text:
March 13 2013 Issue
U.S. Companies Asked to Submit Initial TPP Short Supply Requests by March 31
The Obama administration is giving U.S. interested parties until March 31 to submit any requests for short supply designations as part of the ongoing Trans-Pacific Partnership Agreement negotiations. The TPP short supply proposal put forward by U.S. negotiators includes separate short supply designations structured on a permanent and temporary basis. All short supply designations would be established as of the date negotiations are concluded, which means that no additional short supply requests would be allowed after the TPP takes effect.
Once the TPP enters into force, textile and apparel goods containing fibers, yarns or fabrics that are designated to be in short supply and meet all other requirements would qualify for preferential tariff treatment even though the input does not originate from a TPP country. If a fabric is designated in permanent short supply for a particular apparel product, that product could qualify for TPP preferences if it is cut and sewn in one or more TPP countries and meets any other additional requirements, such as assembly with TPP-origin sewing thread.
The TPP is a free trade agreement being negotiated between 11 countries: the United States, Australia, Brunei, Canada, Chile, Malaysia, Mexico, New Zealand, Peru, Singapore and Vietnam. Governments other than the U.S. may engage in their own internal short supply analyses with a different deadline. The participating countries have announced their intention to conclude the agreement by the APEC summit to be held in Bali on Oct. 1-8.
CBP Unveils New C-TPAT System of Records
The U.S. Department of Homeland Security has unveiled a proposal to establish a new Customs-Trade Partnership Against Terrorism system of records, known as the DHS/CBP-018 C-TPAT System of Records. This new system of records collects and manages information, including personally identifiable information, about prospective, ineligible, current or former trade partners in C-TPAT and other entities and individuals in their supply chains. The system also collects and maintains information, including personally identifiable information, regarding members of a foreign government secure supply chain program that have been recognized by CBP through a mutual recognition arrangement or a comparable arrangement as being compatible with the program. The C-TPAT program provides a security link portal that allows partners and applicants to access and manage their information.
CBP notes that it is publishing this new system of records notice to notify the public about the system, permit trade partners access to the information they provide, and offer a description of how and where information is collected and maintained. The new system of records will be effective on April 12 unless comments are received that result in a contrary determination.
DHS has issued a separate notice in the Federal Register seeking input by April 12 on a related proposal that would exempt portions of this new system of records from one or more provisions of the Privacy Act because of criminal, civil and administrative enforcement requirements. CBP believes that these exemptions are needed to protect information relating to DHS activities from disclosure to subjects or others related to these activities.
USTR Welcomes Progress on U.S.-Taiwan Trade and Investment Issues
The Office of the U.S. Trade Representative announced on March 10 that the recent discussions held by U.S. and Taiwanese officials in Taipei within the framework of the bilateral Trade and Investment Framework Agreement yielded "numerous results" and "positive outcomes." Among other things, the USTR welcomed Taiwan's agreement to conduct bilateral technical exchanges to facilitate the establishment of science-based maximum residue levels for pesticides in order to allow Taiwan to enjoy the benefits of new, safer and more environmentally-friendly products. The meetings also produced new joint statements on investment principles and information and communication technology services and saw the launch of new TIFA working groups on investment and technical barriers to trade.
The U.S. commended Taiwan for approving amendments to the Trade Secret Act in order to increase deterrent penalties for trade secret misappropriation and welcomed a two-year pilot program that will help create a more stable market for medicines in Taiwan's health care system. In addition, Ambassador Demetrios Marantis asked his counterparts to ensure that Taiwan's food safety measures, including those related to meat exports, are based on science and comply with international standards.
FDA Extends Deadline for Comments on Draft Qualitative Risk Assessment of Low-Risk Activity/Food Combinations
The Food and Drug Administration has extended from Feb. 15 to May 16 the deadline for interested parties to submit comments on a document entitled "Draft Qualitative Risk Assessment of Risk of Activity/Food Combinations for Activities (Outside the Farm Definition) Conducted in a Facility Co-Located on a Farm" that the agency made available for public comment on Jan. 16. The purpose of this document is to provide a science-based risk analysis of those activity/food combinations that would be considered low risk. The FDA issued this document to satisfy the requirements of the FDA Food Safety Modernization Act to conduct a science-based risk analysis and used the results of that analysis to propose to exempt certain food facilities (i.e., those that are small or very small businesses that are engaged only in specific types of on-farm manufacturing, processing, packing or holding activities identified as low-risk activity/food combinations) from the proposed requirements for hazard analysis and risk-based preventive controls.
Company Agrees to Pay $400,000 Penalty for Failure to Report Defective Play Yards
The Consumer Product Safety Commission has provisionally accepted an agreement under which an Illinois company would pay a $400,000 penalty to settle charges related to defective play yards. Any interested person may ask the CPSC not to accept this agreement or otherwise comment on its contents within 15 days after its publication in the Federal Register.
The CPSC states that the play yards at issue are defective because their side rail can fail to latch properly and can unlatch unexpectedly when a child pushes against it, posing a fall hazard to children. From about January 2000 through July 2009 the company received approximately 350 reports of the play yard collapsing, resulting in 21 injuries to young children. The company did not report that information to the CPSC until January 2009 and a recall of about one million play yards was finally conducted in July of that year.
Federal law requires manufacturers, distributors, and retailers to report to CPSC immediately (within 24 hours) after obtaining information reasonably supporting the conclusion that a product contains a defect which could create a substantial product hazard, creates an unreasonable risk of serious injury or death, or fails to comply with any consumer product safety rule or any other rule, regulation, standard or ban enforced by CPSC.
In agreeing to the settlement the company denies the CPSC's allegations that its play yards contained a defect that could create a substantial product hazard, or that it knowingly violated the reporting requirements of the Consumer Product Safety Act. Pursuant to the CPSA, the CPSC must consider the appropriateness of the penalty in relation to the size of the business of the person charged, including how to address undue adverse economic impacts on small businesses. The company at hand is a small business as set forth in the Small Business Administration guidelines regarding size of business.
APHIS Meeting to Explore Strategic Priorities, Budgetary Issues and Pending Program Restructuring
The Department of Agriculture's Animal and Plant Health Inspection Service will hold a public meeting on April 11 to share information about the agency's budget, pending program restructuring and potential impacts, and cross-agency strategic priorities. APHIS is seeking to continue to improve its business strategy and program delivery methods to enhance the agency's overall effectiveness in the face of a number of new and evolving challenges, including decreasing budgets, fewer available resources, and the continued need to successfully balance science, policy and public opinion. The agency notes that it wants to engage its stakeholders in a dialog early in the process as it begins to consider new opportunities and rethink current business practices.
APHIS is especially interested in stakeholder perspectives regarding (i) public and private partnerships, such as disease management and eradication programs that benefit from state and industry contributions; (ii) non-regulatory solutions, such as compliance and education programs that can be used, when appropriate, to achieve results without the need for rulemaking; (iii) new technology that encourages commerce or enhances APHIS' ability to protect American agriculture; and (iv) customer service improvements that make it easier for stakeholders to do business with APHIS.
APHIS will accept input on these and other related matters by May 13.
New Section 337 Investigation on Certain Integrated Circuit Devices
The International Trade Commission has initiated a Section 337 investigation of certain integrated circuit devices and products containing the same made in Finland, South Korea and Taiwan. The scope of this proceeding includes mobile smartphones containing certain types of integrated circuits. The complainant alleges that the respondents have infringed certain U.S. patents and is requesting the ITC to issue exclusion and cease and desist orders on subject merchandise.
State Dept. Seeks Input on Shrimp Exporter's/Importer's Declaration
The State Department is seeking comments by May 13 on the extension without change of the shrimp exporter's/importer's declaration (Form DS-2031). This information collection is necessary to document shrimp imports pursuant to the State Department's implementation of Section 609 of Public Law 101-162, which prohibits the entry into the United States of shrimp harvested in ways that are harmful to sea turtles. Respondents are shrimp exporters and government officials in countries that export shrimp to the United States. The DS-2031 form is to be retained by the importer for a period of three years subsequent to entry, and during that time is to be made available to CBP or the State Department upon request.
New Maritime Agreements Filed, Input Sought on Another Agreement
The Federal Maritime Commission has issued notice that the following new agreements has been filed. Interested parties may submit comments by March 25.
West Coast of South America Discussion Agreement – The amendment excludes Peru from the membership of four of the parties.
U.S. Pacific Coast-Oceania Agreement – The amendment (i) deletes A.P. Moller-Maersk A/S as a party to the agreement; (ii) adjusts the vessel provision and allocation provision of the agreement; (iii) adjusts the scope of the two service strings operated under the agreement; (iv) adjusts the maximum size of the vessels which can be deployed by the parties; and (v) restates the agreement.
MSC/CSAV Ecuador – North Europe Vessel Sharing Agreement – The agreement authorizes CSAV and MSC to cross-charter space to each other in the trade from ports in North Europe to ports on the U.S. East Coast on the one hand, and from ports on the U.S. East Coast to Panama and Ecuador on the other hand.
