only results in common with previous studies of gut microbiota from wild and laboratory reared female Lu. longipalpis and laboratory reared female P. duboscqi [18]–[20], [55], included the prevalence of Gram negative bacteria and the identification of A. baumanni, which in this study was found in NEVL males. Although previous reports established an essential basis for phlebotomine gut microbiota current knowledge, in these studies bacteria were identified using standard bacteriological methods. Consequently, those descriptions did not consider the remaining 99% of unculturable environmental microbes [56]. Hence the differences with this study, which applied a culture independent unbiased high-throughput approach that bypassed cloning of environmental DNA.
Interestingly and in accordance with results from this study, in previous reports the proportion of bacteria isolated from wild dipterans has been low. Studies on the midgut microbiota of wild mosquitoes, isolated bacteria from less than 50% of the specimens and the numbers of bacteria varied between individuals [57]–[58]. In a more recent study which used culture dependent and independent screening of field-collected Anopheles, bacteria were found in 15% of the mosquitoes, few of the mosquitoes harboured more than one bacterial species and only one species was found in more than one mosquito [23].
Only one bacterial type was found in EVL females (SS1), which corresponded to an unculturable bacterium originally isolated from cow faeces (Figure 2). Furthermore, a sequence match against RDP [52] indicated high similarity with Alistipes sp., a Gram negative anaerobic bacteria found in human faeces (Figure 2). Five bacterial species were found in NEVL females (PP1), four of which were Gram positive (Figure 2). Of these species, R. pickettii, A. flavithermus, G. kaustophilus and S. coelicolor, were originally isolated from contaminated lake sediment, waste water [59], deep-sea sediment [60] and soil [61], respectively. Interestingly, A. flavithermus and G. kaustophilus are thermophilic. Even though R. pickettii 12D was originally isolated from contaminated lake sediment, it is a ubiquitous microorganism found in water and soil [62] and is emerging as an opportunistic pathogen found in a wide variety of clinical samples [63]. P. acnes [64] is a universal inhabitant of human skin and is found at high population densities on the fat-rich areas of the face, scalp and upper trunk [65]. Four bacterial types were found in NEVL males (PP2), 50% of which were Gram negative (Figure 2). One of these bacterial types was the multidrug-resistant A. baumannii [66], which is recovered from natural environments and has emerged as an important opportunistic pathogen worldwide [67]. Another of the bacterial types corresponded to uncultured Veillonella sp. isolated from human skin [68]. The other two bacteria were uncultured bacterial types originally recovered from environmental samples. In one case, BLASTN analysis indicated homology both to an uncultured bacterium from a water sample (Atlantic Ocean) and to Leifsonia xyli, a sugar-cane pathogen [69]. The other bacterial sequence corresponded to a proteobacterium clone isolated from wetland soil [70].
In summary, bacteria identified in this study are ubiquitous in the diverse environments these sandflies frequent (faeces, soil, water, sediment, plants, human skin) and which were present in both sampling sites (Figure 1, Table 1). Hence, possibly they were indicative of the behavioural patterns and feeding habits of these sandflies and are probably part of their transient microbiota. However, more in depth research is required to determine these interactions.
Four different species of fungi were found in NEVL Lu. longipalpis (PP2 and PP1), which differed between males and females (Figure 2). In previous reports for P. papatasi and P. tobbi, mycoses with a high incidence rate were found in the guts and malpighian tubes of wild specimens. Similar fungi cultured from guts of laboratory reared P. papatasi were identified as A. sclerotiorum and S. cerevisiae [25]. Microsporidians, which are highly pathogenic for some insects [71], have also been found parasitising neotropical sandflies [32], [72]. The two species identified in this study in NEVL females were P. conglomerata [73], a plant pathogen (mildew), and C. bertholletiae [74], a common soil fungus and a rare cause of zygomycosis in humans. On the other hand, the species found in NEVL males were M. verticillata [75], a genus commonly found in soil and a zygomycete which also causes zygomycosis in animals, and T. irritans [76], which belongs to a genus of foliar pathogens [77]. Given the very high vegetation density in the Lapinha Cave area (Figure 1, Table 1), a possible scenario is that plant pathogenic spores adhered to the sandflies' hairy surface during sugar-feeding on infected plants. This suggested Lu. longipalpis has a putative capacity of casual dispersal of plant pathogens, among others (see below). In conclusion, fungi identified in this study are found ubiquitously in the environments frequented by sandflies (plants and soil), which were abundant in the sampling site (Lapinha Cave), and so were probably indicative of their sugar-feeding habits and behavioural patterns.
