text:
COVE CITY � The Thursday night Cove City Friends of the Library meeting turned into a celebration to recognize Elinor Hawkins, the library�s founder, with the state�s highest civilian honor � induction into the Order of the Long Leaf Pine.
COVE CITY � The Thursday night Cove City Friends of the Library meeting turned into a celebration to recognize Elinor Hawkins, the library�s founder, with the state�s highest civilian honor � induction into the Order of the Long Leaf Pine.
About 75 people attended the surprise event, but thousands of people in North Carolina have heard the voice of Hawkins reading to them some of the first stories they ever heard.
She was the voice of Telestory Time, now on the air for more than 55 years, starting from 1952 to 1958 on WFMY-TV Channel 2 in Greensboro, and again in 1963 on Channel 12 in New Bern where it continues to this day on WCTI-TV.
The Saturday morning �Reading Can Be Fun� hour has encouraged many children to enjoy reading and use of the Craven-Pamlico-Carteret Regional Library System to expand their lives in many directions.
But some of them may not know the impact she has had on the availability of the books they read themselves as she came to rural coastal Carolina and brought the resources of libraries and access to information about almost anything through the regional library system she created.
Under her leadership, eight new libraries were constructed in the three counties and for 42 years she established and directed a regional library system.
Thursday�s venue was small but fitting for the grand dame of books for this area of Eastern North Carolina.
More lovely and high spirited that the stereotypical �madam librarian,� encouraging reading has been a passion and her gracious but composed manner that helped get the facilities and resources.
�I am overwhelmed,� said Hawkins, after Cove City Library Board Chairwoman Lynn Gonzalez, presented the plaque. �I loved my work for 42 years but could never have done it without my family, my loving husband Carroll, my son John � who grew up in the library,� his wife and their children Stephen and Leslie. She named a long list of others, both living and now dead, including �my mother, who said �Never give up.��
She had special thanks for 50-year regional library system secretary, the late Hayden Jones, and the first public library board chairman Bill Sheron, as well Cove City librarians Nancy Chase, Barbara Avery and Becky Willis.
At 82, like a walking book, off the top of her head Hawkins recounted the pages of library system facilities growth, from a closet in Cove City Hall, the 1770 John Wright Stanley House in New Bern, the old train station in Carteret County, the Woman�s Club building in Vanceboro, the Sheriff�s office in Havelock, a locked cabinet in the Newport Fire House, and a small room in the N.C. Aquarium in Bogue Banks.
�I love my work and I never expected an award like this and I am very thankful,� Hawkins said.
Awarding the honor certifying �the integrity, learning, and zeal� of Hawkins was in the last chapter of former Gov. Bev Perdue, as she signed it Dec. 31, 2012. She sent regrets that she could not attend the presentation.
Born in Morgantown, W. Va. Elinor Hawkins came to Eastern North Carolina after marrying Cove City native Carroll Hawkins. They met while she met while a graduate student in library science at the University of North Carolina-Chapel Hill.
He was by her side Thursday after more than 60 years of marriage. Now a retired N.C. Department of Revenue employee, he and Elinor Hawkins spent a few years in Greensboro before put down roots in his farming community home where they helped many see a larger world through books and community service.
New Bern-Craven County Librarian Joanne Straight said, �The Order of the Long Leaf Pine is a well deserved honor, recognizing Elinor Hawkins� many years of service and commitment to the citizens of North Carolina. She worked tirelessly to improve libraries services for Craven, Pamlico and Carteret counties.�
Sue Book can be reached at 252-635-5665 or firstname.lastname@example.com. Follow her on Twitter@SueJBook.


text:
We live in very challenging times. As we go through school we are taught that if you work hard and go to college, then you will get a good job and live happily ever after.
We live in very challenging times. As we go through school we are taught that if you work hard and go to college, then you will get a good job and live happily ever after.
Sadly, the reality is life for twenty somethings isn't quite that simple. The job market is still bad enough that many of the jobs traditionally reserved for recent college graduates are being filled by people with more experience.
There seems to be a proverbial log jam in the job market. Employers are not promoting or expanding at fast enough rates to provide jobs for young professionals.
The cascading effect is people with bachelor's degrees are forced to take jobs that either do not match their degree or do not require a degree at all. This problem is not exclusive to people with bachelor's degrees. Law school graduates can expect to be competing against literally hundreds of applicants for entry level jobs; most will not in fact find employment that requires a law degree.
Absent dramatic changes in the next few years, the above problem will cause the student loan bubble to burst. It is easy to understand why this will happen. If graduates cannot find good paying jobs, then they will not be able to make their student loan payments.
The amount of student loan debt has been on the rise for years. College tuition increases explain some of this. However, it is not the only cause for student loan debt.
Sadly many students, particularly those who attend community colleges, borrow much more than they actually need to cover tuition costs. This money theoretically is to be spent on other school expenses, but in reality is spent on frivolities such as big screen TV's, video games, clothes, etc.
Most of the student loan debt is financed by the Federal Government. So, when people are unable to pay back these loans taxpayers are on the hook for the expense.
How can we solve these problems? The simple and easy answer would of course be to unleash an economic boom. Unfortunately, at least in the near term, that seems unlikely to happen so let's focus on the things we can control.
First and foremost we need to stop pushing every man, women, and child toward a college education. The reality is there are plenty of good paying jobs that do not require a degree and not everyone is meant to go to college. Our education system needs to focus more on skilled trades.
When Governor Engler was in office an emphasis was put on vocational trades. There were many courses offered in these areas and some students were able to come out of high school with an actual skill.
The Granholm Administration got away from this. Instead of expanding and improving these programs they switched the focus back to strictly preparing students for college.
What's needed to rectify this situation is a policy and culture change. We need to not simply pay lip service to skilled trades programs; we need to make them a focal point of high school education. Skilled trades classes need to be placed on the same level as math, science, and reading.
Making this change will help our education system more accurately reflect our society. Not every student is the same. Thus, we should not expect a cookie cutter solution to work in our education system.For those that do choose to attend college; improvements to that system can be made as well.
Chief among them would be changes to the courses required to obtain a college degree. For the typical bachelor's degree, a student is required to complete exactly 30 credits in their major and 90 other credits for a total of 120 credits.
So, exactly 25% of a student's time, money, and effort is spent on courses directly related to their major. The other 75% is spent on prerequisites, electives, and other college-wide requirements. Simply taking the 30 credits required for the major is not an option; the degree requires the full 120 credits for completion.
Is it any wonder why a degree costs so much?
Advocates of the current system will submit that the other requirements make for a more well-rounded graduate and citizen. To that I would draw from my experience. I took all of these classes and I can say with certainty I learned a lot more from real life than I ever did from these classes.
Most of the time I would simply grit my teeth and write what I knew the professor wanted to hear in an effort to get through the course. Is that really what college should be about?
Should students, parents, and taxpayers be paying for courses that only serve the purpose of advancing a particular universities or person's worldview?
If we reform this system by cutting the amount of credits required for a college degree in half, imagine the impact this would have on our society. Students would be graduating in half the time at half the cost with the same amount of course work in their major.
Unless we address these issues I fear young people will struggle to get ahead for generations to come.
James Lower is an active member of the Ionia County Republican Party and former County Commissioner. He holds a Bachelor's Degree in Economics from Michigan State University and is a current MBA candidate at Grand Valley State University.

subset: commoncrawl


text:
Cover Preview:
Mr. Bean's Holiday (2007) R1 DVD CoverCategories: Movie DVD Covers & Labels
This cover has been viewed 200 times. Vote & check the gallery bellow or leave a comment.
4260 × 28520 votes
Cover Preview:
This site uses Akismet to reduce spam. Learn how your comment data is processed.


text:
See what's available at your address
Get the latest entertainment for less with our Fetch + premium channel pack bundle.
Get Fetch now
Low call rates with no frills or hidden costs. It's great value, plain and simple.
View Phone plans
Our SIM-only mobile plans let you keep your old mobile number, while giving you beefy call and data value.
View Mobile plans
Connect better with
award-winning service
We love the 'net... So we've made it our job to connect more Australians to faster broadband, phone and web services and backed it all up with friendly customer service staff who know our products inside out.
View all of our awards.


text:
Laramie's Ivinson Memorial Hospital, Now UCHealth Affiliate
AURORA, Colo. (AP) — University of Colorado Health has reached a management agreement making Ivinson Memorial Hospital in Laramie, Wyo., an affiliate of UCHealth.
The agreement was signed in October but took effect Tuesday.
It allows Ivinson and University of Colorado Health to share expertise and resources, but Ivinson will remain an independent hospital run by the seven-member board of the Albany County Hospital District.
Ivinson's top executives will be employed by UCHealth. Ivinson retains responsibility over its own finances.
UCHealth combines Memorial Hospital in Colorado Springs, Poudre Valley Hospital, Medical Center of the Rockies, Colorado Health Medical Group and University of Colorado Hospital.


text:
smartparts needed a new banner ad design and created a contest on 99designs.
A winner was selected from 39 designs submitted by 13 freelance designers.
see description
We need to design a poster for a shop, to be put outside on the street, to attract customers.
Physical dimensions are 60cm wide and 85cm high.
Resolution of the file should be suitable for high-quality print.
Design should have 8 sections, as follows:
1.TITLE - It has to contain the phrase "PC and Laptop Repair and Accessories"
2. GENERAL IMAGE - something or somebody cheerful with a laptop, something suggesting that buying and repairing the laptop inside the shop will make the customer very happy
3. SUPPORTED BRANDS - It has to contain the phrase "Supported Brands" and contain images of laptop brand names, such as Toshiba HP Sony IBM Dell Samsung Apple Acer.
4. BATTERY - It has to contain the word "Batteries" and the following image.
5. AC ADAPTER - It has to contain the word "AC Adapters" and the following image.
6. LCD SREEN - It has to contain the word "LCD Screens" and the following, or similar, image.
7. MEMORY - It has to contain the word "Memory" and the following, or similar, image.
8. TELEPHONE - It has to contain the phrase "tel.: 0207 255 3505"
An example of layout can be found here.
However, you do not have to stick to it if you have something better in mind.
The design has to capture attention, has to make people want to go inside.
That is all, good luck.
- see above
- see above


text: I have a number of VPN clients connecting to a Ubuntu 12.04 server (This was set up using Zentyal 3.0). I need to connect many more. I would like to be able to address the clients from the server by client hostname. The Openvpn server gives all the clients IPs in the 184.108.40.206/24 range. The Server running Openvpn has a 192.168.1.20 address. The DNS server is up and running but it does not resolve the host names of the clients on the VPN. How can I set up the DNS on the server so that I can ping, ssh etc., and connect to the Clients by hostname, from the server, rather than by IP address. Thanks, Jodel