The FMC has also formally requested that the parties to the below listed agreement provide additional information by March 28.
Consolidated Chassis Management Pool Agreement – The Ocean Carrier Equipment Management Association and its member lines; the Association's subsidiary Consolidated Chassis Management LLC and its affiliates; CCM Holdings LLC; CCM Pools LLC and its subsidiaries; Matson Navigation Co.; and Westwood Shipping Lines.
Ex-Im Bank Asked to Finance Exports of Commercial Aircraft
The Export-Import Bank of the United States has received an application for final commitment for a long-term loan or financial guarantee to support the export of U.S.-manufactured commercial aircraft to the Philippines. These aircraft will be used for long-haul passenger air service between the Philippines and destinations in Asia, Canada and other routes. Comments on this application are due no later than April 8.
Foreign Regulatory Changes Could Affect Exports of Various Products
According to the National Institute of Standards and Technology, the WTO has been notified of regulatory changes that may affect exports of specific products to the following countries. For information on how these restrictions may affect your business, contact ST&R.
Argentina – register of medical technology producers and products – procedures for registration applications
Argentina – approval of draft resolution amending Chapter XV, Article 1194, of the Argentine Food Code
Chile – draft technical regulation relating to the Food Health Regulations, Supreme Decree No. 977/96 (comments due by April 21)
Chile – draft regulation establishing national quality specifications for Grade B diesel (comments due by April 1)
China – interim provisions on regulating the registration of repackaged IVD products and notice regarding the issuance of classification sub-catalog of IVD products (comments due by May 11)
Colombia – draft resolution of the Ministry of the Environment and Sustainable Development prohibiting the manufacture in and importation into Colombia of household refrigerators, freezers and refrigerator/freezer combinations containing or requiring for their production or operation the hydrochlorofluorocarbons listed in Group I of Annex C to the Montreal Protocol
Costa Rica – technical regulations establishing labeling requirements for footwear and parts thereof when sold separately in the territory of Central American countries (comments due by May 1)
France – order approving certain provisions supplementing and amending the safety regulations governing fire and panic hazards in buildings open to the public (comments due by April 30)
France – draft order implementing Article L. 5232-1 of the Public Health Code relating to personal stereos (April 30)
Guatemala and Honduras – technical regulations establishing labeling requirements for footwear and parts thereof when sold separately in the territory of Central American countries (comments due by April 25)
Lithuania – order of the Minister of Environment on the list of construction products subject to regulation
Mexico – implementation of amendment to Mexican Official Standard NOM-005-SCFI-2011 establishing specifications and test and verification methods for systems for measuring and dispensing petrol and other liquid fuels
Mexico – draft Mexican Official Standard PROY-NOM-163-SEMARNAT-ENER-SCFI-2012 on carbon dioxide emissions for new light-duty vehicles (comments due by April 22)
Nicaragua – partial amendment of Nicaraguan Mandatory Technical Standard No. 03 023-12 Second Revision on slaughtered, ready-to-cook chickens (raw), whole or cut in pieces, and chicken offal
Saudi Arabia – draft technical regulations for barley malt, fried potato, food packages (Part 2: plastic package – general requirements), black tea, sunflower seeds, coriander whole or ground, dates syrup (dibs altamr), pumpkin seeds (white pulp), frozen liver, maamol and chilled marinated meats (comments for food packages were due by March 8; comments for all other products are due by May 8)
Saudi Arabia – GCC draft technical regulations for general requirements for bakery products
Uganda – final draft standards for fortified food grade salt, fortified edible oils and fats, fortified sugar, fresh sweet potatoes and dried sweet potato chips (comments due by May 5, May 6 or May 8, depending on the product)


text: Meta Rate My Team PokéBase - Pokémon Q&A Login Register All Activity Questions Unanswered Tags Users Ask a Question Rules Chat What are some Pokemon that are mixed sweepers? 1 vote 22,881 views Could someone tell me which Pokemon ( with movesets) represent the role of a mixed wall I suck at asking questions I know facepalm mixed-sweeper asked Jul 23, 2013 by aceofspades Sweepers or walls? commented Jul 23, 2013 by JarJar~ sweepers commented Jul 23, 2013 by aceofspades Please log in or register to add a comment. Please log in or register to answer this question. 5 Answers 2 votes Best answer Errm. your question is mixed sweepers. Your description of the question is mixed walls o_O Mixed Sweepers: Kinda depends on your definition of sweeper but I'm going to do it as in Mixed attackers First off; Salamence @ Life Orb Ability: Moxie EVs: 64 Atk / 192 SpA / 252 Spe Naive nature (+Spe, -SpD) - Draco Meteor - Outrage - Earthquake - Fire Blast Salamence... a Pokemon once deemed in the Ubers tier. Not anymore and this gives him the perfect chance to wreck ou :P Draco Meteor does massive damage as does Outrage and Earthquake/Fire Blast provide coverage and take care of pesky things like Heatran and Ferrothorn Dragonite @ Life Orb Ability : Multiscale EVs: 76 Atk / 252 SpA / 180 Spe Mild Nature -Hurricane -Thunder -Aqua Tail -Roost This guy is a mixed rain attacker. I run him on my rain team and hs godly, wrecks any team unprepared, and even some prepared ones since they largely expect physical D-Dance dragonite with lum bery. Infernape @ Life Orb Ability: Iron Fist EVs: 64 Atk / 252 SpA / 192 Spe Naive Nature -Close Combat -Fire Blast -HP Ice -Grass Knot Mixed attacker Infernape. So cool :P. Close Combat gets the Iron Fist boost, while Infernape's good SpA stat lets it abuse the rest for excellent coverage and damage Hydreigon @ Expert Belt Ability : Levitate EVs: 4 HP / 252 SpA / 252 Spe Mild Nature -Draco Meteor -Fire Blast -Superpower -Roost/Dark Pulse Time to get wrecked with mixed Hydreigon :P I use this guy and his great. Expert Belt helps bluff a choice set, so nothing on the opponent's team is safe. Roost or Dark Pulse is your choice really, survivability or even more coverage & power Kingdra @ Life Orb Ability : Swift Swim Nature: Naughty EVs: 252 Atk / 24 SAtk / 232 Spe -Dragon Dance -Waterfall -Outrage -Hydro Pump Ermygerd Kingdra. Nuff said lol. Though he isn't as good without rain, and since drizzle is banned with swift swim in ou =/. But he still has his merits - after a Dragon Dance some players may decide to bring in a defensive wall and attempt to stall/cripple Kingdra - they aren't going to like a Hydro Pump into their face provided it is at least nuetral Tyranitar @ Focus Sash Ability : Sand Stream EVs: 94 SpA/ 152 Atk / 252 Spe Hasty Nature -Fire Blast -Ice Beam -Stealth Rock -Crunch Tyranitar mixed attacker lead ;o. Excellent attack stat, decent SpA, so becomes quite viable. Fire Blast and Ice Beam help get rid of some usual counters (Ferrothorn hai >:D) Stealth rocks to set up and Crunch just hits ghost/psychic types for a ton of damage Landorus @ Life Orb Ability : Sand Force EVs:252 Atk / 4 SAtk / 252 Spe Naive Nature -Rock Polish -Hidden Power [Ice] -Stone Edge -Earthquake Rock Polish to boost speed. This guy hits hard, kills and HP Ice is used to get rid of some of his counters - hello gliscor and hippowdon. :P Might add some more later :P Hope I helped so far. And yes, most mixed attackers are actually dragon types (at least the good ones) since Dragon types often have the best overall attacking stats, but some exceptions like Jirachi who have all rounded stats work answered Jul 23, 2013 by Sempi selected Jul 23, 2013 by aceofspades Thank you so much commented Jul 23, 2013 by aceofspades Please log in or register to add a comment. 2 votes These are a couple of Pokemon that are somewhat less famous or utilized mixed sweepers, and among some of my favorites. [email protected] orb Ability: clear body Nature: naughty Evs: 232 atk 252 sp atk 24 spd -Meteor Mash -Psychic/Grass Knot -Hidden Power Fire -Hammer Arm/Ice Punch/Earthquake This guy is a good mixed tank, thanks to great offensive stats and defensive typing. Talk about four moveslot syndrome though. Ou tier [email protected] Orb Ability: Flash Fire EVs: 252 Atk / 4 SAtk / 252 Spd Naive Nature - Fire Blast - Pursuit - Sucker Punch - Hidden Power [Grass] Frail but dangerous, Houndoom is quite a nice mixed attacker in UU. Pursuit and Suckerpunch make your opponent tread carefully, while fire blast provides some more muscle power. Hp grass for coverage. [email protected] orb Ability: Teravolt Nature: Hasty Evs: 200 atk 56 sp atk 252 spd -Ice Beam -Outrage/Dragon Claw -Fusion Bolt -Focus Blast/Earth power/ Hidden power fire None other than the rejected Uber, base 170 atk and 120 sp atk in Ou? That's right. Take advantage of these ridiculous offensive stats to dish out pain to the Ou tier. Just remember to compensate for Kyurem's rather lackluster speed and defensive typing. answered Jul 23, 2013 by Dudeicolo very helpful commented Jul 23, 2013 by aceofspades Please log in or register to add a comment. 