Protist sequences were only found in EVL male and female specimens, of which the vast majority were found in males (Figure 2). Nearly 90% of the identified apicomplexans corresponded to coccidians (genera Cryptosporidium, Eimeria, Sarcocystis and Besnoitia, 88.7%) and the rest to gregarines (genera Ascogregarina and Psychodiella, 7%) and haemosporidians (Plasmodium spp., 4.3%). The absence of leishmanial sequences was not unexpected considering the rate of infection of sandflies with Leishmania is generally very low (0.01–1%) [78], even in endemic areas [5].
Gregarines have been reported in over 20 species of sandflies and Ascogregarina spp. have only been described in mosquitoes [79]. Given A. taiwanensis sequences were found in EVL males in this study, this could indicate that the parasite also infects Lu. longipalpis. Genus Psychodiella comprises 3 species with host specificity to phlebotomine sandflies: P. chagasi, P. saraviae and P. mackiei [35], [79]. In the New World, only P. chagasi and P. saraviae have been found parasitising Lutzomyia spp. and P. chagasi seems to infect a large range of neotropical species [33]–[36]. In this study, P. chagasi sequences were found in EVL males. The exact pathology caused by gregarines is unknown, but in Lu. longipalpis the parasite can reduce longevity and egg production and the level of parasitaemia can reach over 80% in laboratory colonies [80]. Notwithstanding, the use of P. chagasi as a control method in the field has not been considered efficient because the parasite seems to have a limited range and a minimal effect on sandfly biology under natural conditions [81]. The fact that in this study P. chagasi was found in randomly caught wild specimens, suggested it could be a more efficient control method under natural conditions than what was previously reported.
The free-living oocyst stage of coccidians is discharged by infected animals through their faeces. Sandflies are found around human habitations and breed in specific organic wastes, exploiting the accumulation of organic matter produced by domestic animals and poor sanitary conditions such as faeces, manure, rodent burrows and leaf litter [5]. Since the EVL sampling site (Posadas) was a worst case-scenario homestead which included dense vegetation, various domestic animals and abundant organic matter (Figure 1, Table 1), this could account for the presence of these parasites in EVL males and females. On the other hand, female sandflies suck blood from different animal species including humans, bovines, pigs, equines, dogs, opossums, birds, various rodents and reptiles [5], [82] and, additionally, some Plasmodium spp. that parasitise lizards have been found in sandflies [37]. In the field it is common to see lek-like aggregations of males and females assembled on or near hosts where blood feeding and mating occur [83]–[84]. This behaviour could account for the presence of haemosporidian sequences (blood borne parasites) in EVL males. Nevertheless, as these sequences were found only in males, this suggested males, and not females, would be the primary source of Plasmodium spp. Furthermore, P. falciparum was recently identified by PCR in faecal samples from gorillas [85] and considering the EVL sampling site had a significant amount of organic matter (Figure 1, Table 1), it is highly feasible that males acquired these microorganisms from human faeces. Alternatively, EVL males could have acquired these microorganisms by contact with other vectors bearing P. falciparum (i.e., Anopheles spp.) during transportation. In any case, these results suggest that, due to their behavioural patterns, Lu. longipalpis could be implicated in the casual dispersal of parasites of medical and veterinary importance.
Human sequences were found in males and females from both locations, whereas chicken sequences were found in NEVL females and EVL males and lizard sequences were only found in NEVL females (Figures 1 and 2). Lu. longipalpis is ubiquitous in dwellings where sanitary conditions are poor and domestic animals, such as dogs, chickens and pigs, are kept in and around the houses. In this kind of environment, the sandfly tends to congregate at outdoor sites, including animal sheds, where leks easily form on abundant, stationary hosts [7], [84]. In this context, as previously mentioned, the EVL location (Posadas) was a worst case-scenario homestead that kept dogs, chickens and a cat, had dense vegetation and a nearby spring (Figure 1, Table 1). In the NEVL location (Lapinha Cave), a chicken was kept to attract sandflies and as a source of food (Figure 1, Table 1). Furthermore, 35 species of lizards can be found in the Minas Gerais region [86]. As female sandflies blood feed on different animal species such as birds, reptiles and humans [5], [82], the presence of these sequences in females was not unexpected. Contrariwise, it was unexpected to find human and chicken sequences associated with males. Nonetheless, this could be due to their previously mentioned behavioural patterns of aggregation and courtship, where male sandflies are often seen over the host where they form leks, attracting females for a blood meal and increasing their chance for mating [83]–[84]. Alternatively, the trap itself was another area of close contact between male and female sandflies and with other potential vectors of medical importance. Consequently, males could have acquired these sequences by contact during transportation.