text:
Sunday, August 17, 2008
Pickled Sliced Cucumber Salad Recipe
I've given you so many sweet recipes recently that it's definitely time to share a lovely savoury recipe instead.
Here's a quick pickled cucumber salad that would keep nicely a week or so in your fridge. It's gutsy, has got lots of flavor and crunch, and tastes lovely. I nibbled on it straight from a bowl, but it would make a nice side salad to some grilled meat as well.
The only downside is that you only get about two 500 ml jars out of this amount. If you give one to your mum or a dear friend, then you're just left with one jar yourself, which is no way enough ... For best result, use small crisp cucumbers and not the long supermarket ones.
Pickled Sliced Cucumber Salad
(Marineeritud kurgisalat)
1 kg small crisp cucumbers
2 large onions
1 garlic clove, finely chopped
1 Tbsp sugar
1 Tbsp sea salt
1 Tbsp vinegar (30%)
freshly ground black pepper
1 tsp Dijon or Estonian mustard
1 to 2 large dried dill "blossoms" (ask from the market)
Slice the cucumber and onions into thin slices - I used my kitchen mandoline. Mix all ingredients in a large bowl, then cover with cling film and leave to stand at room temperature (that's 18-20 C) for about 5 hours.
Shake the bowl or mix the salad couple of times, so the seasonings would distribute evenly.
Divide into sterilised jars and close the jars (there should be enough liquid now to cover the cucumber slices).
Keeps for a week or two in the fridge.
9 comments:
- Thredahlia said...
Küüslaugukurgid :) - jube mõnusad, kas või keset talve! Mul on selle tarvis alati isegi paar tilliõisikut sügavkülmas.
- 8:12 pm, August 17, 2008
- Kitchen Flavours said...
Wow, fresh and healthy looking salad. Nice pic also. First time here in your blog. U have rocking blog.
- 8:56 am, August 18, 2008
- [eatingclub] vancouver || js said...
This is great! I've been looking at pickled cucumbers these past few days: for some reason, I have the urge to pickle some cucumbers (never done it before) and this is certainly a lovely salad.
- 9:24 am, August 21, 2008
- Jeanne said...
Funny - I've been thinking of making a pickled cucumber salad recently, despite the distinctly un-summery weather we're having! Might make this for a BBQ in 2 weeks time - thanks!
- 7:08 pm, August 22, 2008
- Elizabeth said...
This looks like a great and refreshing summer salad! If I can't find dried dill blossoms, would chopped fresh dill be a good substitute or something else? Thanks!
- 8:38 pm, August 23, 2008
These were easy to prepare, and have been coming out of the refrigerator every evening for dinner. I used dill seeds and freeze dried dill weed.
- 1:34 am, August 26, 2008
- gastrogeek said...
this looks just like the cucumber salad my austrian best friend's mother used to make when we were children. I can't wait to make it.
- 11:16 pm, September 21, 2008
- viagra online said...
I love to eat salads, because are more healthy than if you eat something fried. I recommend you Chili Lime Fruit Salad, that's always I eat at lunch, and that as this one is really nutritive.
- 11:10 pm, July 09, 2010
- Genie said...
This is perfect for the few cucumbers coming out of my garden right now. Thanks so much for the recipe! It sounds a bit like what my mom used to make in the summers when I was a kid. It was a mix of vinegar, water, and sugar with sliced cucumbers and onion in it. Left in the fridge for a day... We all loved it.
- 4:36 pm, August 14, 2015


text:
Manufacturer: HellermannTyton
Part No: SNP2410C2
Description: Snapper Hose Clamps, .991"/1.115" Min/Max Diameter, POM, White, 100/pkg
Country Of Origin: US
HellermannTyton snapper hose clamps are designed to provide a quick, ratcheted closure reducing installation time. The design also allows the clamp to be easily removed and re-applied when necessary. Snappers are made of high performing materials providing stability in a variety of diverse environments. Available in 32 standard sizes.
Add to Cart
Add to Sample Cart
Add to Quote Cart
Add to Parts List
This product is no longer in stock.
Please notify me when available.


text:
12.11.12: The CCRI Knights men's basketball team traveled to Haverhill, MA to play the Knights of Northern Essex Community College in a Region XXI non-league game. The Knights of CCRI defeated the Northern Essex Knights 84-70. With the victory CCRI improves to 7-4 on the season and 3-2 in Region XXI play.
Knights' Co-Captain Isaac Medeiros (Johnston, RI) had his best night as a CCRI Knight in a battle of Knights. Medeiros charted a monster double-double with game highs in both points with 28 and rebounds with 15 to lead the Knights of CCRI over the Knights of Northern Essex CC. CCRI jumped out to a quick 7-0 lead to start the game and closed the first half with an 18-6 run to go into halftime leading 47-35. CCRI rode the hot shooting of Medeiros who had 15 of his 28 points in the first half. He had help in the first half from Rob Hazard (North Providence, RI) and Co-Captain Desmond Williams (Torrington, CT) who each scored 11 first half points. CCRI utilized multiple pressure defenses to build their lead up to 22 points with the score 61-39 just five minutes into the second half. Northern Essex would not go quietly as they chipped away at the lead to get it down to 13 points 76-63 with just 2:43 remaining in the game. CCRI closed the game with an 8-7 run to win by the final score of 84-70.
Leading the way for the Knights was Co-Captain Isaac Medeiros who was unstoppable scoring 28 points while ripping down 15 rebounds to go with two blocked shots, two steals and an assist followed by Co-Captain Desmond Williams with 19 points, including four 3-pointers, four assists and a blocked shot. Also helping CCRI was Rob Hazard with 13 points and three assists and Co-Captain Bryan Yarce (Cranston, RI) added eight points, four assists and two steals.
Head Coach Rick Harris commented after the game: "We continued to show improvement on both offense and defense tonight. The team has responded well to this stretch of four games in six days."
The Knights next game will be Saturday when they will travel to Charlestown, MA to play the Bulldogs of Bunker Hill Community College at 6:00pm for another Region XXI non-league game.
The Community College of Rhode Island, New England's largest comprehensive community college, enrolls nearly 18,000 students in credit courses and thousands more in non-credit and job training classes. Its athletic program boasts 12 varsity programs and an academic support program for all student-athletes.
This page developed and maintained by CCRI Athletics. Send comments and suggestions to firstname.lastname@example.com.


text:
June 25, 2008
Do you feel like a frumpy mother and want to become a hip mom? Is your skin sagging, hairstyle drab or clothing stuck in a time warp? As we age, we want to continue to feel and look young, but it helps to know a few secrets. Dr. Phil is joined by Robin, who reveals her tips for looking younger, feeling better and getting slimmer.
Lisa, Sally, Joanne, Evelyn and Karlyne are moms in their 40s and 50s who say they are noticing the effects of age, and their hair, skin and weight are causing them to look older than they feel. Robin arranges for this group of moms to meet with her team of experts to get on their way to discovering the fountain of youth.
Learn how to jump-start your transformation!
Surprise Trip
After weeks of working hard on their new fitness and beauty regimens, the five women are whisked away in a limousine. They have no idea their next stop is Robin's front door for a girls-only champagne brunch!
See the other surprises Robin has in store.
Shopping Lesson
Robin is joined by her daughter-in-law, Erica, and Entertainment Tonight fashion guru Cojo for a shopping trip to L.A.'s famous store, Kitson, where she teaches you how to look fantastic and youthful at any age.
If you're over 40 and think you're too old to dress young and hip, think again!
Cojo's Anti-Aging Style Tips Cojo offers his wardrobe dos and don'ts. Plus, he shows solutions for clothing disasters that add decades to looks.
Are you making mistakes when you get dressed?
Mom Makeovers Revealed!
After five weeks of eating healthier, working out more and taking better care of their skin, the women receive the final touches to their new looks, including an age-appropriate, fashionable outfit selected for for them by Robin and Cojo. See which mom took off 18 inches!
The women reveal their age-defying makeovers for the first time on Dr. Phil's stage.
Robin's Secrets Revealed
To help the five moms lose weight, restore their youthful appearance and get their hormones in check, Robin arranged for them to visit the experts she personally uses.
See the team enlisted to help, and find out their specialties.


text:
Breaking Bones
Unless you're NFL legend Ronnie Lott ... you can't feel Rashad Johnson's pain -- but you can see it ... 'cause now there's a photo of the incredibly gruesome finger injury the NFL safety suffered Sunday.
If you haven't heard ... the Arizona Cardinals player crushed his left middle finger during the game against the New Orleans Saints -- but didn't realize he'd actually lost the tip until he took off his glove ... and partial digit was left behind.
Late Monday ... Johnson tweeted a pic of the photo, saying ... "Blessed to be able to still go out and do what I love this season. #thankful #trulyblessed"
Yes, he's planning to play this weekend -- which will seem even more amazing once you see the pic of the bloody finger (if you dare).


text:
Axis & Allies miniatures terrain, european front
Flag -tokens to indicate target area
Wooden bridge WIP
Flag -tokens to indicate target area
Wooden bridge WIP
Pacific terrain is addition to my AAM terrain collection. A lot of the terrains used in these games are some, but there are several new components which make the terrain look completely different.
This terrain is going to show you step by step how I'm doing my winter war gaming terrains for Axis & Allies miniatures. I'm going to post the photos for start, and write more text later about creation process. You can find some details about the terrain creation process in Axis & Allies miniatures ww2 town terrain, WIP -post.
This post shows you detail about constructing models and terrain bases for ww2 town terrain meant to be used with Axis & Allies miniatures. You can see photos of the ready terrain in another post. You can see details about construction phases by bringing the mouse pointer on the photo.
My earlier victory area flags have been quite popular, and because of a request, I've made a new set of flags. I haven't been able to print them myself, so the photo below is from dano903 at http://www.axisandalliesunderground.com and http://community.wizards.com . If you're interested in these flags prepared and based, ask him!