1 vote Sometimes you don't even need a Mixed attacker on your team. Choiced Pokemon can Trick a wall to help eliminate it. And at times you can even rely on sheer force to bust through walls with things like Choice Band Terrakion, Choice Band Haxorus, and Choice Specs Tornadus. But here are some great Mixed attackers: Jirachi @Expert Belt Serene Grace 152 Atk, 204 SpA, 152 Spe Ivs: 31/31/31/31/31/30 Naive (+Spe, -SpDef) -Iron Head -Fire Punch -Hidden Power Ice -Thunderbolt Hydreigon @Life Orb Levitate 104 Atk, 240 SpA, 164 Spe Hasty (+Spe, -Def) / Naive (+Spe, -SpDef) -Draco Meteor -Fire Blast -Earthquake -Outrage Salamence @Life Orb Intimidate 100 Atk, 176 SpA, 232 Spe Rash (+SpA, -SpDef) / Naive (+Spe, -SpDef) -Draco Meteor -Fire Blast -Brick Break / Earthquake -Roost You can also post an RMT to help us find what would fit on your team easier. answered Jul 23, 2013 by WoonderWaffle Please log in or register to add a comment. 1 vote Infernape @ Life Orb / Expert Belt Trait: Iron Fist EVs: 252 Spd / 180 Atk / 76 SpA Naive / Hasty Nature Overheat Close Combat Thunder Punch U-Turn / Mach Punch Overheat is a powerful Fire type STAB and can be used with no drawback due to it being the only special attack in the set. Close Combat is STAB and gets good coverage. Thunder Punch is for Flying and Water types. U-Turn is to make a quick escape and Mach Punch is priority. Salamence @ Life Orb Trait: Moxie / Intimidate EVs: 64 Atk / 192 SpA / 252 Spe Naive / Hasty Nature Draco Meteor Outrage Fire Blast Earthquake Draco Meteor and Outrage are dual Dragon STABs and no wall who only works on one side is safe. Fire Blast takes care of Steel types such as Forretress, Skarmory, and Ferrothorn. Earthquake is for doing more damage against Jirachi and hitting . Those are my favorites. Hope I helped! :) answered Jul 23, 2013 by JarJar~ Please log in or register to add a comment. 1 vote I'm just gonna go ahead and post a Mixed Hydreigon set that I like (really, guys? No U-turn?). @ Expert Belt/Life Orb Trait: Levitate EVs: 200 Atk / 56 SpA / 252 Spe Naive Nature (+Spe, -SpD) - Draco Meteor - Fire Blast/Flamethrower - Superpower - U-turn Personally, this is one of my favorite Pokemon to use as it is so easy to take something down using it. It can break right through walls and checks to a common Hydreigon get punched right in the face. Heatran switch into your Draco Meteor? Boom. Superpower. Now lets get onto the set itself. Hydreigon can either utilize a Life Orb to quickly rack up damage (on itself and its opponents), while an Expert Belt can bluff a Choice set. The ability Levitate, which is your only option, gives your Hydreigon an immunity to Ground along with the Psychic imunity. The given EVs allow Hydreigon and Nature allow Hydreigon to run marathons and hit hard physically and specially. Draco Meteor is so goddamn powerful and Hydreigon doesn't even worry much about the SpA drop thanks to it carrying two physical moves. Fire Blast or Flamethrower. You either get power or accuracy and better PP respectively. These moves will still really mess up Pokemon like Ferrothorn among others. Superpower does the same as Draco Meteor, but instead lowers the Atk and Def by one stage and is generally less powerful. U-turn over EQ because you're already hitting too many things super-effectively, but now you have much more momentum riding in your team and now you can beat Celebi, no problemo. Pros: - Its a mixed sweeper! It hits on the both sides of the physical and special spectrum. - Moves with bad side effects like Draco Meteor and Superpower are almost shrugged off. - One of the best checks around for Celebi. Cons: - Even though it is above average, its attack requires a good investment to meet its special. - Weaker defenses combined with a nature that will reduce its SpD or Def along with a Life Orb (if you choose that item) means Hydreigon won't be around forever. - It's dark type weakness gives it a fatal weakness to very common fighting type moves. answered Jul 23, 2013 by melcakes Please log in or register to add a comment. Related questions Are mixed sweepers really necessary? Give me a list of mixed sweepers from gen 1-4 What are the advantages/disadvantages to a mixed sweeper to a special or physical sweeper? What is a good nature and ev spread for mixed Hydreigon? What is the best set for a mixed standard Latios? What is a good EV spread for a mixed Latios? What is a good EV spread for a mixed Absol? What is a good nature for a Mixed Sweeping Infernape? Useful Meta Posts How to change your username How to add a user avatar Why was my post hidden? The points system Question etiquette Flags vs Downvotes Anime FAQ

subset: commoncrawl


text:
Founded in 2013 by documentary filmmaker Tess Motherway, the Dublin Doc Fest has become a staple in innovative and independent documentary filmmaking. The latest screening, hosted by the Irish Georgian Society, includes everything from silent artistic productions, to biting journalistic investigations, to sombre humanity profiles. Whilst every film showcased could merit its own article, I will focus on my personal picks from the festival.
Ryan Ralph's Love and Other Drags exemplifies the currency and innovation this festival prides itself in. The film documents the life Stephen, a 24 year old drag queen from Belfast, and his dreams of stardom and performing in his persona, the Lady Portia Di' Monte. Dismantling gender roles and societal stereotypes, the film focuses on the duality of Stephen's life; the mundane and the extraordinary. It challenges the perspective of the viewer, especially those who would dismiss people like Stephen as outcasts or societal exceptions, and does so with unique humor and intellect.
Another standout production is Ciaran Cassidy's The Last Days of Peter Bergmann, a sombre look into the suicide of a mystery man at Rosses Point in Sligo, and his efforts to ensure he would never be identified. Created primarily through CCTV footage and witness testimony, the viewer is brought into the world of 'Bergmann' as he meticulously dismantles all clues of his past life and identity, before ending his life. Indeed, by the conclusion it dawns on the viewer that the image of Bergmann on the CCTV is the only evidence he existed at all, depriving us of closure, and underscoring that there are no happy endings.
The final film is Gordie, created almost exclusively by Traolach Ó Murchú. This piece documents the reflections of a man and his traumatic childhood, as he recalls the sexual terror committed upon him by his now deceased brother. The unflinching subject matter is complimented by its fantastic camera work, with shots of snowy landscapes and childlike perspective low-shot angles bringing us uncomfortably close to the horrific realism of the event. The piece ends with the man's sombre forgiveness of his brother, with a lingering shot of the gravestone, and the ambient stunned silence of the audience.
The Dublin Doc Fest was hosted by the Irish Georgian Society at the City Assembly House on Saturday the 28th February.


text:
XD please... do it
Ok, I have 2 eggs (Rowlet and Popplio), working on hatching 'em now.
I just got a froakie and a level 52 skarmory from wonder trade O.O
Trying to connect to you now...unfortunately they don't have the best natures, but hopefully they'll do you proud regardless.
I'm having issues trying to find you from the plaza link trade. I did add your friend code and it all looks good.
Connecting to Internet!! D:
Got you, you can just give me whatevers, I dont mind.
You tried to give me magikarp!! X3
Hey, how did you evolve that Evee to the leafy green version? My guide says to go to the moss covered rock in the jungle but I'm not sure where that is. How far into the game are you? What island?
Dude, how do you have so many Eevees...I have a hard time finding them, lol. I also have a Formantis if you'd like that and don't have one already. They are cute.
Leafon, super strong attacker! Rock is in lush jungle
I think you traded an eevee with a Max Elixir. It's in my inventory as new right now and I don't have any of those.
But there are a couple different rocks in the jungle...does it matter?
Uh I dunno. I was actually just looking for a random pokemon to give you and I found an eevee
I'm going to make you a VIP. Hey, how were you able to remove your hat?
...you may be new to the game but you know more than I do.
I dunno, I got a hair cut o.o How long have you been playing?
Hmm...not quite a month yet? I'm at the third island but I tend to be rather lackadaisical about my story progression because I check out all the nooks and crannys I can, grinding various pokemon to level up.
I'm about to beat the second island!
All my Lv.100 Pokemon so far ^^
And yeah i think i didnt need to say anything xD ........
I would kill for that Porygon-Z.
you mentioned me!


text:
|Home | About | Journals | Submit | Contact Us | Français
How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression.
Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN.
We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.
The dark and nutrient poor cave environment exerts substantial pressure upon cave-dwelling animals. Perhaps as a consequence of these limited resources, cave-adapted animals from most major phyla exhibit a remarkable convergence in morphological and physiological changes related to cave life, including features that are both constructive (the lengthening of legs, fins and antennae, the appearance of novel behaviors, and elaboration of non-visual sensory systems) and regressive (the reduction or loss of vision and pigmentation) [1,2]. Although these changes have been studied in a diverse set of cave animals, the genetic and evolutionary mechanisms responsible for them remain poorly understood.