A total of ten different plant species were identified in males and females from both locations. Capsicum annuum (bell pepper), Elaeis guineensis (African oil palm) and Solanum tuberosum (potato) were identified in three of the four Lu. longipalpis samples. Juglans hindsii (Northern California walnut) was found in both EVL samples and Artemisia annua (sweet wormwood) was found in both male samples. Rhapidophyllum hystrix (needle palm) and Brassica napus (rapeseed) were only found in EVL females and Vitis vinifera (grapevine) was only found in EVL males. Oryza sativa (rice) and Nicotiana tabacum (tobacco) were only found in NEVL females and males, respectively (Figure 2). As adults from both sexes feed on sugars from different plant sources [12], this diversity could be indicative of the different feeding preferences and/or food source availability. Moreover, as sugar meals are not composed primarily by cells, plant RNA could also have originated from other sources. Namely, pollen dispersed by wind could have adhered to the sandflies' hairy surface or, alternatively, these vectors could be casual pollinators during sugar-feeding.
For a more comprehensive understanding of these results, a few limitations of the chosen approach should be considered. In the first place, homology searches are circumscribed to the number and quality of sequences in the databases at the time of analysis. The relatively high number of sequences which showed no significant hits (∼14%) was a clear indication of this. Moreover, if the query corresponds to a given organism that has not yet been sequenced, the hit will probably coincide with the most related organism found in the database. Notwithstanding and given this situation, the results from the homology search will provide a close approximation to the real case-scenario. Another aspect is that, similarly to a previous study [48] and in order to obtain as much environmental data as possible, specimens were neither surface cleaned nor dissected to extract their guts. As they were not surface cleaned, some (or all) of the identified taxa could have been surface contaminants, acquired during transportation by contact with other captured species, i.e. hymenopterans, lepidopterans and mosquitoes, or during manipulation in the lab. In the latter case, even though samples were manipulated with extreme care, this was still a potential source of contamination. Nevertheless, if contamination occurred during manipulation, it was plausible to expect the same contaminating species in males and females from the same location (when specimens were identified and separated according to sex) or from both locations (when total RNA was extracted). The only species present in all four samples was Homo sapiens and, consequently, contamination during manipulation was a possibility for these reads. Notwithstanding, as some biological control agents act by surface contact, such as Beauveria bassiana [39], [87], and since the ultimate goal of this study was to identify possible biological control agents for this neglected infectious disease vector, had the specimens been surface cleaned, this information could have been lost together with other valuable environmental data. On the other hand, as the gut was not separated from the rest of the specimen and, consequently, they were not analysed independently, it was not possible to classify the observed taxa in putative surface contaminants and gut inhabitants, among others. Therefore, the possible role of putative permanent gut residents could not be inferred, such as influence on the insect development cycle or on the parasite transmission ability. Nevertheless, even a careful extraction process would not preclude the possibility of cross-contamination between the gut and the rest of the specimen and/or loss of information. In this sense, the chosen approach ensured that no data was lost and, notwithstanding the aforementioned limitations, enabled the identification of taxa that could putatively influence sandfly development and which have become the target of ongoing studies to determine their significance and location in the sandfly.
Finally, the diversity of bacterial, fungal, protist, plant and metazoan sequences found in this study in wild adult Lu. longipalpis from endemic and non-endemic locations, mostly confirmed their feeding habits and behavioural patterns. Nevertheless, it also suggested that these vectors could possibly be a chance source of dispersal of various animal and plant diseases, such as coccidiosis and malaria. This is particularly significant since the geographical distribution of this vector is undoubtedly expanding [7]. The fact that RNA was obtained from these animal and plant pathogens would indicate that they were biologically active, but this cannot be determined with the present results and further studies must be performed to establish the significance of these findings. The identification of gregarines in wild Lu. longipalpis specimens could indicate that these parasites are a more efficient control method under natural conditions than what was previously suggested [81]. This is specially meaningful as studies on biological control of phlebotomines are still scarce and its practical application seems to be limited to the adult VL vector stage [16]. The employment of biolarvicides in the field is difficult due to the diversity of habitats in which this vector can reproduce and evidence that Lu. longipalpis larvae appear to be thinly dispersed and not concentrated in any particular microhabitat [17]. Nevertheless, as the number of samples analysed in this study was limited, a greater number of specimens must be studied to establish the significance of these results. Current studies are underway to analyse the presence and establish the significance of the taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those taxa implicated in the biological control of this vector and to the etiologic agents of animal and plant diseases.