text:
For 2012 subscriptions, there will be no price increases for the journals, and conditions for customers will remain at their current level. As of 2012 online access will be via our new platform De Gruyter Online which will be launched at the end of 2011. (19/9/11)
For 2012 subscriptions, there will be no price increases for the journals, and conditions for customers will be the same as for 2011. (29/9/11)
This page is primarily for the information of CAUL/CEIRC members who wish to subscribe to this publisher's products and/or services. It provides information about offers to CAUL and CAUL-specific pricing, and links to relevant pages on the publisher's own web site. For other information, please go directly to Berkeley Electronic Press.
See also Offers to CAUL/CEIRC - Licences - Technical/Sales Support - Statistics
bepress 2011 titles list with pricing & collection (6/3/11)
bepress 2010 journals titles list, with pricing (7/1/11)
Alphabetical Journal List (bepress, 7/1/11)
Digital Commons a hosted institutional repository platform that comes with a unique suite of applications. Digital Commons is delivered as a single, integrated solution and fully hosted by bepress. There are no servers to install/maintain allowing clients focus on the repository content and faculty outreach/services and not on fast-changing technology.
The many advantages of Digital Commons include such features as:
Services include:
Bepress can provide you with a production-ready Digital Commons within 2-3 weeks.
bepress 2011 licence, included in order form (7/1/11)
bepress 2011 response to CEIRC survey re licence conditions (7/1/11) [Members only]
De Gruyter acquires 67 journals from Berkeley Electronic Press (19/9/11 - see spotlight)
Contact: Helen Rohr de Gruyter Asia–Pacific (29/9/11)
Please note that all 2012 renewals, claims and correspondence regarding these journals should be sent to the following address:
Walter de Gruyter Inc.
North American Subscription Office
PO Box 830350
Birmingham AL 35202-0350
Email: firstname.lastname@example.com
Members-only documents may be viewed from the display at the end of this page.
For current pricing see Walter de Gruyter eJournals pricing for CAUL (31/10/12)
bepress ResearchNow Full Access 2011 proposal to CAUL (7/4/11, updated 8/4/11, confirmed for 2012, 3/11/11) [Members only]
bepress ResearchNow Full Access 2011 pricing for CAUL (6/3/11, confirmed for 2012 3/11/11) [Members only]
bepress Journals Price List and Subscriptions (12/9/07)
bepress 2008 Prices and News (12/9/07)
Berkeley Electronic Press eJournals offer to CAUL (via DA, 22/4/08, updated 26/6/08)) [Members only]
Order form and license terms (23/4/08)
Package discount pricing 2005 (30/9/05)
2005 Journals List (30/9/05)
Preliminary offer to CAUL (29/4/03)
Digital Commons
For previous offers to CAUL, see the ProQuest page (23/7/08)
Copyright 2009 Council of Australian University Librarians


text:
I got the Uruk Hai Army set and did some army building, ending up with this:
Lord Godfrey now actually has a Northern Garrison to command.
The grand total of my High Elf faction. The middle sorceress will probably be peach-ified once I get the parts.
It's hard to think of Uruk-Hai as anything else but I think I managed to customize them pretty well. I also think the Uruk armour looks hilarious and good on skeletons.
Close-up of the Uruk swords for GoD.
Re-assembled Scallywags and the two leaders of my reformed Half-Elf Barbarian faction.
Essentially Elves who mixed with Barbarians, some retain the Elf peach skin while others retain the beardlessness.
Some are more Elf-like than barbarian, like these archers.
These swordsmen are pretty much the Elite.


text:
Palm Desert Felony Charges Defense Attorneys
Felony vs. Misdemeanor
In general, a misdemeanor crime is any crime that may carry a punishment that may include up to a year in jail, fines and forfeiture, but does not carry a sentence in a federal or state prison. A felony conviction, on the other hand, is any crime that may result in a prison sentence of one year or more in a state or federal penitentiary. The court standards for evidence and defense are typically different for misdemeanors and felony charges, as well. This is where a Palm Desert Felony Charges Defense Attorneys can help, give us a call to go over your legal rights.
Misdemeanor charges may include major traffic infractions that result in an accident, violations of municipal and county statutes, and lesser property crimes. Felony crimes are likely to involve a more complex process of pre-trial motions and court dates. Federal felony charges involve a different standard of evidence and timeline for legal motions than California state felony charges.
Whether you have been charged with a misdemeanor or felony crime in Palm Desert, your Palm Desert Felony Charges Defense Attorneys will need experience to ensure that you have the best possible opportunity to protect your rights and your future. Make no mistake, a conviction for a misdemeanor might not include a serious prison sentence, but the conviction will have a long-term effect on your record.
What is the difference between a felony and a misdemeanor?
Felony: Felonies expose the individual charged to the potential of a prison sentence and prison sentences can be as long as a life sentence. Felonies may also carry additional ancillary sanctions such as eliminating the ability to vote or extended probation periods.
Felony DUI: Under California law, a felony DUI actually refers to a serious misdemeanor DUI charge that can be the result of driving while intoxicated, and causing an accident that results in injury or death of another person. In addition, a driver with a record of repeat DUI convictions may be charged with a felony DUI, even if no accident or injury resulted from the offense. Under California law, a felony DUI charge also requires proof of negligence resulting in the accident. A conviction may result in a prison sentence.
Misdemeanors: Usually charged by a written complaint and may include up to one year in jail and a fine up to $1,000. Typical misdemeanor crimes include charges such as DUI, disorderly conduct, solicitation of prostitution, and domestic violence. Misdemeanor crimes can be raised to the level of a felony charge if the District Attorney feels that sufficient evidence exists to prove significant harm to a victim or property. You have the right to waive your right to appear in person, and your attorney may appear to represent you at court hearings.
Contact John Patrick Dolan A Palm Desert Felony Charges Defense Attorneys
If you or a loved one is charged with a felony or misdemeanor criminal offense in the Palm Desert area, it is important that you seek the council from an experienced Palm Desert Felony Charges Defense Attorneys specialist immediately. Attorney Dolan knows the local court system, police agencies and prosecutors. Contact our offices to schedule a free consultation about your misdemeanor or felony charge right away. He is available in his office (760) 775-3739 or on his cell (562) 824-4007 whenever you need him.