Although natural selection is probably involved in the evolution of constructive cave adapted phenotypes [3], the evolutionary forces driving regressive changes are less certain. Darwin suggested that eye degeneration - one of the most conspicuous traits found in cave animals - evolved due to disuse [4]. This idea was refined by others to implicate neutral mutation and genetic drift as a consequence of relaxed selection for vision in the cave environment [5,6]. The neutral hypothesis was favored until recently when the results of new genetic and developmental studies supported the adaptive evolution of eye regression in cave animals. Three competing hypotheses have been proposed for the adaptive evolution of eye regression: (1) direct natural selection against eyes to conserve energy in the resource poor cave environment [7]; (2) indirect selection against eyes to open sufficient space for the elaboration of constructive characters [2,8-10]; and (3) indirect selection against eyes due to the enhancement of traits that are negatively linked to optic development by antagonistic pleiotropy [11,12]. Distinguishing among these hypotheses has been difficult since the genetic basis of eye reduction is unknown in cave-dwelling animals.
The teleost Astyanax mexicanus is an excellent model organism for studying the evolution of traits associated with cave life, including eye regression [5,13-16]. Within the past few million years, at least five independent colonizations by two different migrational waves of eyed surface fish have established 29 geographically isolated Astyanax cavefish populations in northeastern Mexico [17-20]. After subsequent radiation underground, the founder cavefish populations became isolated in separate caves and evolved eye regression, reduced pigmentation or albinism, enhanced sensory systems and behavioral changes associated with cave life [5,11,12,21-32]. Despite this isolation, Astyanax surface fish and cavefish are interfertile in the laboratory, allowing the evolution of constructive and regressive traits to be studied by genetic analysis.
Previous studies reported that eye degeneration in Astyanax is triggered by lens apoptosis and dysfunction due to expanded sonic hedgehog (shh) gene expression along the embryonic midline [25,26]. Additionally, shh hyper expression was shown to increase jaw width and taste bud number, and to mediate the expansion of the forebrain and hypothalamus [11,22]. In a recent study, Elipot et al. found that shh modifies the hypothalamic serotonergic network and increases foraging efficiency in cavefish by shifting behavior from fighting to foraging [32]. These experiments support the hypothesis that eye regression in Astyanax has evolved at least in part as a result of indirect selection against eyes in favor of increased feeding efficiency through pleiotropy of the shh genes. However, recent genetic studies have discovered 8 to 12 quantitative trait loci (QTL) involved in eye reduction in Astyanax cavefish [5,7,33], but none that are linked to either the shhA or shhB genes, suggesting that upstream modulators of the SHH signaling system and/or other genetic factor(s) may be important in eye regression (for review, see ref. [3]).
In the present study, we launch an alternative approach to address the evolutionary mechanisms involved in eye degeneration. Assuming a positive relationship between visual decay and the evolution of constructive changes in other sensory systems - as has been proposed by many previous investigators [2,8,10,34,35] - we investigated the genetic basis of a constructive trait, the vibration attraction behavior (VAB), to evaluate its possible relationship to eye degeneration. VAB is the swimming of cavefish toward an oscillating object, a behavior that has evolved repeatedly in different Astyanax cavefish populations [27,36,37] and which may be present in Amblyopsid cavefish as well [38,39]. VAB is mediated by an increase in the number and size of cranial superficial neuromasts (SN) in cavefish [27]. Although VAB is usually absent in surface fish, a small proportion of those raised in the laboratory can show a weak form of VAB. In wild populations of surface fish, VAB is presumably deleterious since it may be easily detectable by predators [40]. In contrast, VAB is adaptive in cavefish since it increases foraging in an environment devoid of light with sparse food and no macroscopic predators [27,40].
Here we report the results of genetic analyses of VAB, SN enhancement and eye size. Using this approach, we discovered that the QTL underlying VAB, a specific class of SN located within the cavefish eye orbit (EO), and reduced eyes form two distinct clusters of overlapping QTL that together explain a significant portion of the genetic variation underlying these traits. By ablation of EO SN, we discovered that these sensory receptors contribute to VAB. Moreover, by shh overexpression, we also showed that induction of eye degeneration in surface fish did not induce VAB or increase EO SN, suggesting that these traits are part of an antagonistic system impacting eye formation that is independent of SHH signaling, and that the extra space opened by eye regression is insufficient to promote the appearance of these constructive traits. Therefore, we propose that the adaptive evolution of VAB and EO SN enhancement has contributed to eye degeneration in Astyanax cavefish.
To determine the phenotypic and genetic relationship among eye size, VAB and SN enhancement, we first estimated the number of genetic factors that control these traits. We crossed a female Texas surface fish with a male Pachón cavefish and raised about 130 F1 individuals. One pair of these F1 progeny was crossed to generate 384 F2 individuals for phenotypic and genetic analysis. We then measured the degree of VAB, SN number and size at suborbital bone 3 (SO-3 SN) [41], SN number within the eye orbit (EO) and overall eye size in each individual. The distribution of VAB and SN scores among the P0, F1 and F2 generations suggests that all four traits are heritable and controlled by one or more genetic factors (Figure 1A-D). Classic Castle-Wright estimates of the effective number of loci for VAB, SO-3 SN number and diameter, and EO SN number were 2.0 ± 1.0, 3.9 ± 1.2, 0.2 ± 0.1 and 20.9 ± 22.1, respectively (mean ± standard error of the mean, s.e.m.). However, because the F2 individuals from this cross did not recapitulate the entire distribution of cavefish VAB phenotypes (Figure (Figure1A),1A), we crossed another pair of F1 progeny from the same P0 to generate an additional F2 family. We used marker-assisted selection to choose three pairs of F2 individuals from this family that were hetero- or homozygous for cavefish alleles at two putative VAB and three SO-3 SN QTL loci (see Methods). We then crossed these F2 in order to generate 91 F3 individuals that recovered the entire cavefish VAB phenotype, although the distributions for SO-3 and EO SN remain somewhat restricted (Figure 1A-D). In combination with the original 384 F2, these additional F3 were included to increase our ability to detect correlations and QTL for the VAB, SN and eye size phenotypes.
Since SN are the sensory receptors that facilitate VAB [27], we expected the number of SN to be strongly correlated with the level of VAB in our hybrid families. In a previous study using cavefish, surface fish and their F1 progeny, we found a significant positive correlation between VAB and the number of SN at SO-3, which are located in the cranial region immediately ventral to the EO [27]. However, in the present study using both F2 and F3 hybrids, we found that the correlation between these two traits was not significant (r = 0.13, P = 0.052; Figure Figure1G),1G), although the correlation between VAB and SN diameter at SO-3 was significant (r = 0.14, P = 0.044; Figure Figure1H).1H). This new result suggests that the number of SN at SO-3 may not be the most important parameter determining VAB. After further examination of SN within the cranial regions of surface fish and cavefish, we found that SN are located in the EO in cavefish (yellow dotted line in the bottom of Figure Figure1N)1N) and in F2 and F3 hybrids with reduced eyes, but not in surface fish (yellow arrow in the bottom of Figure Figure1L,1L, M: F3; Figure Figure1K:1K: surface fish). In contrast to the results for SO-3 SN, we found that the number of EO SN was positively correlated with VAB (r = 0.29, P < 0.001; Figure Figure1F)1F) and negatively correlated with eye size (r = -0.44, P < 0.001; Figure Figure1J),1J), and that eye size was also negatively correlated with VAB (r = -0.26, P < 0.001; Figure Figure1I).1I). These results suggest that VAB may be mediated by an increase in EO SN and that both traits are negatively correlated with eye size.
To determine the number and position of the genetic loci controlling VAB, SN number and eye size, we first constructed a genetic linkage map of the Astyanax genome from 246 markers genotyped among 384 F2 individuals. Most of the markers represent randomly-distributed microsatellite polymorphisms, but we also included 28 markers representing candidate genes for SN development, behavioral variation and other previously mapped traits (for example, ngn1, eya1, nrg2, the 5ht serotonin receptors, the mao serotonin enzyme, the sert serotonin transporter, shh, pax6 and oca2) [7,23,42]. Our linkage map recovered 27 linkage groups (LG) spanning 1,513 cM (Figure (Figure2).2). Since Astyanax have 25 haploid chromosomes [43], this map likely covers the entire genome. Our map is comparable to a previously published Astyanax genetic map [23] constructed from a cross between a Mexican surface fish and Pachón cavefish. Small discrepancies in marker order and inter-marker distance between this previous map and ours are likely due to the use of different surface fish populations.