Supporting Information
Table S1.
This table lists all the reads that showed significant hits in the homology database searches (1e-50 cutoff E-value). Reads are grouped according to the sample they were identified in (PP1, SS1, PP2 or SS2). The information provided for each read includes: read accession number (number assigned to each read by NCBI SRA), read number (number assigned to each read when the samples were sequenced), hit accession number (of the hit with the highest E-value), a brief description of the hit and its E-value as indicated by the homology search.
We gratefully acknowledge the Research Network for Leishmaniasis in Argentina (Red de Investigación de Leishmaniasis en Argentina, REDILA), the Posadas Municipality Quality of Life Department (Secretaría de Calidad de Vida, Municipalidad de Posadas) and the dedicated assistance of Dr. Ma. Soledad Santini and Mr. Enrique Adolfo Sandoval who captured, identified and separated the wild male and female Lu. longipalpis specimens from Posadas and provided detailed information on the capture location. We gratefully acknowledge the Laboratory of Medical Entomology of Dr. Paulo Pimenta from the Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, FIOCRUZ, for capturing, identifying and separating the wild male and female Lu. longipalpis samples from Lapinha Cave and for providing detailed information on the capture location. We also thank an anonymous reviewer for very helpful suggestions, corrections and comments on a previous draft of the manuscript.
CBM, LAD and RVRP are members of the CONICET research career (Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina).
Author Contributions
Conceived and designed the experiments: CBM. Performed the experiments: CBM. Analyzed the data: CBM. Contributed reagents/materials/analysis tools: CBM LAD RVRP. Wrote the paper: CBM. Developed the custom applications used in the analysis: LAD. Critically revised the manuscript: LAD RVRP.
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|A Tale about a Tax: Social Security
|Steyn: Just Shrug, It's a UN Atrocity
by Christopher Chantrill
February 14, 2005 at 3:00 am
IF YOU ARE a Brit leftie, you must believe that selection or streaming in the government school system is evil. Now the Blair government is starting to think of returning to some sort of streaming and selection in the government schools. Of course, the whole process will be properly controlled by government experts and elaborate procedures and safeguards. And it must all be done without raising the hackles of the Labour Party faithful.|
Christopher Chantrill blogs at www.roadtothemiddleclass.com. His Road to the Middle Class is forthcoming.
But I saw a man yesterday who knows a fellow who had it from a chappie
that said that Urquhart had been dipping himself a bit recklessly off the deep end.
Freddy Arbuthnot
Dorothy L. Sayers, Strong Poison
Civil Societya complex welter of intermediate institutions, including businesses, voluntary associations, educational institutions, clubs, unions, media, charities, and churchesbuilds, in turn, on the family, the primary instrument by which people are socialized into their culture and given the skills that allow them to live in broader society and through which the values and knowledge of that society are transmitted across the generations.
Francis Fukuyama, Trust
Tear down theory, poetic systems... No more rules, no more models... Genius conjures up
rather than learns... Victor Hugo
César Graña, Bohemian versus Bourgeois
We have met with families in which for weeks together, not an article of sustenance but potatoes had been used; yet for every child the hard-earned sum was provided to send them to school.
E. G. West, Education and the State
When we began first to preach these things, the people appeared as awakened from the sleep of agesthey seemed to see for the first time that they were responsible beings, and that a refusal to use the means appointed was a damning sin.
Finke, Stark, The Churching of America, 1776-1990
When we received Christ, Phil added, all of a sudden we now had a rule book to go by, and when we had problems the preacher was right there to give us the answers.
James M. Ault, Jr., Spirit and Flesh
A writer who says that there are no truths, or that all truth is merely relative, is asking you not to believe him. So dont.
Roger Scruton, Modern Philosophy
As far as the Catholic Church is concerned, the principal focus of her interventions in the public arena is the protection and promotion of the dignity of the person, and she is thereby consciously drawing particular attention to principles which are not negotiable...
[1.] protection of life in all its stages, from the first moment of conception until natural death; [2.] recognition and promotion of the natural structure of the family... [3.] the protection of the right of parents to educate their children.
Pope Benedict XVI, Speech to European Peoples Party, 2006
At first, we thought [the power of the West] was because you had more powerful guns than we had. Then we thought it was because you had the best political system. Next we focused on your economic system. But in the past twenty years, we have realized that the heart of your culture is your religion: Christianity.