text:
Schistosomiasis affects more than 200 million people globally. The pathology of schistosome infections is due to chronic tissue inflammation and damage from immune generated granulomas surrounding parasite eggs trapped in host tissues. Schistosoma species are unique among trematode parasites because they are dioecious; females require paring with male parasites in order to attain reproductive maturity and produce viable eggs. Ex vivo cultured females lose the ability to produce viable eggs due to an involution of the vitellarium and loss of mature oocytes. In order to better understand schistosome reproductive biology we used data generated by serial analysis of gene expression (SAGE) to identify uncharacterized genes which have different transcript abundance in mature females, those that have been paired with males, and immature females obtained from unisexual infections. To characterize these genes we used bioinformatics, transcript localization, and transcriptional analysis during the regression of in vitro cultured females. Genes transcribed exclusively in mature females localize primarily in the vitellocytes and/or the ovary. Genes transcribed exclusively in females from single sex infections localize to vitellocytes and subtegumental cells. As female reproductive tissues regress, eggshell precursor proteins and genes involved in eggshell synthesis largely have decreased transcript abundance. However, some genes with elevated transcript abundance in mature adults have increased gene expression following regression indicating that the genes in this study function both in eggshell biology as well as vitellogenesis and maintenance of female reproductive tissues. In addition, we found that genes enriched in females from single sex infections have increased expression during regression in ex vivo females. By using these transcriptional analyses we can direct research to examine the areas of female biology that are both relevant to understanding the overall process of female development and worm pairing while determining novel therapeutic approaches directed at the maturation of female schistosomes.
Author Summary
Schistosomiasis is a chronic, debilitating disease that affects over 200 million people globally. The pathology associated with schistosomiasis is caused by host immune responses to parasite eggs. Therefore, it is imperative to identify pathways responsible for controlling worm reproductive biology. Schistosome females must be in constant contact with male parasites in order to achieve reproductive maturity. The process of pairing and reproductive maturation in female worms is poorly understood, in part, because it does not occur outside of the host. In addition, when female schistosomes are removed from their mammalian host they regress to an immature state. In this study our goal was to characterize a unique set of genes in Schistosoma mansoni whose transcript abundance differs in mature and immature female worms. We found that the genes with higher transcript abundance in sexually mature female worms were expressed in female reproductive tissues, while those transcripts enriched in sexually immature worms were present in sub-surface somatic cells. Transcript abundance of the selected genes changed dramatically when females were removed from their host. These findings inform new approaches to study female worm biology and will provide insights into the processes of worm pairing and reproductive maturation.
Citation: Cogswell AA, Kommer VP, Williams DL (2012) Transcriptional Analysis of a Unique Set of Genes Involved in Schistosoma mansoni Female Reproductive Biology. PLoS Negl Trop Dis 6(11): e1907. https://doi.org/10.1371/journal.pntd.0001907
Editor: Malcolm K. Jones, University of Queensland, Australia
Received: August 6, 2012; Accepted: October 2, 2012; Published: November 15, 2012
Copyright: © 2012 Cogswell 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: These studies were supported by National Institutes of Health-National Institute of Allergy and Infectious Diseases (NIH-NIAID) award R21AI097529. 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.
Schistosomiasis, caused by infections with trematode parasites from the genus Schistosoma, affects more than 200 million people worldwide [1]. Cercariae released by intermediate host snails penetrate the skin of the mammalian definitive host where they develop into schistosomula. Schistosomula migrate first to the lungs and then to the liver sinusoids. In the liver juvenile parasites begin blood feeding and pair prior to migrating to the mesenteric or rectal veins (S. mansoni and S. japonicum) or to the perivesical venous plexus of the bladder (S. hematobium) where they mature into adult parasites. Paired worms produce eggs that pass out of the host in the urine or feces. These eggs can also become trapped in immune-generated granulomas in host tissues, primarily the liver, bladder wall and intestinal epithelium, causing a strong Th2 immune response resulting in the pathology associated with schistosomiasis. Since eggs are central to the pathology of schistosomiasis and the transmission of the parasite to the intermediate host it is essential to improve our understanding of worm reproductive biology.
Schistosomes are unique among trematodes because they are dioecious, with worm sex being determined chromosomally [2]. Furthermore, females must pair with a male in order undergo complete sexual development and the production of infectious eggs. After pairing with a male, the female reproductive structures, mainly the ovary and the vitelline glands, undergo terminal differentiation. The vitellarium comprises two thirds of the mature female body and participates in a diverse array of functions. Vitellocytes undergo four distinct developmental phases. Stage 1 cells are the most immature and localize to the distal regions of the vitellarium farthest away from the vitelline duct. They possess few lipid droplets and ribosomes, both requirements for the synthesis of eggshell precursor proteins. The progenitor-like state of Stage 1 vitellocytes suggests that they are a stem cell population that undergoes mitosis and differentiation when females pair with males [3], [4]. Stage 2 and 3 vitellocytes represent intermediate stages of differentiation in which they are directed to a terminal cell fate, but are still synthesizing the necessary components required for mature vitellocyte functions. Stage 4 vitellocytes are terminally differentiated and are the largest cells in the vitellarium. They possess large lipid droplets and a large number of ribosomes. These cells contain vesicles with proteins that are required for eggshell synthesis, including shell precursor proteins and tyrosinase, an enzyme that cross-links the precursor proteins to form the eggshell, and are localized proximal to the vitelline duct allowing for their release into the duct which they traverse to reach the ootype where they merge with an oocyte and egg biogenesis begins.
The ovary also undergoes dramatic changes as maturation occurs. Unpaired females posses a highly coiled immature ovary entirely composed of oogonia, immature female germ cells [3]. Upon pairing, the oogonia undergo mitosis and meiosis to produce mature oocytes. Mature oocytes are round in structure and the nucleus is small compared to the cytoplasm [4], [5]. In mature females, the oogonia are restricted to the anterior ovary whereas the oocytes reside in the posterior ovary where they are released into the oviduct. As the oocytes traverse the oviduct they merge with vitellocytes in the ovo-vitelline duct leading to the ootype, where the egg is formed [5], [6].
The ootype, surrounded by the Mehlis' gland, is where the vitellocytes, sperm, and oocyte form an egg. The process of eggshell formation is rapid due to the presence of pre-made precursor proteins, but the signals leading to their release and eggshell formation have not yet been characterized. Egg formation is aided by secretions from the Mehlis' gland [7]. In addition, the vitellocytes are responsible for the synthesis of two subshell layers; the Reynolds' layer, localized proximally to the eggshell, and the von Lichtenberg's envelope, a syncytial epithelium surrounding the developing oocyte [7]. These subshell layers develop along with the miracidium after the egg is excreted from the gonopore.
Single sex schistosome infections do not occur in a natural setting, but can be generated by infecting laboratory hosts with cercariae produced from mono-miracidial infections of snails. Because cercariae are produced by asexual reproduction in sporocysts, the gender of the infecting miracidium determines the gender of the cercariae produced. In this way, we are able to study differences between age-matched female worms that have developed in the absence of male worms (adult female worms from single-sex infections, AFSS) and sexually mature, egg-producing females that have paired with a male and undergone complete sexual development (adult female worms from mixed sex infections, AFMS). There are striking morphological and histological differences between AFSS and AFMS parasites [8], [9]. AFSS are smaller than AFMS, the size difference being largely reflective of the undifferentiated vitellarium. The ovary of AFSS worms is shrunken and contains only oogonia [10] indicating that female germ cells are present but in the absence of unknown, male-derived signals these germ cells are incapable of undergoing divisions to produce oocytes. The vitellarium of AFSS parasites is smaller than in AFMS parasites and contains only stage 1 vitellocytes [8], again reinforcing the notion that female germ cells are present in AFSS, but without proper signals, stage 1 vitellocytes are not able to mature. Biochemical differences include decreased production of eggshell precursor proteins (e.g., p14, p19, p48) and tyrosinase in AFSS compared to AFMS [11]–[14]. After mature worms are removed from their mammalian host and cultured in vitro they lose the ability to produce viable eggs and AFMS reproductive tissue regress to an immature stage similar to those found in AFSS [11], [15], 16. This process is termed "regression".
Sequencing of the S. mansoni genome [17] has allowed research to shift from single gene-directed studies to global studies of various aspects of worm biology and pathogenesis. It is estimated that 40% of schistosome genes have no known function and have no homology to proteins in other genera [17]. Global transcription analysis during worm development using serial analysis of gene expression (SAGE) has been used to identify genes that have different transcript abundance during worm development and differentiation [18]. Here we investigate schistosome female reproductive biology and characterize genes differentially regulated in AFMS and AFSS worms through tissue localization and transcriptional analysis during seven days of regression. We find that many AFMS-enriched transcripts localize to the vitellarium and vitellocytes within the vitelline duct. We also find that while most genes are down-regulated in ex vivo females regardless of the presence of a male, removal of AFMS parasites from the host leads to an increase in transcript abundance for a select set of genes. Overall, these studies demonstrate that the involution of the vitellarium and the loss of reproductive maturity are governed by events occurring early during regression and provide evidence that female reproductive maturity is governed by signals provided both by the male parasite and the host microenvironment.
Materials and Methods
Ethics statement
This study was approved by the Institutional Animal Care and Use Committee at Rush University Medical Center (IACUC number 11-064; DHHS animal welfare assurance number A3120-01). Rush University Medical Center's Comparative Research Center (CRC) is operated in accordance with the Animal Welfare Act (Public Law (P.L.) 89–544) as amended by P.L.91–579 (1970); P.L.94–279 (1976); P.L. 99–198 (1985); and P.L 101–624 (1990), the Public Health Service's Policy on Humane Care and Use of Laboratory Animals (revised, 2002), the Guide for the Care and Use of Laboratory Animals (revised, 2011) and the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training. The CRC is registered with the Animal and Plant Health Inspection Service (APHIS) arm of the United States Department of Agriculture (USDA). The Institution has an Animal Welfare Assurance on file with the National Institutes of Health, Office of Laboratory Animal Welfare (OLAW), A-3120- 01. The facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The CRC is directed by the Senior Director of the CRC, a Doctor of Veterinary Medicine (D.V.M.) and a Diplomate of the American College of Laboratory Animal Medicine (ACLAM), who reports to the Associate Provost and Vice President for Research, who is also the Institutional Official for Animal Care and Use.