Using this linkage map, we performed three types of QTL scans: (1) classic interval mapping with a single-QTL model treating the F2 and F3 separately, as well as combining these F2 and F3 datasets; (2) a modified single-QTL analysis using both F2 and F3 but accounting for kinship among these generations in QTLRel [44]; and (3) multiple QTL mapping with both F2 and F3 as implemented by the function stepwiseqtl in R/qtl [45,46]. By comparing the results of the first and second analyses (Additional file 1, the left and right column), we assessed the consistency of our QTL scans as well as the potential noise caused by using multiple generations. If the results were similar and relatively unaffected by kinship (see variance components in Additional file 2), we then applied the multiple QTL scan in stepwiseqtl since our Castle-Wright estimates suggest that a minimum of two or more loci control most traits (see above). We analyzed six traits using the classic and modified single-QTL models (VAB, SO-3 SN number, SO-3 SN diameter, EO SN number, eye size/SL and, as a control, albinism). We found similar results for all traits except SO-3 SN number and diameter (Additional file 1D, E). These two traits exhibited large variance components due to kinship and we thus dropped them from further analysis. The variance components of the remaining traits were small (Additional file 2). Using stepwiseqtl with both F2 and F3 to analyze albinism and eye size, we were able to detect two previously-reported QTL for albinism at the oca2 gene locus on LG1 [23] (Table (Table1,1, Additional file 1F, G) and for eye size on LG 2 [33]. We conclude that we can safely drop relatedness for the remaining traits and, therefore, show the results for multiple QTL mapping using the combined F2 and F3 datasets.
We initially surveyed QTL for VAB at different vibration stimuli. As reported previously, cavefish show the strongest VAB at 35 Hz and intermediate-levels at 10 and 50 Hz; additionally, some surface fish show an intermediate-level of VAB at a broad range between 5 and 35 Hz [27,40]. By performing an abbreviated QTL scan using the single-QTL model, we detected two significant QTL for VAB, but only with the 35 Hz stimulus (Figure (Figure3A).3A). This indicates that cavefish VAB at 35 Hz has a genetic basis. The same VAB QTL were revealed using stepwiseqtl mapping below.
By performing stepwiseqtl, we detected nine QTL: two each for VAB and EO SN number on LG 2 and 17, and five for eye size on LGs 2, 4, 6, 16, and 17 (Figure (Figure3,3, Table Table1).1). The QTL for VAB and EO SN number were relatively consistent across all types of analyses, allowing for some variation since single-QTL models are not designed to detect QTL for complex traits. The eye size QTL on LG 2 and 17 were also consistent among analyses, but the eye QTL on LG 4, 6 and 16 were not evident in the single-QTL scans (Additional file 1B, left column). Although this result may be expected since eye size is genetically complex in Astyanax [33], these three QTL will need to be confirmed in future analyses. We did not detect significant epistasis among any of these QTL (Figure (Figure3,3, Table Table1).1). For each phenotype, the sum of detected QTL accounted for 19.8%, 18.5% and 40.1% of the total phenotypic variance in VAB, EO SN number and eye size, respectively (Table (Table1).1). Although the QTL for these three traits were found on five different linkage groups distributed throughout the Astyanax genome, the Bayesian credible intervals for six VAB, EO SN and eye size QTL formed congruent or overlapping clusters on LGs 2 and 17 (1 cM overlap at the 47 cM position in LG 2, and 5 cM overlap at the 3 to 8 cM position of LG 17; Figures Figures22 and 3B, C). Particularly noteworthy, the credible intervals for the two most significant eye size QTL overlap with the two QTL for VAB and EO SN number (Figure 3B, C). The observed number of QTL clusters for these three traits is significantly higher than expected by under a Poisson distribution (Χ2 = 98.2, df = 3, P = 3.8 × 10-21) [47]. Since alleles located within short genomic regions are generally inherited together as a unit, these QTL clusters support the physical correlations we found for VAB, EO SN number and eye size (Figure 1F, I, J).
Further examination of these overlapping QTL supports the hypothesis that VAB is enhanced by expanding the number of SN within the EO at the cost of eyes. We found that the cavefish alleles at each QTL shift the VAB, SN and eye size phenotypes in the direction expected given the distribution of these phenotypes between the surface fish and cavefish P0; when VAB level was high there were more SN and smaller eyes than when VAB level was low or absent (Table (Table1;1; see insets of Figure 3B, C; and Additional file 3). None of the 28 candidate genes were associated with any of the 9 QTL. As reported previously [7,33], the shhA locus was not located in or near any of the eye size QTL (see at LG13 of Figure Figure2).2). In addition, alphaA crsytallin (cryaa) and shroom2, which were located within eye QTL in a previous study [3], did not map to an eye size QTL in the present analysis. The significant clustering of VAB, EO SN and eye size QTL on LG 2 and 17 supports the conclusion that the genetic factors responsible for VAB and EO SN enhancement are also responsible for eye regression, either as a result of genetic hitchhiking or pleiotropy.
The results of the quantitative genetic analyses suggest that VAB and EO SN number are phenotypically and genetically linked to eye size in Astyanax cavefish. These results raise the intriguing possibility that VAB is facilitated by an increase in EO SN in cavefish. To test this hypothesis, we compared the level of VAB among cavefish whose area of EO or SO-3 SN were ablated. Following EO SN ablation, cavefish showed a significant decrease in VAB (paired t-test: t9 = 3.66; P = 0.005; Figure 4A, D; ablated areas in the EO region are shown in Figure Figure4B;4B; EO SN numbers before ablation are shown in Figure Figure4C).4C). In contrast, cavefish showed only a minor reduction in VAB following ablation of SO-3 SN, which was not significant (paired t-test: t6 = 0.68; P = 0.524; Figure Figure4D).4D). Although the EO SN ablation included a part of the SO-3 region, as well as some canal neuromasts (Figure (Figure4B),4B), EO SN were consistently removed, whereas SO-3 ablation removed more SN than the total number of EO SN (number of ablated SN in the EO SN ablation: 38.5 ± 7.8; in the SO-3 SN ablation: 98.0 ± 27.1; mean ± s.e.m.; t15 = -2.45, P = 0.027). Thus, even though cavefish have fewer EO SN than SO-3 SN, these ablation experiments provide strong evidence that SN in the EO area are the main sensory receptors responsible for VAB.
Although the genes that increase VAB and EO SN may drive eye regression through genetic hitchhiking or pleiotropy (see also Figure 1I, J), it is still possible that reduced eye size alone may increase VAB. To test this alternative possibility, we further compared the level of VAB among surface fish with experimentally regressed eyes following shhA overexpression [26]. In addition to eye size, expanded shh expression is also known to increase jaw width and taste bud number in cavefish [11], suggesting that this pathway could potentially alter SN number or VAB. Following shhA mRNA overexpression, eye diameter varied considerably among surface fish (horizontal axis on Figure 5A-D), but was not associated with a significant increase in VAB (r = -0.12, P = 0.53), EO SN (r = 0.00, P = 1.00), or SO-3 SN (r = 0.20, P = 0.29) (Figure 5B-D). These results suggest that shh-induced eye regression is insufficient to increase EO SN and VAB and that the molecular pathway decreasing eye size concomitantly with VAB and EO SN enhancement is parallel to the SHH pathway. We conclude that the same or closely linked genetic factors responsible for the enhancement of EO SN and VAB are also responsible for the reduction in eye size (Figure (Figure66).
We have shown that eye regression in Astyanax cavefish is phenotypically and genetically correlated with the evolution of VAB, an adaptive trait controlling feeding efficiency [27], as well as the enhancement of its sensory receptors, SN specifically localized within the EO. Genetic mapping studies revealed clusters of congruent or overlapping QTL on LG 2 and 17 that control eye size, VAB and the number of EO SN. In addition, we have also demonstrated a significant role of the EO SN in VAB, and that the open space created by shh-induced eye degeneration is insufficient to promote VAB or SN enhancement. This experimental result further highlights the importance of the genetic linkage between eye loss and the VAB sensory system. Our results support the hypothesis that selection for an adaptive behavior and its underlying sensory receptors are enhanced in cavefish at the expense of eyes. To our knowledge, this is the first study to report phenotypic and genetic relationships between a regressive phenotype, an adaptive behavior and the sensory system responsible for this behavior.
We have discovered a significant genetic correlation between VAB and EO SN number but not between VAB and SO-3 SN number and size. This result was surprising in light of our previous studies, which found that removal of SO-3 SN reduced (but did not eliminate) VAB [27]. A possible explanation for this discrepancy is that the physical SN ablation method used in our previous study did not discriminate effectively between EO SN and SO-3 SN, which are present in adjacent regions in the cavefish cranium, or that the actual contribution of SO-3 SN to VAB is minor compared to that of EO SN. Our current ablation study supports either explanation by showing that EO SN are the major VAB receptors, whereas SO-3 SN, despite their larger number, have only a minor role in VAB (Figures (Figures11 and and4).4). Although our EO SN ablation experiment also ablated some SO-3 SN and canal neuromasts, we conclude that the latter receptors play a minor role in VAB for two reasons. First, in the SO-3 SN ablation experiment, more SN were ablated than EO SN. Yet, despite the loss of more SN in SO-3, the level of VAB was not attenuated significantly (Figure (Figure4).4). In contrast, deleting a much smaller population of EO SN resulted in a dramatic and statistically significant reduction in VAB (Figure (Figure4).4). Second, in several former studies, the involvement of canal neuromasts in VAB was excluded through three observations: (1) selective SN ablation was enough to decrease VAB even while maintaining canal neuromasts [27], (2) cavefish VAB was tuned to the frequency at the peak sensitivity of SN but not of canal neuromasts [27], and (3) the selective interference with canal neuromasts failed to attenuate the response [37]. So what makes EO SN necessary for VAB? It is possible that there are structural or neural circuitry differences between the two populations of EO and SO-3 SN that may account for our results, or that the EO SN are better positioned above the eye socket to detect fluctuations in water due to the bulging shape of the adipose tissue plug that fills the eye orbits of cavefish [5] or bulging eye balls of F2 and F3 hybrids [48]. However, since the level of VAB did not drop to zero in our EO SN ablation, it seems likely that other SN contribute to VAB as well, possibly including SN at SO-3 and on the dorsal trunk [27].