David Aikman, Jesus in Beijing
But the only religions that have survived are those which support property and the family.
Thus the outlook for communism, which is both anti-property and anti-family, (and also anti-religion), is not promising.
F.A. Hayek, The Fatal Conceit
Conservatism is the philosophy of society. Its ethic is fraternity and its characteristic is authority the non-coercive social persuasion which operates in a family or a community. It says we should....
Danny Kruger, On Fraternity
Families helped each other putting up homes and barns. Together, they built churches, schools, and common civic buildings. They collaborated to build roads and bridges. They took pride in being free persons, independent, and self-reliant; but the texture of their lives was cooperative and fraternal.
Michael Novak, The Spirit of Democratic Capitalism
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©2007 Christopher Chantrill


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Asphalt Fumes
OSHA Standards
State Standards
There are twenty-eight OSHA-approved State Plans, operating state-wide occupational safety and health programs. State Plans are required to have standards and enforcement programs that are at least as effective as OSHA's and may have different or more stringent requirements.
There are currently no specific OSHA standards for asphalt fumes. Exposures to various chemical components of asphalt fumes are addressed in specific standards for the general and construction industries, such as personal protective equipment (PPE). This section highlights OSHA standards, preambles to final rules (background to final rules) and letters of interpretation (official letters of interpretation of the standards) related to asphalt fumes.
General Industry (29 CFR 1910)
- 1910 Subpart I, Personal protective equipment
- 1910.132, General requirements [related topic page]
- 1910.134, Respiratory protection [related topic page]
- Appendix A, Fit testing procedures (Mandatory)
- Appendix B-1, User seal check procedures (Mandatory)
- Appendix B-2, Respirator cleaning procedures (Mandatory)
- Appendix C, OSHA respirator medical evaluation questionnaire (Mandatory)
- Appendix D, Information for employees using respirators when not required under standard (Mandatory)
Construction Industry (29 CFR 1926)
- 1926 Subpart E, Personal Protective and Life Saving Equipment
Letters of Interpretation
- Citations for the wearing of short pants engaged in hot tar and asphalt construction work. (April 17, 1997). Compliance officers will be instructed to balance the need for personal protective clothing, such as long pants, during hot asphalt operations against the need for clothing that is appropriate for severe environmental conditions such as extremely warm weather.
- Natural or mined asphalts do not fall within the scope of the Coal Tar Pitch Volatiles Standard. (May 26, 1983). Natural or mined asphalts do not fall within the scope of the Coal Tar Pitch Volatiles (CTPV) Standard. Again, the CTVP Standard, as stated in 29 CFR 1910.1002, applies to emissions only from products that are residues of distillation processes. Thus, "natural" or mined asphalts, which are not distillation residues, do not fall under the CTPV standard.
- Petroleum asphalt removed from coverage under the Coal Tar Pitch Volatiles Standard. (April 8, 1983). OSHA believes that removal of petroleum asphalt from coverage under the Coal Tar Pitch Volatiles (CTPV) Standard was appropriate based on both scientific and technical considerations. Data indicate that there are qualitative and quantitative chemical differences between the volatiles arising from coal tar and asphalt. OSHA does recognize, however, that asphalt fumes can present a health hazard to employees.
- Search all available letters of interpretation.
Occupational Exposure Levels
There is no OSHA standard or permissible exposure level of asphalt fumes, although a 5 mg/m3 permissible exposure limit (PEL) was proposed in 1992. OSHA's quantitative risk assessment estimated a significant risk of lung cancer among exposed workers at levels as low as 0.2 mg/m3. The American Conference of Governmental Industrial Hygienists (ACGIH) currently recommends a Threshold Limit Value (TLV) of 0.5 mg/m3 as an 8-hour time weighted average. NIOSH currently recommends no more than 5 mg/m3 for a maximum exposure per 15-minute short-term exposure in addition to assigning a carcinogenicity designation. See NIOSH Pocket Guide to Chemical Hazards, Appendix A - NIOSH Potential Occupational Carcinogens for the latest policy and guidelines for this designation.Back to Top


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HOME > Global 30 Japan Education Fair (U.S.A)
Global 30 Japan Education Fair (U.S.A)
1. WelcomeThe Global 30 Project Secretariat is pleased to announce that the Global 30 Japan Education Fair in U.S.A will take place at the Japanese Culture and Community Center of North California (JCCCNC) in San Francisco on October 12, and at the University of California, Berkeley on October 13, 2013. The Global 30 Japan Education Fairs are organized worldwide to allow students, their guardians, teachers, and school counselors to have face-to-face consultations with the faculty members and university staff regarding all aspects of study and life in Japan. This year, we are delighted to bring our Education Fair to the United States for the first time.