Parasites and animals
The Puerto Rican/NMRI strain of S. mansoni and female Swiss-Webster mice were used in all experiments. Mice were euthanized using Nembutal in sterile water with heparin and adult schistosomes were recovered by hepatic portal perfusion [19]. Eggs were purified from the livers of infected mice and hatched in pond water to obtain miracidia for mono-miracidial infections of juvenile Biomphalaria glabrata snails (strain NMRI). Snails were shed in individual wells five weeks post infection and mice were infected with cercariae shed from a single snail via tail exposure as described [19]. Seven weeks post infection mice were euthanized and worms were obtained as described above.
Identification of AFMS- and AFSS-enriched transcripts
Data on global transcription analysis during worm development generated by SAGE [18] was mined to identify transcripts with different steady-state abundance in AFMS and AFSS worms. The SAGE method captures 21-base pair tags that are used to uniquely identify the source gene from within the genome. Tags are isolated from the mRNA pool, concatenated, cloned, and sequenced. The population of tags defines patterns of expression of individual genes and quantification of all tags provides a relative measure of gene expression (i.e., mRNA abundance). A detailed description of SAGE can be found in [18]. SAGE tags were mapped to a predicted transcript using BLAST analysis of the available S. mansoni genomic (http://www.genedb.org/Homepage/Smansoni) and EST (http://compbio.dfci.harvard.edu) databases. To begin, we determined chromosomal location of the SAGE tag and then we identified any coding regions 5′ of the SAGE tag either predicted through GeneDB or confirmed through EST sequencing. To clone the sequences, SAGE tags (Table S1) were used as reverse primers in PCR along with the T3 primer present in pBluescript portion of the S. mansoni λ phage cDNA library to perform modified 5′ RACE PCR [20]. The product was cloned into pCRII vector and transformed into chemically competent Top10 Escherichia coli using the TOPO TA cloning kit (Invitrogen). In cases where the SAGE tag was associated with a predicted transcript we designed gene specific primers for PCR amplification. After sequence confirmation we designed additional primers in order to amplify a region of the gene to be used as a probe. Primers used for cloning, RT-PCR, and riboprobe synthesis are shown in Table S2. Protein domain identification and analysis was done using BLASTp, PSI-BLAST, and Interpro Scan.
Life-cycle stage transcriptional analysis
cDNA was synthesized from total RNA obtained from separated adult male and female parasites from mixed-sex infections, male and female parasites obtained from single sex infections, all seven weeks post infection, and liver-stage parasites 23 days post infection. Worms were homogenized in Trizol (Ambion) using a sterile dounce and RNA was isolated using a chloroform extraction followed by ethanol precipitation. RNA was treated with DNAse (Promega) and cleaned using the Zymo DNA-free RNA kit (Zymo). Concentration and purity of RNA was verified using a Nanodrop spectrophotometer and cDNA was synthesized using the iScript cDNA synthesis kit (BioRad) using random hexamer and oligo(dT) primers. Gene-specific forward primers were designed from the sequences identified above and used with the SAGE tags to amplify transcripts from freshly made cDNA. For genes that were not identified by SAGE, gene specific primers designed from the predicted open reading frames identified in the S. mansoni GeneDB were used in RT-PCR reactions. β-tubulin was used as a control gene. Primers are shown in Table S2. Following cDNA synthesis, RT-PCR was performed using Flexi GoTaq DNA polymerase (Promega) under the following parameters: an initial denaturation step of 95°C 1 min followed by 25–35 cycles of 95°C 30 sec, 48–54°C 1 min, 72°C for 1 min, and a final extension of 72°C for 7 min. Both the cycle number and the annealing temperatures were dependent on the gene of interest (Table S2).
Tissue localization of AFMS- and AFSS-enriched transcripts
Riboprobes were synthesized according to previously published methods [21]. Briefly, probes were synthesized from restriction enzyme digested DNA according to the orientation of the insert in pCRII using the Riboprobe synthesis kit (Promega) labeling with digoxigenin (Roche). Whole mount in situ hybridizations (WISH) were done as described [21]. Briefly, worms were fixed in 4% paraformaldehyde for 30 min and dehydrated in methanol. Following bleaching in 6% hydrogen peroxide in methanol to prevent tanning of the vitellaria, worms were permeabilized using Proteinase K (Ambion), incubated with pre-hybridization buffer (55% deionized formamide, 5× Saline Sodium Citrate (SSC), 1 mg/mL yeast RNA, 1% Tween 20) for 2 hours and then hybridized (pre-hybridization buffer with 10% dextran sulfate) with a riboprobe at 56°C overnight. Excess riboprobe was removed by washing in 2× and 0.2× SSC followed by blocking in K block (5% horse serum, 0.45% fish gelatin, 0.3% Triton-X 100, 0.05% Tween 20 in 1× PBS). Detection of bound riboprobe was done by incubation of worms in anti-digoxigenin alkaline phosphatase-conjugated antibody (Roche) diluted 1∶2000 in K block overnight incubation at 4°C. Removal of unbound antibody was performed by washing in maleic acid buffer (100 mM maleic acid, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 2 hr in six changes of buffer. After washing, specimens were incubated in alkaline phosphatase buffer (100 mM Tris, pH 9.5; 100 mM NaCl; 50 mM MgCl2; 0.1% Tween-20 brought up to volume with 10% polyvinylalcohol solution). Hybridization signals were detected by adding 450 µg/mL nitro blue tetrazolium (Roche) and 175 µg/mL 5-bromo-4-chloro-3-indolyl phosphate (Roche) in alkaline phosphatase buffer. After development, worms were washed in PBS then de-stained with 100% ethanol. Worms were mounted in 80% glycerol and imaged using a Zeiss AxioStar Plus microscope. Image analysis was done using ImageJ software.
Transcriptional analysis of female enriched genes during regression
Adult worms were obtained from infected mice as described above. All parasites were cultured in BME medium [22] supplemented with 0.1% gentamicin (Gibco), 2% fungizone (Fisher), and 1% penicillin/streptomycin (Cellgro) at 37°C/5% CO2. Ten unpaired females or 10 male-female pairs were cultured in 5 ml of medium in 6-well tissue culture plates. For seven day cultures, the media was changed every 48 hr. To relax male parasites and facilitate separation of females from males, worm pairs were briefly incubated in 2.5% Tricane (Sigma) in media. Only females that were paired for the entire duration of the study were used for further analysis. cDNA synthesis from mature and ex vivo cultured worms was performed as described above. Real time quantitative RT-PCR (qPCR) primers for all genes were designed using Integrated DNA Technologies qRT-PCR primer design software (Table S3). qPCR was done in triplicate using a 7900 HT Fast Real Time PCR System (Applied Biosciences) and EvaGreen Sso Fast master mix (BioRad). Expression changes were quantified using the 2−ΔΔCt method [23] normalized to β-tubulin expression. Dissociation curves were performed for each primer pair to verify specific amplification of one product.
Results and Discussion
Gene identification
Nineteen transcripts that were found by SAGE to be present at higher levels in AFMS and two with higher transcript abundance in AFSS were chosen for further analysis. Table 1 lists all genes studied, their SAGE tag counts, their GenBank accession numbers, potential functions or conserved protein domains and literature citations. In this study, the reference ID for each gene is its SAGE tag number. Bioinformatic analysis of these differentially expressed genes demonstrated that they fall into three major categories; genes containing conserved domains that have orthologs with other organisms, trematode specific genes, and genes that do not appear to have orthologs outside of the genus Schistosoma.
Genes with orthologs in other organisms have a diverse array of conserved domains but they fall into four distinct categories: signaling (11779 and 21733), nucleotide metabolism (10927 and 28488), protein-protein interactions (1610, 10401, and 33844) and mRNA regulation (11055). Trematode specific genes (8056, 10403, 10548, 10688, 10763, 11223, and 21110) have orthologs in other Trematodes (e.g., Clonorchis sinensis and Fasciola hepatica) but not in any other genera. Proteins encoded by these genes have not been carefully studied and thus the functions predicted by the domain architecture are not confirmed. Four AFMS genes (10435, 10617, 11088, and 11283) contain no conserved domains and appear to be Schistosoma specific (orthologs only found in S. japonicum). AFSS specific gene 6767 appears to be restricted to the Schistosoma spp., while 15402 has orthologs in a diverse array of eukaryotes. RT-PCR was done to verify expression patterns obtained by SAGE. All transcripts had higher abundance in the expected stage (AFMS or AFSS) (Fig. 1).
The same cDNA was used for the β-tubulin control and the gene specific PCR reactions. Lanes are: 1: mature female, 2: single sex female, 3: mature male, 4: single sex male, 5: liver stage parasites. All gels shown are normalized to 1 µg of cDNA and β-tubulin controls. The image shown is a composite of multiple agarose gels.
To better understand female reproductive biology, we included tyrosinase 1, p14 (an eggshell precursor protein), Fs800, a female associated secretory Cu/Zn superoxide dismutase (FsSOD), and a female specific mucin (FsMucin) [13], [14], [24]–[26] as controls. Fs800 and SOD have both been localized to the vitellaria using in situ hybridization while FsMucin has been localized to the reproductive duct anterior to the vitellaria using immunohistochemistry [21], [24], [26], [27]. Additionally, p14 and tyrosinase 1 have been localized to the mature vitellocytes using in situ hybridization, histochemistry, and immunohistochemistry [14], [21], [28].
SAGE tag 11055 was found to encode a cytoplasmic polyadenylation binding protein (CPEB). CPEBs have not previously been identified or characterized in S. mansoni; however, they are known to play important roles in the regulation of mRNA translation in oocytes in other organisms [29]–[31]. Because genomes in other organisms encode up to four CPEB proteins, we queried the S. mansoni sequence databases to determine if multiple CPEB proteins were present. We determined that there are two additional CPEB genes. CPEB2 is female specific, expressed in both AFMS and AFSS worms but not other stages examined, while CPEB3 was found to be expressed at similar levels in all life cycle stages examined (Fig. 1).
To better study oocyte maturation we examined the transcription of conserved germline helicase 1 (cgh-1), a transcript identified in Caenorhabditis elegans that regulates the expression of maternal mRNAs in developing oocytes [32]. In addition, cgh-1 is directly linked to apoptosis in the germline of C. elegans and is well conserved across invertebrate and vertebrate species [32], [33]. A recent study demonstrated that there is an increase in apoptosis in the vitellarium of regressed S. mansoni females [34]. In S. mansoni, cgh-1 mRNA is present at higher levels in females when compared to males and liver stage parasites (Fig. 1), a finding confirmed by mining the SAGE database (Table 1).
Transcriptional analysis of AFMS specific genes
In order to examine the tissue specific presence of each gene we localized transcripts using WISH. In order to gain a better understanding of the molecular changes that occur as the female reproductive tissues regress to an immature state, we monitored transcript abundance in cultured, ex vivo worms using qPCR. We hypothesized that molecular changes occur prior to the visible involution of the vitellarium and the ovary. To examine both early and late events in regression of the female reproductive tissues we cultured ex vivo worms for one, two, three, and seven days. Females were cultured both in the presence and absence of male worms to determine if pairing status affected transcript abundance during regression.