Our present investigation has also clarified the difference between the strong type of VAB observed in cavefish (10+ approaches towards a vibrating object) and the weaker form previously detected in a small proportion of surface fish raised in the laboratory (> 4 approaches) [27,40]. Cavefish VAB peaks at 35 Hz, whereas surface fish VAB shows a continuous low activity from 5 to 35 Hz [27,40]. In this study, VAB showed two genetic components detectable at 35 Hz, but none at 10 or 50 Hz, suggesting that the weak form of surface fish VAB appears without a major genomic change and could be accomplished through environmental effects and/or developmental plasticity. Considering that at least three cavefish populations (Pachón, Los Sabinos and Piedras cavefish) exhibit a strong form of VAB [27,49], VAB may, therefore, represent a broadly distributed evolutionary trait in cavefish. The elaboration of SN at a new site, the EO, could have been a key factor in the evolution of VAB during adaptation to subterranean habitats [10,40]. Determining the mechanisms underlying EO SN development and VAB among independently evolved cave populations [50,51] could open a path to understanding the origins of neurological and behavioral novelty during evolution.
The major conclusions of this study are that EO SN number is strongly correlated with both VAB and eye size, and that all three traits are controlled by QTL on LGs 2 and 17 with congruent or overlapping Bayesian credible intervals. These results suggest that the gene loci responsible for VAB, EO SN number and eye-size may be subject to possible physical linkage and genetic hitchhiking or may in fact be the same genes with pleiotropic effects. The existence of QTL clusters could facilitate rapid adaptation to environmental changes by concurrently fixing multiple beneficial traits. Previous QTL analyses have also shown tight linkages and overlapping QTL for several other cavefish traits, including eye size, lens size, taste bud number, tooth number, melanophore number and chemical sense ability [7,33]. This observation suggests that adaptation to cave life in Astyanax is the result of tradeoffs among many phenotypic traits, possibly through functional shifts within pluripotent neural crest cells which contribute to most of the above traits through developmental processes [52]. However, one must note that widespread clustering of QTL does not necessarily entail a phenotypic tradeoff. In a previous scan of QTL in Astyanax, Protas et al. reported the overlap of six QTL for eye size and melanophore number (MelE), yet the phenotypic correlation between these traits is only r = -0.05 [7,33].
The present investigation provides a clear example of a tradeoff between constructive traits (VAB and EO SN) and a regressive trait (eye size) in parallel to the SHH-signaling pathway. The short genomic distance between the QTL for eye regression, EO SN and VAB [11] could be a key factor in promoting the rapid adaptation of Astyanax to a novel environment. In other rapidly evolving animals, such as African cichlids and three-spined sticklebacks, QTL clusters have also been shown to control groups of potentially advantageous traits, including sex determination and body color, and body shape and plate number [53,54].
We have discovered a significant genetic correlation among eye size, VAB and EO SN number. Our results, therefore, support the hypothesis that eye loss has evolved in Astyanax cavefish as a result of indirect selection against eyes due to a trade-off between two constructive traits, VAB and EO SN enhancement, based either on genetic hitchhiking or pleiotropy. There has been a long-standing debate on the neutrality or benefit of eye regression in cave animals [1,2,4,34,35]. Since cave-adapted arthropods exhibit reduced eyes and elongated sensory antennae [1,2,8], the loss of eyes as a result of selection for increased tactile sensitivity is an attractive general hypothesis for the convergent evolution of eye regression in cave fauna.
Astyanax mexicanus surface fish used in this study were laboratory raised descendants of original collections made in Balmorhea Springs State Park, Texas, and cavefish were laboratory raised descendants of original collections from Cueva de El Pachón (Pachón cavefish) in Tamaulipas, Mexico. We generated all hybrid progeny from an original mating between one pair of cavefish and surface fish [11,41,55,56]. One pair of F1 hybrids from this cross was mated to generate 384 F2 individuals for phenotypic and genetic analysis, while another pair of F1 hybrids was crossed to generate a second group of about 200 F2 progeny. These F1 hybrids were randomly selected. We genotyped this second set of hybrids at markers 017a, 215d, 145a, 204d and 218e, which are near potential VAB (017a and 215d) and SN (145a, 204d and 218e) loci identified by a preliminary QTL scan using 384 F2 individuals. To overcome a reduced rate of spawning in this group, we chose three pairs of F2 progeny that were either homozygous or heterozygous for the cavefish alleles at the marker loci and crossed them to generate 91 F3 progeny. Although these hybrids exhibited a reduced rate of spawning, there was no noticeable difference in their survivability. Hybrids were fed living Artemia larvae and were maintained individually in 500 ml tanks or in groups of approximately 10 in 3 L tanks. We phenotyped each hybrid once for SN number and eye size and two or three times for VAB when they were one to two years old. Because of the limitations of tank size [57], hybrids of different ages approximate the same standard length (approximately 3 cm) and there is no positive relationship between age and SN number at either SO-3 or the EO (Additional file 4). Additionally, the results of our QTL scans did not differ when including age as a covariate (data not shown). The animal procedures used in this study were approved by the University of Maryland Animal Care and Use Committee and conform to NIH guidelines.
We isolated genomic DNA from fin-clips using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA) or the quick extraction protocol [58]. We genotyped fish for 226 genome-wide polymorphic microsatellite loci described previously [23] and for polymorphisms within or near 20 candidate genes for SN number and behavioral variation. We successfully genotyped 93,208 (95.6%) of these microsatellite markers among 382 F2 and 11,375 (95.5%) of 125 selected microsatellite markers among 91 F3. Candidate gene homologues were identified in the fugu, medaka, stickleback, tetraodon and zebrafish genomes. Degenerate primers were designed to match the most conserved amino acid regions using CODEHOP [59] or SCPrimer [60]. We performed degenerate PCR on genomic DNA extracted from the fin-clips of P0 surface fish and Pachón cavefish using Takara LA Taq DNA polymerase (Clontech, Mountain View, CA, USA) and the following touch-down program: 1 cycle at 94°C for 90 sec; 3 cycles each at 94°C for 20 sec, 63°C for 20 sec and 68°C for 2.5 minutes; 3 cycles each at 94°C for 20 sec, 61°C for 20 sec and 68°C for 2.5 minutes; 3 cycles each at 94°C for 20 sec, 59°C for 20 sec and 68°C for 2.5 minutes; 30 cycles at 94°C for 20 sec, 57°C for 20 sec and 68°C for 2.5 minutes; and finally 1 cycle at 72°C for 4 minutes, followed by a 4°C incubation. PCR fragments were characterized by either direct sequencing with the same degenerate primer used for PCR or by conventional sequencing after sub-cloning into the pCRII plasmid using the TOPO TA Cloning Kit (Invitrogen, Grand Island, NY, USA). The homology of each candidate gene fragment was confirmed by blastn. After sequencing each candidate gene region in surface fish and Pachón cavefish, the sequences were extended with primers designed from known sequences using the GenomeWalker™ Universal Kit (Clontech), and aligned using either Vector NTI (version 7, InforMax, Bethesda, MD, USA) or Sequencher™ (version 5, Gene Codes Corp., Ann Arbor, MI, USA) software to identify microsatellite and single nucleotide polymorphisms (SNPs). Microsatellite polymorphisms were genotyped as described by Protas et al. [23] after designing primers in Primer3Plus [61]. SNPs were genotyped by either Taqman® (Roche Applied Science, Indianapolis, IN, USA) or HybProbe (Roche Applied Science) technology. Primers and fluorescent probes were designed using Primer Express (version 3.0, Applied Biosystems, Carlsbad, CA, USA) or LightCycler® Probe Design Software 2.0 (Roche Applied Science), respectively. The primer and probe sequences for these candidate genes are provided in Additional file 5.
We assayed VAB as described previously [27,49]. Briefly, four or five days before the beginning of an assay, individuals were acclimated in a cylindrical assay chamber (Pyrex 325 ml glass dish, 10 cm diameter × 5 cm high, Corning, Corning, NY, USA) filled with conditioned water (pH 6.8; conductivity approximately 600 μS). During the assays, vibration stimuli were created using a 7.5 mm-diameter glass rod vibrating at 35 Hz using a Leader LG1301 function generator (Leader Instruments Corp., Cypress, CA, USA) with an audio speaker (Pro Speakers, Apple, Cupertino, CA, USA). The number of approaches (NOA) to the vibrating rod was video recorded during a three-minute period under infrared illumination (880 nm wave length, BL41192-880 black light, Advanced Illumination, Rochester, VT, USA), and counted using ImageJ 1.42q software (NIH, Bethesda, MD, USA).