The Fair is organized in close cooperation with the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government. The Global 30 Project was initiated by MEXT five years ago to draw more qualified international students to Japan. The member universities of the Global 30 Project offer degree programs in English (Bachelor, Master's, Professional Master's and Doctorate Degrees).
2. Date, Time, and VenueSan Francisco Date: October 12, 2013 (Sat) Time: 10:00 am – 4:00 pm *Doors open at 9:45am Venue: Japanese Cultural and Community Center of Northern California (JCCCNC) 1840 Sutter St, San Francisco, CA 94115 Directions * Street parking is available around the center with normal street restrictions. Additional public parking is available at the Japan Center garage and on Webster Street. University of California, Berkeley Date: October 13, 2013 (Sun) Time: 10:00 am – 4:00 pm *Doors open at 9:45am Venue: University of California, Berkeley Clark Kerr Campus Conference Center Directions / Campus Map * Parking is available and the cost is $10.00 for a parking permit. The parking permits can be purchased on the day of the event.
3. Program(1) University Booths (Plenary Hall) Representatives from the Global 30 universities will be available at their booths for any questions that prospective students and family may have. You are welcome to visit and speak with them regarding programs, admission procedures, scholarships, and advantages of studying in Japan.
(2) Study in Japan: Information Sessions (Lecture Room) Concurrently with the one-on-one sessions, a series of information sessions will take place at the breakout room. We welcome your participation in this Q & A panel session to become familiar with all aspects of studying in Japan. *The program is subject to change without prior notice
The Global 30 Program Overview
Member Universities of the Global 30 Project
Session I: Student Life and Career Development (Student Life /Japanese Language Course/ Housing/Career Development and Counseling Service etc)
Session II: How Do I Apply? (Application Requirements/Admission Process/Tuition Fees/ Scholarships etc)
4. Who Should Attend
High school students interested in pursuing undergraduate degrees at Japanese universities
University students interested in pursuing graduate degrees at Japanese universities
Parents, Guardians, School Counselors and Teachers (Japanese proficiency not required)
5. Attending Universities
Member universities of the Global 30 Project: Tohoku University, University of Tsukuba, The University of Tokyo, Nagoya University, Kyoto University, Osaka University, Kyushu University, Keio University, Sophia University, Meiji University, Waseda University, Doshisha University, and Ritsumeikan University


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A place to learn about the latest and upcoming books, contests, author interviews, book reviews, and all around fun.
Thursday, June 28, 2012
Pictures of You
Revuew by Nancy
After the death of her philandering husband, Eva saw no reason to stay at home while his relatives glared at her. She had never fit in their circle and, while packing up Stephen's things, she had found a ticket to Brazil for an expedition in search of a topaz stone. Why not? It's not as if she had a good reason to stay.
At the airport, she spies a good-looking man who stops her breath. Then she is met by a taxi driver and hurried on to the next leg of her journey – the small town where the expedition will begin. She is dropped off in front of a house where the housekeeper doesn't speak English – nor do the neighbors. After walking the entire village, she ends up where she started, hot, dehydrated and a bit angry. Turns out this is the right home, after all.
Eva and her companions soon meet their leader, Roberto de Carvalho, the mysterious man at the airport! She wakes up in his bed with him kissing her. Not the tour introduction she had imagined! Maria had put her up there to rest and couldn't explain that it was the boss' room.
She is overwhelmed by the town, the jungle, the food and by Roberto himself. Until he opens his mouth and calls her a "black widow" searching immediately for a new husband. She is the only woman on the trip and, through her passion for photography, immediately becomes the video person for them all.
They are searching for the Epsinhaco Topaz from a map given to Roberto by an old friend. Do they find it? Possibly. Will Roberta stop nagging Eva for every little thing? NEVER! She is falling in love and he could care less. This is an e-book unveiling of some of Barbara Delinsky's early works and, while I don't think I've read her before, I am looking forward to more of her titles coming my way. The characters are wonderfully done, the tropic scenery beautifully described and the storyline is exceptional.
Labels: Barbara Delinsky
1 comment:
- Harvee Lau said...
I'd read it for the setting and descriptions, though the love story sounds predictable. The mystery may be more interesting, however. Glad you liked it!
- June 28, 2012 at 5:46 AM
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