In a previous study we found that tyrosinase 1 mRNA localized to vitellocytes in the vitellarium, the vitelline duct, and the ovo-vitelline duct [21]. By contrast, p14 transcripts are only detected vitellocytes in the vitellarium and vitelline duct, but not in vitellocytes the anterior ovo-vitelline duct (Fig. 2A). Prior research found that eggshell precursor proteins and tyrosinase 1 are packaged in granules within vitellocytes [7], [13], [14], [35], [36]. Our results suggest that p14 is packaged into granules by maturing vitellocytes prior to reaching the ovo-vitelline duct, while tyrosinase 1 is expressed later. In agreement with prior reports [34], the abundance ofp14 transcripts decreases as female parasites undergo regression regardless of the pairing status of the female (Fig. 3). We also find that tyrosinase 1 exhibits a similar decrease in transcript abundance following in vitro regression of the female (Fig. 3).
Whole-mount in situ hybridizations with riboprobes specific to each gene are shown. Panel A: p14; PanelB: FsMucin; Panel C: Fs800; Panel D: 10688; Panel E: 10548; Panel F: 11223; Panel G: 21110; Panel H: 11779; Panel I: 28488; Panel J: 10763; Panel K: 10401; Panel L: 21733; Panel M: 33844; Panel N: 10927; Panel O: 10403; Panel P: 8056; Panel Q: 8987; and Panel R: 10617, Panel S: 10435; Panel T: 11088; Panel U: 11283; Panel V:, 11055/CPEB1; Panel W: CPEB2; Panels X and Y: cgh-1, the boxed region in (X) is shown at higher magnification in (Y). Tissues indicated are vitellarium (arrowhead), the vitelline duct and the ovo-vitelline duct (black arrow), and the ovary (*). All images are representative of >30 female worms. Scale bars are 100 µm for all panels except Y (50 µm). Images were taken on a Zeiss AxioStar Plus and analyzed with ImageJ software.
Transcript abundance was measured by quantitative real time PCR and analyzed using the 2−ΔΔCt method. Fold changes are measured relative to S. mansoni β-tubulin and normalized to uncultured, mature females. Black bars represent relative abundance in females from a single sex infection (AFSS:AFMS). Gold bars show gene expression changes in females that have been cultured with males (from left to right) for 1, 2, 3, and 7 days. Blue bars show gene expression level changes in unpaired females (left to right) at 1, 2, 3, and 7 days in culture.
FsMucin mRNA has a different localization pattern than p14 or tyrosinase 1 and is detected mainly in the vitelline and the ovo-vitelline ducts in the duct epithelium rather than the vitellocytes within the duct (Fig. 2B). It has been suggested that FsMucin acts as a protective barrier to the duct epithelium in the vitelline and ovo-vitelline ducts that prevents premature eggshell formation [27]. Although FsMucin has a different localization pattern than p14 and tyrosinase 1, FsMucin has a similar decrease in transcript abundance following in vitro regression of female worms and these decreases in abundance were independent of the presence or absence of a male worm (Fig. 3).
Several of the trematode specific genes (10548, 21110, 11223, and 10688) contain a trematode eggshell synthesis (TES) domain [13], which is also present in Fs800 (Fig. 4) [13], [24]. TES domains are found in a diverse array of trematode species including F. hepatica, C. sinensis, and Opisthorchis viverrini [13]. Proteins with this domain comprise a unique class of eggshell proteins that are found in the eggshell, but are not classified as eggshell precursor proteins from the p14, p19, or p48 families. Unlike eggshell precursor proteins, TES domain containing proteins contain a lower percentage of glycine and tyrosine residues (6.1% Y, 6.5% G, on average) when compared with eggshell precursor proteins p19 (11.6% Y and 9.6% G), p14 (11% Y and 44% G), and p48 (26% Y and 15% G) [37]. Within the TES domain there is a conserved sequence (G-X4-G-X18–20-G-X3–6-S-X12–19-F). However, the function of proteins is unknown. Like tyrosinase 1, Fs800 transcripts localize to the vitellarium and vitellocytes in the vitelline duct and the ovo-vitelline duct (Fig. 2C), a finding that confirms results from in situ hybridizations on worm sections [24]. The other TES domain containing proteins (10688, 10548, 11223, and 21110) have a similar transcript tissue distribution bearing more similarity to tyrosinase 1 than p14 suggesting that TES proteins are packaged in granules later than the eggshell precursor proteins (Fig. 2D–G). Overall, this indicates that Fs800-like proteins have a different function in eggshell synthesis than p14. With the exception of 21110, all TES genes show decreased transcript abundance following in vitro regression (Fig. 3). 21110 shows a transient increased transcript abundance in the first two days of culture followed by a rapid decline in both paired and unpaired females after three in culture (Fig. 5).
The signal peptide domains are overlined with a solid line, while the TES domain is overlined with a dashed line. Alignments were generated using ClustalW and BoxShade. Residues in black are identical in highlighted proteins while residues in grey are conservative changes.
Relative transcript abundance was measured by quantitative real time PCR analyzed using the 2−ΔΔCt. Expression of S. mansoni β-tubulin was used as an endogenous control and female worms from a mixed sex infection without in vitro culture were used for normalization. Black bars represent the relative abundance in unpaired, sexually immature females compared to paired, sexually mature females (AFSS:AFMS). Gold bars represent expression in AFMS females paired with a male after 2, 3, and 7 days (from left to right) in culture. Blue bars represent AFMS females cultured without a male after (from left to right) 2, 3, and 7 days, respectively.
Transcripts of several genes with conserved domains found in diverse range of species (10395, 10401, 1610, 10763, 24488, and 11779) localize to different female reproductive structures. 10395, a previously characterized SOD, has a unique localization pattern with transcripts being detected in immature vitellocytes [21]. Because 10395 encodes a Cu/Zn SOD protein and is present in immature vitellocytes, it may be functioning to detoxify superoxide radicals generated early in vitellogenesis due to high metabolic requirements of the rapidly differentiating cells. In ex vivo cultured female parasites the abundance of 10395 mRNA decreases (Fig. 3). During regression vitellocytes become less metabolically active and require less SOD activity, a finding supported by the decreased abundance of 10395 transcripts as the vitellarium regresses in vitro. However, prior research was not able to biochemically demonstrate SOD function in this protein [25], [26].
11779 transcripts are detected in mature vitellocytes and the vitelline and ovo-vitelline ducts (Fig. 2H). 11779 contains a diacylglycerol acetyltransferase domain (DAGAT) (Fig. S1A). DAGAT domain containing proteins catalyze the terminal step of triacylglycerol formation. In addition, these enzymes maintain steady state levels of the signaling component diacylglycerol [38]. 11779 shows decreased transcript abundance in response to reproductive regression (Fig. 3). Because only mature vitellocytes contain lipid droplets made up of triacylglycerol and other lipids, we hypothesize that the decreased 11779 transcription is reflective of the decreased synthesis of lipid-droplet containing mature vitellocytes.
28488 transcripts localize only to mature vitellocytes in the vitellarium and in the vitelline duct and are not detected in vitellocytes in the ovo-vitelline duct (Fig. 2I). 28488 contains a type I phosphodiesterase/nucleotide pyrophosphatase domain that is similar to human ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (Fig. S1B). In humans and other organisms, these proteins function as phosphodiesterases as well as phospholipases, catalyzing the production of lysophosphatidic acid. The synthesis of lysophosphatidic acid and its subsequent excretion into extracellular fluid induces growth factor responses including cell proliferation [39]. 28488 is predicted contain a signal peptide and to be targeted to the secretory pathway or the cell membrane (Fig. S1B). It may be functioning in the vitellarium to generate lysophosphatidic acid, inducing vitelline cell proliferation, a process that would be required in the vitellarium, but less in the vitelline and ovo-vitelline ducts. 24488 also has decreased transcript abundance as female parasites regress in vitro regardless of the pairing status of the female (Fig. 3). Together, these results may indicate that both the maturation and proliferation of vitelline cells are affected by in vitro regression of the vitellarium.
10763 has weak similarity (4.48e-05) to antistasin (pfam02822), which can inhibit trypsin family proteases [40] (Fig. S1C). 10763 transcripts are detected in vitellocytes in the vitellarium and in the ovo-vitelline duct (Fig. 2J). 10763 transcripts are decreased in the vitelline duct prior to merging with the ovo-vitelline duct (Fig. 2J). During in vitro regression of female parasites the 10763 transcripts decrease in abundance (Fig. 3).
10401 has a unique transcription pattern with mRNA localizing to the vitellocytes both in the vitellarium, in the ducts, and also in the ovary (Fig. 2K). 10401 protein contains a C-type lectin domain (Fig. S1D). C-type lectins play a variety of roles in mediating cell-cell interactions. 10401 transcript abundance rapidly decreases after one day in culture regardless of the pairing status of the female (Fig. 3).
Interestingly, 1610, encoding a tetraspanin protein, has an mRNA localization pattern similar to tyrosinase 1 [21]. Twenty eight tetraspanin genes are present in the S. mansoni genome (17), the most well characterized being Tsp2 [41]. These proteins have four transmembrane domains with a large extracellular loop between the 3rd and 4th transmembrane segments. 1610 is a member of the CD63-like tetraspanin group (Fig. S1E). Tetraspanins act as "molecular facilitators" on the cell surface by interacting with laterally located proteins forming a scaffold that can aid in the formation and stability of signaling complexes [42]. 1610 also has decreased mRNA abundance following regression in vitro regardless of the pairing status of the female parasite (Fig. 3).
21733 encodes a protein containing a RING (Really Interesting New Gene [43]) finger domain at its carboxyl terminus (RING-variant domain (smart00744) C-X2-C-X10–45-C-X1-C-X7-H-X2-C-X11–25-C-X2-C, where X is any amino acid) (Fig. S1F). RING finger domain containing proteins have a conserved Zn binding domain enriched in cysteine and histidine residues. This domain binds two Zn atoms in a 'cross brace' arrangement [43]. RING finger domain containing proteins are exclusively found in eukaryotes, and generally interact with E2 ubiquitin conjugating enzymes. 21733 transcripts are detected in the vitellocytes in the vitellarium, the vitelline duct (not in the ovo-vitelline duct), and the posterior ovary (Fig. 2L). A very large increase in 21733 transcript abundance (∼50 fold) during regression may indicate an increase in ubiquitin targeted protein degradation occuring during the involution of reproductive tissues (Fig. 5).
33844 encodes a protein very rich in proline and glutamine residues, with P+Q = 45% of total (Fig. S1G). 33844 is predicted (SecretomeP 2.0, NN-score = 0.858) to be a non-classically secreted protein. 33844 transcripts localize to vitellocytes in the vitellarium and the vitelline duct (Fig. 2M). The transcription of 33844 is greatly increased (∼60 fold) in females undergoing regression (Fig. 5).
10927 encodes an initiation factor 2B subunit (eIF2B) (Fig. S1H). These proteins function in guanine nucleotide exchange between guanine diphosphate to guanine triphosphate (GDP to GTP) to facilitate translation initiation by eIF2B in a number of proteins involved in vitellogenesis [44]. 10927 transcripts are detected in vitellocytes in the vitellarium, vitelline/ovo-vitelline ducts, and the ootype (Fig. 2N). 10927 does not exhibit large changes in mRNA abundance during ex vivo culture (Fig. 5).
10403 contains a signal peptide domain (predicted by SignalP) and two transmembrane domains flanking a brain-specific membrane anchored protein domain (pfam12280, E-value: 3.69e-37) (Fig. S1I). This protein domain contains many glycosylation sites and is only found in eukaryotes [45]. 10403 transcripts are detected in all vitellocytes in the vitellarium and vitelline duct but not in the ovo-vitelline duct (Fig. 2O). 10403 shows a transient increase in transcript abundance after one day in culture followed by a return to levels seen in mature female parasites, but not silenced to levels detected in AFSS worms (Fig. 5).