Eye size was determined from photographs of each fish by digitally measuring the diameter of each eyeball along its rostral-caudal axis using ImageJ. We standardized these measurements by dividing them by each fish's standard length (SL), the length of the body from the tip of the snout to the base of the caudal fin.
Neuromasts were vital stained as described previously [27,49]. Briefly, fish were immersed in 25 μg/ml 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI; Invitrogen, Eugene, OR, USA) [62] dissolved in conditioned water for one hour, followed by immersion in ice-cold 66.7 μg/ml Ethyl 3-aminobenzoate methanesulfonate salt (MS222, Sigma) in conditioned water. The specimens were viewed under a fluorescence microscope (Axioskop 2 equipped with 2.5× Plan-Neofluar lens with a numerical aperture of 0.075 and a FITC filter set; Zeiss, Göttingen, Germany); we then photographed the fish with a Zeiss Axiocam CCD camera. Neuromasts were quantified on images of DASPEI-stained fish using ImageJ software. SN were counted both in the epidermis over the cranial third suborbital (SO-3) bone [41] and within the orbit epidermis dorsal to the line of the suborbital canal neuromasts (indicated by red dashed line in Figure 1L-N). To determine SN size, the long diameter of the 10 largest SN in the same area was measured and averaged.
Following marker genotyping, we used MapManager QTXb20 [63] software to construct an initial linkage map of the SNP and microsatellite markers using 384 F2 fish with complete genotype data and the Kosambi map function. We then used the program R/qtl [45] to identify statistically unlikely events, such as double crossovers in adjacent intervals or single crossovers in a small interval under maximum likelihood estimation [64], to refine marker order and to estimate inter-marker distances.
We performed QTL mapping using two strategies: (1) multiple interval mapping via the function stepwiseqtl in R/qtl [45]; and (2) single-QTL model mapping accounting for relatedness in the program QTLRel [44]. All logarithms of the odds (LOD) significant thresholds were determined following 2,000 permutation tests after single-QTL model mapping with scanone implemented in R/qtl or scanOne in QTLRel. The 95% Bayesian credible intervals for QTL positions, which are analogous to 95% confidence intervals, were obtained using R/qtl. For multiple QTL mapping, we first calculated genotype probabilities for any missing genotype using the calc.genoprob function before performing the forward/backward model selection function implemented in stepwiseqtl. Following model selection, we chose the model with the maximum penalized LOD score generated from 1,200 permutation tests [65]. For QTLRel mapping, we first generated pedigree charts for the F2 and F3 hybrid families and then calculated condensed identity coefficients and estimated variance components with the cic function. We used these variance components when scanning the genome for QTL using the scanOne function (see Additional file 2). The genome-wide significant thresholds (P < 0.05) for this latter analysis were calculated from 2,000 permutation tests.
SN were ablated by a modification of our previous method [27]. Prior to ablation, we measured VAB and counted neuromasts stained with DASPEI. Only cavefish showing a high VAB level (more than 16 NOA) were selected for SN ablation. SN were ablated by applying Vetbond non-toxic tissue adhesive (3M, St. Paul, MN, USA) either to the EO or the SO-3 region using a micro nylon loop, a traditional embryological tool [58]. After application of tissue adhesive to one side, cavefish were exposed to air for 10 seconds, and tissue adhesive was then applied to the same area on the opposite side, followed by a second cycle of air-drying. The treated fish were placed in a 10 cm-diameter cylindrical chamber containing conditioned water at room temperature. Within a day of active swimming the tissue adhesive usually peeled off the body, resulting in a void in the underlying field of SN. After four to six days of acclimation/recovery, SN ablated fish were subjected to the VAB assay followed immediately by staining for one hour with 25 μg/ml DASPEI.
Overexpression was carried out by injecting zebrafish shhA mRNA (20 to 80 pg) into two to four cell stage embryos as described previously [26]. Capped shhA mRNA was synthesized from the pSP64T plasmid using the mMESSAGE mMACHINE® SP6 Kit (Life Technologies, Grand Island, NY, USA).
Correlation studies were conducted using IBM SPSS 20.0.0 software (IBM, Somers, NY, USA). Castle-Wright estimates were performed according to Lynch and Walsh [66]. Significance of QTL clustering was performed using a goodness-of-fit [47]. We divided the 1,500 cM Astyanax genome into 50 bins of 30 cM each and counted the number of bins with 0, 1, 2 and 3 QTL. We then compared this observed distribution to that expected from a Poisson distribution where the average number of QTL expected per bin is 0.18 (9 QTL divided by 50 bins of 30 cM each).
DASPEI: 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide; EO: eye orbit; LG: linkage groups; LOD: logarithms of the odds; MelE: melanophore number above the eye; NOA: number of approaches; QTL: quantitative trait locus; s.e.m.: standard error of the mean; shh: sonic hedgehog; SL: standard length of fish; SN: superficial neuromast; SNPs: single nucleotide polymorphisms; SO-3: suborbital bone 3; VAB: vibration attraction behavior.
The authors declare that they have no competing interests.
MY and WRJ conceived the project and designed the experiments. MY performed behavioral assays, vital dye labeling (DiI), morphometrics, cloning, most of the genotyping, QTL mapping, SN ablation studies, and a part of the shh-injection experiments and analyses, and generated the figures. YY performed some of the shh-injection experiments. KEO participated in the QTL mapping analyses. MY wrote the manuscript with significant contributions from KEO and WRJ. All authors read and approved the final manuscript.
LOD scores from single-QTL mapping analyses with or without accounting for relatedness among the F2 and F3. (A-F) LOD scores computed with classic single-QTL interval mapping using the F2 dataset (blue lines), F3 dataset (green lines), or F2 + F3 combined dataset (red lines) in R/qtl (left column) and QTLRel (right column). In QTLRel, condense identity coefficients were calculated to adjust background variation according to kinship among the F2 and F3 generations. This analyses found (A) two QTL by the classic scan with the F2 and the F3 dataset (left column), or one QTL (right column) for VAB, (B) five QTL with the F2 and the F3 dataset (left at LG 2, 9, 17 and 23), or three QTL (right at LG2 and 17) for eye-size, (C) one QTL for EO SN number in both, no significant QTL for either (D) SO-3 SN number or (E) SO-3 SN diameter, and (F) one QTL for albinism in the both methods. (G) The LOD score from multiple QTL mapping of albinism also identified a single QTL at the oca2 locus, as described previously [23]. The horizontal lines indicate genome-wide significance of P < 0.05 based on 2,000 permutation tests.
Variance Components calculated in QTLRel software. The phenotypes of VAB, Eye size, EO SN number and albinism exhibited small variance components for both the additive and the dominance genetic matrixes, whereas SO-3 SN number and SO-3 SN diameter exhibited large variance components at the dominance genetic matrix. AA: additive genetic matrix; DD: dominance genetic matrix; and EE: the residual matrix.
Scatterplots of phenotypic values against each genotype at the two cluster loci. Scatterplots of phenotypic values of VAB (top row), eye size/SL (middle row) and EO SN number (bottom row) were shown at the marker position of 17b (left column, VAB and EO SN number), 222d (left column, eye size), and 17a (right column, all three traits) at linkage group 2 and 17. Blue dots and bars: F2 generation, Green dots and bars: F3 generation. Bars indicate mean ± s.e.m. Sf/Sf, surface fish homozygote; Sf/Cf, heterozygote; and Cf/Cf, cavefish homozygote. The cavefish alleles at each QTL cluster shift the distributions of VAB, SN and eye size phenotypes in the direction toward higher VAB level, more EO SN and smaller eyes.
Phenotypic distributions between approximately one- and two-year-old fish used in current study. The boxplot of phenotypic distributions were compared between approximately one- and two-year-old F2 fish. The increased VAB levels in two-year-old fish (top) was not associated with the increases of EO (middle; no significant difference: n.s.; The Mann-Whitney non-parametric test was applied since phenotypic values were not normally distributed), or SO-3 SN number (bottom; significantly decreased in two-year-old fish). N were indicated in the boxes.
Candidate gene primer and probe sets used in this study. Primers for the microsatellite markers were designed to amplify 150 to 350 base-pair genomic fragments containing length polymorphisms between surface fish and cavefish. Primers and fluorescent probes for Taqman and HybProbe genotyping methods were designed to detect single nucleotide polymorphisms between these two morphs.