8056 has weak similarity to β/γ crystallin domain containing proteins [46] (pfam00030, E-value: 0.054) (Fig. S1J) and its transcripts are found in vitellocytes within the vitellarium, vitelline duct, and the ovo-vitelline duct (Fig. 2P). 8056 transcript abundance fluctuates during regression (Fig. 5). Further study of chaperone proteins and their functions in vitellogenesis and eggshell formation will allow us to determine the exact functional significance of this unique expression pattern.
Transcripts from several genes that have no significant orthology to proteins outside of the Schistosoma genus, 8987, 10617, and 10435, localize to vitellocytes in the vitellarium, the vitelline duct, and the ovo-vitelline duct (Fig. 2Q, R, and S, respectively). 8987 and 10617 are weakly similar in protein sequence to TES domain containing proteins (Fig. S1K). However, they lack the signal peptide sequence and the conserved internal motif (Figure 4). 10435 does not show orthology to any conserved domains after BLAST or PSI-BLAST comparison but has an orthologous sequence in S. japonicum (Fig. S1L). Transcript abundance of these genes decreases during regression (Fig. 3).
11088 transcripts are detected in vitellocytes in the vitellarium and the vitelline duct (Fig. 2T), while 11283 transcripts are detected only in the vitelline duct epithelium (Fig. 2U). Both 11088 and 11283 only have orthologs to uncharacterized S. japonicum proteins (Fig. S1M and S1N, respectively). Without genome information available for more Trematodes, it remains to be seen if these genes are specific to schistosomes or if they have orthologs in other trematode species that have not yet been sequenced.
CPEBs play multiple roles in female reproductive biology
Cytoplasmic mRNA polyadenylation is a widely conserved mechanism to control translation, and cytoplasmic polyadenylation element binding proteins (CPEBs) have been shown to function in translational control in oocytes [29], [30], [47]. CPEBs bind a cytoplasmic polyadenylation element (CPE) located in the 3′ UTR of some mRNAs. In Xenopus, CPEB is phosphorylated at Ser 174 by Aurora A, a serine/threonine kinase, allowing it to bind other factors that attract poly (A) polymerase to the end of the mRNA thus promoting polyadenylation and translational activation [29]. Without phosphorylation by Aurora A, CPEB remains bound to the CPE, stabilizing the mRNA but not permitting polyadenylation and translation. In this way, oocytes are able to synthesize and store mRNAs that will be required once the oocyte begins to mature [31], [48].
The S. mansoni genome encodes three CPEB genes (Table 1 and Fig. S1O). From the SAGE data, CPEB1 (11055) was identified as having increased mRNA abundance in AFMS (Table 1). CPEB1 transcripts localize to the posterior ovary (Fig. 2V). CPEB2 transcripts are detected in both AFMS and AFSS worms (Fig. 1) and localize to the posterior ovary and the vitellarium of AFMS worms (Fig. 2W). These results indicate that these CPEBs are playing different functions in S. mansoni. CPEB1/11055 may play a role in oocyte maturation as in Xenopus [29], [48]. Based on the transcription of CPEB2 in vitellocytes and its transcription in mature and AFSS worms, we can infer that CPEB2 is functioning in multiple pathways. The large increase in CPEB1/11055 transcript abundance during regression (Fig. 5) may indicate a role in translational repression of oocytes when females undergo reproductive regression. As oocytes cease to mature, there is an increased need to maintain translational repression of mRNA by CPEB. In contrast, CPEB2 transcript abundance generally decreases during ex vivo culture (Fig. 3). Further study of CPEB function in S. mansoni will be needed to determine the full extent of translational control mediated by CPEB in the life cycle of this parasite.
cgh-1 controls germline apoptosis in the ovary and vitellarium
Because cgh-1 mRNA is present at higher levels in mature females than other life cycle stages (Fig. 1 and Table 1) we hypothesized that cgh-1 may be controlling apoptosis in oocytes similar to its function in C. elegans (Fig. S1P). In support, cgh-1 transcripts are detected at high levels in the posterior ovary, where mature oocytes are located (Fig. 2X, Y). Additionally we found that cgh-1 transcripts are present in vitellocytes, indicating that cgh-1 may also be controlling apoptosis of vitellocytes (Fig. 2X). When female worms are cultured with a male, cgh-1 transcripts increase in abundance after two days in culture (Fig. 5), while in female worms cultured without a male an increase occurs after three days in culture. Despite the transient increases in cgh-1 transcript abundance following regression, we still find overall decreases in cgh-1 transcript levels by seven days in culture. Increased rates of apoptosis have been shown to occur in the vitellarium within five to seven days in culture [34]. Overall, our results suggest a role for cgh-1 in maintenance of germline apoptosis in the ovary and the vitellarium.
AFSS enriched transcripts may function in male-female parasite interactions
In addition to transcripts enriched in mature females, we also identified two transcripts highly enriched in females obtained from single sex infections; 6767 and 15402. As shown in Figure 1, both 6767 and 15402 transcripts are exclusively present in females from single sex infections, confirming the results of SAGE (Table 1) [18]. 6767 is a micro-exon gene (MEG) (Fig. 6) [17]. These genes are composed of alternatively spliced, very short exons that produce a wide range of variant peptides. MEGs have been localized in juvenile parasites to secretory organelles surrounding the esophageal gland and MEG-2 encoded proteins are secreted by eggs and thought to mediate interactions between the egg and the host immune response [49]. Schistosome MEGs show no homology with genes from other organisms. We mapped 6767 to S. mansoni a genomic fragment SC_0319. Other characterized 2.1.i MEGs map to SC_0446. 6767 has the same gene structure as other MEGs of 12 exons with a long N-terminal exon containing 55 amino acids, 10 smaller, symmetrical exons ranging in size from 6–21 amino acids, and a longer C-terminal exon (Fig. 6B). 6767 also has an N-terminal leader sequence and no membrane spanning regions (Fig. 6A). These results indicate that 6767 is most likely a secreted protein, similar to other MEG-2 proteins. In AFSS worms, 6767 transcripts are detected in subtegumental cytons and in stage 1 vitellocytes, the only vitellocytes present in these worms (Fig. 7A, B).
Panel A: Alignment of MEG-2 family members. Proteins include an ESP-15 (CAZ37453.2) and 6767, a MEG-2 family member that is enriched in females from single sex infections. Alignments were generated using ClustalW and Boxshade. Panel B: Exon map of 6767 on S. mansoni genome fragment SC_0319. Exons are designated a-l, sizes of exons are listed below exons.
Panel A: 6767 localized in AFSS worms. Panel B: higher magnification view of the boxed area in image A. 6767 localizes to vitellocytes (arrowhead) and subtegumental cytons (arrow). Panel C: 15402 localized in AFSS worms. Panel D: higher magnification view of the boxed area in image C. 15402 localizes in a punctate staining pattern in subtegumental cytons (arrows in D). Panel E: 6767 localized in AFMS worms following in vitro culture for 3 days. Panel F: higher magnification view of the boxed area in image E. 6767 transcripts are detected in the ovo-vitelline duct (red arrows). Panel G: 15402 localized in AFMS worms following in vitro culture for 3 days. Panel H: higher magnification view of the boxed area in image G. 15402 transcripts are detected in the ovo-vitelline duct (red arrows) and in a punctate staining pattern surrounding the ootype (red arrowhead). Scale bars are 100 µm in A,C, E, F and G and 50 µM in B, D, and H. All images generated with a Zeiss AxioStar Plus microscope.
Although 6767 transcripts are present at very low levels in AFMS worms, their abundance is increased ∼50 fold when these worms are removed from their host and placed in culture in the presence or absence of a male worm (Fig. 8). Following reproductive regression of AFMS worms, 6767 transcripts are detected in vitellocytes within the ovo-vitelline duct (Fig. 7E, F). Because 6767 is a member of a larger gene family and detection of other MEG transcripts by cross-hybridization may have occurred, we performed WISH on ex vivo adult females without in vitro culture and were not able to detect any signal (data not shown).
Relative abundance was measured during regression using the 2−ΔΔCt method. S. mansoni β-tubulin was used as an endogenous control and female worms from a mixed sex infection without in vitro culture were used for normalization. Black bars represent the relative abundance in unpaired, sexually immature females compared to paired, sexually mature females (AFSS:AFMS). Gold bars represent abundance in females paired with a male after 1, 2, 3, and 7 days (from left to right) in culture. Blue bars represent females cultured without a male after (from left to right) 1, 2, 3, and 7 days, respectively.
15402 may be a Schistosoma claudin gene (Fig. S1Q). The claudin genes encode a family of proteins important in tight junction formation and function [50]. Four transmembrane spanning regions are predicted in the protein encoded by 15402 as in other claudin proteins. In AFSS worms, 15402 transcripts were localized to subtegumental cytons, indicating that the protein product of 15402 may be present in the tegument (Fig. 7 C–D). Like 6767, 15402 gene expression increases (∼20 fold) in response to female reproductive regression (Fig. 8). 15402 transcripts were found in vitellocytes in the ovo-vitelline duct and in cells surrounding the ootype in AFMS worms cultured in vitro for three days (Fig. 7 G–H). 15402 expression in mature vitellocytes and in cells surrounding the ootype suggests that it has multiple functions in female reproductive biology.
In this study we have characterized genes that are differentially transcribed in S. mansoni female worms based on their transcript tissue localization and changes in transcript abundance during regression. Although previous studies have examined female reproductive regression through the lens of egg production [8], [12], [34], [51], [52], this is the first study to catalog previously uncharacterized, female enriched transcripts using a regression model yielding a better understanding of female reproductive biology. In addition, we used bioinformatic approaches to define the conserved domains and protein architecture of these genes; providing us with additional information used to cluster female specific genes. We have shown that many AFMS enriched genes are transcribed in vitellocytes and can be clustered based on their presence late in vitellogenesis (in the ovo-vitelline duct) or at earlier stages (present in vitellarium or posterior vitelline duct). Several genes were found to be transcribed in the ovary exclusively or in the ovary and vitellocytes. We found that the AFMS-enriched transcripts cluster into two groups following in vitro regression: 67% of genes have decreased transcript abundance (Fig. 3), while 33% of the genes have increased transcript abundance following regression (Fig. 5). The changes in mRNA abundance observed were independent of male-female pairing. Transcripts with decreased abundance in response to regression localize to the vitellarium, vitellocytes within the vitelline and ovo-vitelline ducts, and the vitelline and ovo-vitelline duct epithelium. Based on protein predictions and localizations, this decrease in abundance may be indicative of their functions in egg biogenesis or synthesis and maintenance of mature vitellocytes. Transcripts that localize to the ovary or the ovary and vitellarium show increased abundance following female reproductive regression. These genes are predicted to encode proteins with diverse functions including oocyte maturation, apoptosis, protein degradation, and vitellocyte-vitellocyte interaction. The genes that are induced by reproductive regression may be essential for maintaining reproductive status in females. Additionally, we examined transcripts that are enriched in AFSS infections. These two genes, 6767 and 15402, may function in the interactions that occur during worm pairing, and may form the basis of an innovative approach to target worm pairing and maturation. Overall, this investigation of S. mansoni female reproductive biology illustrates that many genes present in mature females are devoted to diverse processes ranging from nucleotide metabolism to eggshell biology. By using this approach, we can direct research to examine the areas of female biology that are both relevant to understanding the overall process of female development and worm pairing while determining novel therapeutic approaches directed at female schistosomes.
Supporting Information
Figure S1.
Multiple alignments of genes identified by SAGE. Alignments were generated using ClustalW and BoxShade servers. Residues in black are identical in highlighted proteins; residues in grey are conservative changes. (A) Alignment of 11779 highlighting the diacylglycerol acetyltransferase domain with orthologs in Schistosoma japonicum (AAW27748.1), Clonorchis sinensis (GAA40664.2), and humans (NP_079374.2). (B) Alignment of 28488 showing the type I phosphodiesterase/nucleotide pyrophosphatase domain (overlined in black) and orthologs in S. japonicum (CAX74907.1), C. sinensis (GAA50667.1), and humans (BAA08260.1). (C) Alignment of 10763 showing the antistasin domain. (D) 10401 alignment showing the C-type lectin domain (overlined) with orthologs in S. japonicum (AAW25606.1), C. sinensis (GAA50649.1), and humans (NP_006030.2). (E) Alignment of the CD63-like tetraspanin 1610 with orthologs in S. japonicum (AAW25074.1) and humans (NP_001771.1). The CD63-like domain is overlined and conserved cysteine residues are indicated (*). (F) Alignment of the RING finger domain in 21733 with S. japonicum (AAW25925.1), C. sinensis (GAA30053.2), and human (NP_848545.1) proteins. Conserved residues are starred (*) and the RING finger domain is overlined. (G) 33844 alignment showing the P/Q rich, putative cell surface domain present in Aspergillus fumigates (EDP52841.1) and S. japonicum (AAW25874.1). (H) Alignment of 10927 with S. japonicum (CAX70293.1), C. sinensis (GAA50666.1), and human (NP_001405.1) orthologs showing the translation initiation factor 2 domain. (I) 10403 aligned with orthologs in S. japonicum (CAX72358.1), C. sinensis (ADZ13680.1), and humans (NP_036241.1) with the brain specific membrane anchored protein domain (pfam12280) overlined in black. (J) 8056 and the orthologs in S. japonicum (CAX69522.1), C. sinensis (GAA49010.1), and humans (AAA52109.1). The β/γ crystallin domain was identified by PSI-BLAST; one Greek key motif is overlined in black. (K) Alignment of 8987 and 10617 showing weak similarity to Fs800, a trematode eggshell domain containing protein with orthologs in S. japonicum (Sjapon8987, CAX70772.1 and Sjapon10617, CAX71239.1). (L) Alignment of 10435 with its ortholog in S. japonicum (AAX24261.2). (M) Alignment of 11088 with its ortholog in S. japonicum (AAW26070.1). (N) Alignment of 11283 with its ortholog in S. japonicum (AAM76790.1). (O) Alignment of Xenopus laevis CPEB1 (AAI70313.1) with 11055 (CPEB1), CPEB2, and CPEB3 from S. mansoni. The conserved RNA recognition motifs are overlined (RRM1 and RRM2). (P) Alignment of cgh-1 proteins from S. mansoni and Caenorhabditis elegans (NP_498646.1). (Q) Alignment of 15402 and its ortholog in S. japonicum (XP_002573676.1) with the human Claudin protein (NP_001414.1); the transmembrane domains (TM) are overlined in black. Abbreviations: Sjapon, Schistosoma japonicum; Csin, Clonorchis sinensis; Afumi, Aspergillus fumigates; Xlaev, Xenopus laevis; Hsapien, Homo sapiens.
Table S1.
Primers designed from SAGE tags for 5′RACE PCR to clone genes differentially expressed in female worms.
Table S2.
Primers listed were used for cloning genes into pCRII vector for riboprobe synthesis and reverse transcriptase PCR.
Table S3.
Primers used for quantitative real time PCR.
We thank Palita Sriratana and Heelai Wardak for excellent technical assistance. Schistosome-infected snails were supplied by Dr. Fred A. Lewis, Biomedical Research Institute, Rockville, MD under National Institutes of Health (NIH), National Institute of Allergy and Infectious Disease (NIAID) contract HHSN272201000005I. We thank Dr. Philip LoVerde for the S. mansoni cDNA library.
Author Contributions
Conceived and designed the experiments: AAC DLW. Performed the experiments: AAC VPK. Analyzed the data: AAC DLW. Wrote the paper: AAC DLW.
- 1. Andrade ZA (2009) Schistosomiasis and liver fibrosis. Parasite Immunol 31: 656–663.
- 2. LoVerde PT, Kuntz RE (1981) Chromosome numbers of some schistosomes. J Parasitol 67: 726.
- 3. Tinsley RC (1983) Ovoviviparity in platyhelminth life-cycles. Parasitology 86: 161–196.
- 4. Gonnert R (1955) Schistosomiasis studies. II. Oogenesis of Schistosoma mansoni and the development of the eggs in the host organism. Z Tropenmed Parasitol 6: 33–52.
- 5. Neves RH, de Lamare Biolchini C, Machado-Silva JR, Carvalho JJ, Branquinho TB, et al. (2005) A new description of the reproductive system of Schistosoma mansoni (Trematoda: Schistosomatidae) analyzed by confocal laser scanning microscopy. Parasitol Res 95: 43–49.
- 6. LoVerde PT (1976) Scanning electron microscopy of the ova of Schistosoma haematobium and Schistosoma mansoni. Egypt J Bilharz 3: 69–72.
- 7. Neill PJ, Smith JH, Doughty BL, Kemp M (1988) The ultrastructure of the Schistosoma mansoni egg. Am J Trop Med Hyg 39: 52–65.
- 8. Erasmus DA (1973) A comparative study of the reproductive system of mature, immature and "unisexual" female Schistosoma mansoni. Parasitology 67: 165–183.
- 9. Popiel I, Erasmus DA (1982) Schistosoma mansoni: The survival and reproductive status of mature infections in mice treated with oxamniquine. J Helminthol 56: 257–261.
- 10. Neves RH, Costa-Silva M, Martinez EM, Branquinho TB, Carvalho JJ, et al. (2004) Phenotypic plasticity in adult worms of Schistosoma mansoni (Trematoda:Schistosomatidae) evidenced by brightfield and confocal laser scanning microscopies. Mem Inst Oswaldo Cruz 99: 131–136.
- 11. Popiel I, Cioli D, Erasmus DA (1984) The morphology and reproductive status of female Schistosoma mansoni following separation from male worms. Int J Parasitol 14: 183–190.
- 12. Atkinson KH, Atkinson BG (1980) Biochemical basis for the continuous copulation of female Schistosoma mansoni. Nature 283: 478–479.
- 13. Ebersberger I, Knobloch J, Kunz W (2005) Cracks in the shell–zooming in on eggshell formation in the human parasite Schistosoma mansoni. Dev Genes Evol 215: 261–267.
- 14. Fitzpatrick JM, Hirai Y, Hirai H, Hoffmann KF (2007) Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases. FASEB J 21: 823–835.
- 15. Irie Y, Tanaka M, Yasuraoka K (1987) Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro. J Parasitol 73: 829–835.
- 16. Shaw JR, Erasmus DA (1977) Schistosoma mansoni: Differential cell death associated with in vitro culture and treatment with Astiban (Roche). Parasitology 75: 101–109.
- 17. Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, et al. (2009) The genome of the blood fluke Schistosoma mansoni. Nature 460: 352–358.
- 18. Williams DL, Sayed AA, Bernier J, Birkeland SR, Cipriano MJ, et al. (2007) Profiling Schistosoma mansoni development using serial analysis of gene expression (SAGE). Exp Parasitol 117: 246–258.
- 19. Lewis F (2001) Schistosomiasis. Curr Protoc Immunol Chapter 19: Unit 19.1.
- 20. Le Paslier MC, Pierce RJ, Merlin F, Hirai H, Wu W, et al. (2000) Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library. Genomics 65: 87–94.
- 21. Cogswell AA, Collins JJ 3rd, Newmark PA, Williams DL (2011) Whole mount in situ hybridization methodology for Schistosoma mansoni. Mol Biochem Parasitol 178: 46–50.
- 22. Mann VH, Morales ME, Rinaldi G, Brindley PJ (2010) Culture for genetic manipulation of developmental stages of Schistosoma mansoni. Parasitology 137: 451–462.
- 23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25: 402–408.
- 24. Reis MG, Kuhns J, Blanton R, Davis AH (1989) Localization and pattern of expression of a female specific mRNA in Schistosoma mansoni. Mol Biochem Parasitol 32: 113–119.
- 25. Hong Z, Kosman DJ, Thakur A, Rekosh D, LoVerde PT (1992) Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni. Infect Immun 60: 3641–3651.
- 26. Hong Z, LoVerde PT, Thakur A, Hammarskjold ML, Rekosh D (1993) Schistosoma mansoni: A Cu/Zn superoxide dismutase is glycosylated when expressed in mammalian cells and localizes to a subtegumental region in adult schistosomes. Exp Parasitol 76: 101–114.
- 27. Menrath M, Michel A, Kunz W (1995) A female-specific cDNA sequence of Schistosoma mansoni encoding a mucin-like protein that is expressed in the epithelial cells of the reproductive duct. Parasitology 111: 477–483.
- 28. Bobek L, Rekosh DM, van Keulen H, LoVerde PT (1986) Characterization of a female-specific cDNA derived from a developmentally regulated mRNA in the human blood fluke Schistosoma mansoni. Proc Natl Acad Sci USA 83: 5544–5548.
- 29. Hake LE, Richter JD (1994) CPEB is a specificity factor that mediates cytoplasmic polyadenylation during xenopus oocyte maturation. Cell 79: 617–627.
- 30. Kang M, Han S (2011) Post-transcriptional and post-translational regulation during mouse oocyte maturation. BMB Reports 44: 147–157.
- 31. Mendez R, Richter JD (2001) Translational control by CPEB: A means to the end. Nature Rev Mol Cell Bio 2: 521–529.
- 32. Navarro RE, Shim EY, Kohara Y, Singson A, Blackwell TK (2001) Cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans. Development 128: 3221–3232.
- 33. Boag P, Atalay A, Robida S, Reinke V, Blackwell TK (2008) Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis. J Cell Biol 182: 543–557.
- 34. Galanti SE, Huang SC, Pearce EJ (2012) Cell death and reproductive regression in female Schistosoma mansoni. PLoS Negl Trop Dis 6: e1509.
- 35. Eshete F, LoVerde PT (1993) Characteristics of phenol oxidase of Schistosoma mansoni and its functional implications in eggshell synthesis. J Parasitol 79: 309–317.
- 36. Seed JL, Bennett JL (1980) Schistosoma mansoni: Phenol oxidase's role in eggshell formation. Exp Parasitol 49: 430–441.
- 37. Chen LL, Rekosh DM, LoVerde PT (1992) Schistosoma mansoni p48 eggshell protein gene: Characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene. Mol Biochem Parasitol 52: 39–52.
- 38. Shi Y, Cheng D (2009) Beyond triglyceride synthesis: The dynamic functional roles of MGAT and DGAT enzymes in energy metabolism. Am J Physiol Endocrinol Metab 297: E10–8.
- 39. Goding JW, Grobben B, Slegers H (2003) Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim Biophys Acta 1638: 1–19.
- 40. Lapatto R, Krengel U, Schreuder HA, Arkema A, de Boer B, et al. (1997) X-ray structure of antistasin at 1.9 Å resolution and its modelled complex with blood coagulation factor Xa. EMBO J 16: 5151–5161.
- 41. Loukas A, Tran M, Pearson MS (2007) Schistosome membrane proteins as vaccines. Int J Parasitol 37: 257–263.
- 42. Pols MS, Klumperman J (2009) Trafficking and function of the tetraspanin CD63. Exp Cell Res 315: 1584–1592.
- 43. Freemont PS (2000) RING for destruction? Curr Biol 10: R84–7.
- 44. Clemens MJ (2001) Initiation factor eIF2 alpha phosphorylation in stress responses and apoptosis. Prog Mol Subcell Biol 27: 57–89.
- 45. Elson GC, de Coignac AB, Aubry JP, Delneste Y, Magistrelli G, et al. (1999) BSMAP, a novel protein expressed specifically in the brain whose gene is localized on chromosome 19p12. Biochem Biophys Res Commun 264: 55–62.
- 46. Graw J (1997) The crystallins: Genes, proteins and diseases. Biol Chem 378: 1331–1348.
- 47. De Moor CH, Meijer H, Lissenden S (2005) Mechanisms of translational control by the 3′ UTR in development and differentiation. Semin Cell Dev Biol 16: 49–58.
- 48. Tay J, Hodgman R, Sarkissian M, Richter JD (2003) Regulated CPEB phosphorylation during meiotic progression suggests a mechanism for temporal control of maternal mRNA translation. Genes Dev 17: 1457–1462.
- 49. DeMarco R, Mathieson W, Manuel SJ, Dillon GP, Curwen RS, et al. (2010) Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts. Genome Res 20: 1112–1121.
- 50. Hewitt KJ, Agarwal R, Morin PJ (2006) The claudin gene family: Expression in normal and neoplastic tissues. BMC Cancer 6: 186.
- 51. Basch PF, Rhine WD (1983) Schistosoma mansoni: Reproductive potential of male and female worms cultured in vitro. J Parasitol 69: 567–569.
- 52. Fitzpatrick JM, Hoffmann KF (2006) Dioecious Schistosoma mansoni express divergent gene repertoires regulated by pairing. Int J Parasitol 36: 1081–1089.