We thank Z Sanford, R Fernandez, J Giarrusso, D Norman, S Ahmed, S Siddiqui, L Younger and J Park for fish maintenance; R Fernandez, D Norman, S Ahmed and J Park for genotyping and phenotyping; K Broman for helpful comments on QTL mapping; and A Parkhurst for technical assistance. A Japan Society for the Promotion of Science Postdoctoral Fellowship to MY and NSF (IBN-05384) and NIH (R01-EY014619) grants to WRJ supported this research.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: Hello DIS Dads. I'm taking my wife and daughter to the World for Christmas after being away from them for 6 months. I get back home early in December but during that time we will be in the process of moving to a new state. My wife has had her hands full holding down the fort. She is getting the house packed up and taking care of everything that goes along with selling a house. I would like to do something or get her something to show her how much I appreciate all the hard work she has done in the time that I have been gone. I would like to surprise her with it when we get to Disney. So DIS Dads...any ideas on how to show her how much I appreciate what she has done? She does not have lavish tastes or expectations. I know she would appreciate the gesture more than how much I actually spend on her but I have no problem dropping a pretty penny. Thanks in advance.


text:
Stock markets diverge, all eyes on TrumpMon, 27 Feb 2017 7:12 PM
by Danny McCord
World stock markets diverged Monday, as a recent Trump-inspired rally showed signs of fragility.
A record 11th successive all-time high close for New York's Dow on Friday came as investors nevertheless are growing worried that the recent buying -- fuelled by expectations that US President Donald Trump will introduce economy-boosting measures -- may have gone too far.
Treasury Secretary Steven Mnuchin's warning that growth might not hit the four percent Trump promised, as well as uncertainty around the new president's plans to slash taxes and spend big on infrastructure, sent the dollar tumbling at the end of last week, before recovering ground Monday.
"The markets are continuing to find a way to squeeze out new highs and post small gains on a daily basis, a large portion of which appears to be attributable to the apparently substantial but as yet unknown stimulus measures from the Trump administration," said Craig Erlam, senior market analyst at Oanda trading group.
Elsewhere, the London Stock Exchange on Sunday said it is "highly unlikely" it will be able to meet antitrust conditions set by Brussels for its tie-up with Deutsche Boerse, throwing the merger into doubt.
The announcement by LSE, which also operates the Milan stock exchange, is the latest twist in its longstanding attempt to merge with the German stock exchange operator.
The LSE said it had examined the European Commission's request to divest its majority stake in Italian trading unit MTS, concluding it could not commit to such a request.
LSE and Deutsche Boerse were both trading down 2.6 percent around the half-way mark in Monday's European trading, "as it becomes clear that the merger... is on a knife-edge", noted Neil Wilson, senior market analyst at ETX Capital.
"The regulatory hurdles were always a risk and with Brexit there are additional hurdles to clear that seem close to insurmountable now," he added.
- 'Fraying at the edges' -
Despite Monday's focus on the LSE, "this week it's really about President Trump, his address to a joint sitting of Congress and his tax plan", said Greg McKenna, chief market strategist at AxiTrader.
"I get a sense traders want to believe in him -- hence the stocks rally -- but (with haven investment) gold up and rates down suggest there is some fraying at the edges of market ebullience."
Markets are hoping the speech on Tuesday will provide more details about Trump's tax pledge, which he said this month would be "phenomenal".
- Key figures around 1130 GMT -
London - FTSE 100: UP 0.1 percent at 7,253.12 points
Frankfurt - DAX 30: UP 0.2 percent at 11,822.70
Paris - CAC 40: DOWN 0.1 percent at 4,842.25
EURO STOXX 50: UP 0.2 percent at 3,310.37
Tokyo - Nikkei 225: DOWN 0.9 percent at 19,107.47 (close)
Hong Kong - Hang Seng: DOWN 0.2 percent at 23,925.05 (close)
Shanghai - Composite: DOWN 0.8 percent at 3,228.66 (close)
New York - Dow: FLAT at 20,821.76 (close)
Euro/dollar: UP at $1.0579 from $1.0561
Pound/dollar: DOWN at $1.2411 from $1.2463
Dollar/yen: UP at 112.27 yen from 112.13 yen
Oil - Brent North Sea: UP 39 cents at $56.38 per barrel
Oil - West Texas Intermediate: UP 27 cent at $54.26


text:
2012. Oh my goodness.
I promised myself I'd do some special things this year, and I'm already falling behind. New year's resolutions have never stuck to me; they seem to fade from view after about a week or two, if that long. Because of that, I won't call them resolutions, but instead projects. There, that's less daunting.
I need to keep record of them. Sure, I feel kind of stupid having to keep physical evidence of the promises I'd kept to myself, but at least I have something to look back on, pass or fail. But failure isn't an option this time.
The first of my projects is so incredibly easy: Take a picture everyday. I love taking pictures, I take them all the time, but it seems to be in spurts; I'm very inconsistent. I want to take better photos and more of them, the only way to learn is by doing, right? Lisa Bettany's posts have inspired me more than ever to become a professional photographer, if only for personal enjoyment. I have a fantastic smartphone in a mature (and still growing) ecosystem, pair all of that with the best lens in a cellphone and I have absolutely no excuse not to pursue photography more extensively.
The second project in my list can be done anywhere; Write more, and more frequently. Ideally I'd like to write at least once a day, but I'm sure a time will come when that just isn't doable due to some clash of scheduling. I have my iPhone with me all the time, and if it's Pages or Notes, I am more than capable of jotting a few lines down anytime inspiration hits.
The third, which I'm sure I'll have fun with, is Project 365. Essentially, it's a challenge to write on one topic, everyday, for a year. If I had to pick one topic, it'd be something about iOS. I love iOS, but I also love design and art. I don't see myself being able to confine to one subject, but I think I'm going to base my Project 365 around analyzing myself. Faults, strengths, things I'd like to change, goals and the like.
The question now is just how much of this I can keep to. Challenge accepted.


text:
"This base of this recipe is called Puyallup Fair Scones (Puyallup Fair Scones (Fisher Scones))...I of course had to mess with a good thing and here's the recipe I came up with. These are even better! Here's some of the research on scones that I found...using butter gives the best flavor, but shortening preserves the scones better after the first day. Hence the 1/2 butter, 1/2 shortening, a good compromise. Using heavy cream keeps the scones moister than milk. The recommended flours (for taste and gluten content) are first, Pillsbury Unbleached Enriched All-Purpose Flour (with a 10 - 11% gluten content), second King Arthur Unbleached Enriched All-purpose Flour (with an 11% gluten content). Chilling the butter and shortening in the freezer for 20 minutes or so helps the fat not "melt" when mixing. You can use your hands or a pastry blender to mix the fat and flour, but using a food processor also keeps the fat intact and not "melted" from the heat of your hands. Mix as little as possible so that the gluten does not over develop and make the scones tough. Sifting is critical. 2 1/2 cups unsifted flour weighs more than 2 1/2 cups sifted flour and would make a tougher scone. If you want to get really into it, "they" say that you should place a measuring cup on top of some parchment/wax paper and actually sift the flour into the cup, use the paper to catch the excess."
Mrs. G's Fair Scones
1 recipe photo
READY IN:35mins
YIELD:8 scones
Ingredients Nutrition
- 2 1⁄2 cups unbleached all-purpose flour
- 2 teaspoons baking powder, double acting (make sure your baking powder is still active!)
- 2 tablespoons sugar
- 1⁄2 teaspoon salt
- 3 tablespoons shortening, chilled
- 3 tablespoons unsalted butter, chilled
- 3⁄4 cup heavy cream
- 3 tablespoons heavy cream
- Sift and measure the flour.
- Re-sift with other dry ingredients.
- Work shortening into dry ingredients with the fingers.
- Add rasinins to flour-fat mixture and mix thoroughly (you may omit raisins).
- Add milk to mixture. (If you are omitting the raisins, add another 2 Tablespoons of milk).
- Turn out on to a floured board and divide into two equal pieces.
- Roll or pat each into a round and to the thickness of biscuits (3/4 inch to a full inch).
- Cut into wedge shaped pieces like a pie and bake about 15 minutes at 450 degrees on an ungreased baking sheet.
- To serve like they do at the fair; split open but do not cut clear through. Fill with jam and close.
get the genius kitchen app.
Watch on your iPhone, iPad, Apple TV, Roku, or Fire TV.Learn More


text:
Dierker, Fastaia to represent Astros at Draft
Dierker, Fastaia to represent Astros at Draft
Dierker will be given a different set of duties next month when he and Astros part-time scout Ed Fastaia, a former New York firefighter who was a first-responder on 9/11, will be representing the team during the first day of the First-Year Player Draft, June 4, in Secaucus, N.J.
Dierker and Fastaia, who is representing the team for the second year in a row, will be sitting at the Astros' Draft table and will phone in the pick, which will be announced by Commissioner Bud Selig. The Astros have the No. 1 overall pick.
The annual First-Year Player Draft takes place this year from June 4-6, beginning with the first round and Comp Round A on Monday at 6 p.m. CT. The first night of the event will be broadcast live on MLB Network and streamed live on MLB.com. Rounds 2-40 will also be streamed live on MLB.com on June 5-6.
MLB.com's coverage, sponsored by CenturyLink, will include Draft Central, the Top 100 Draft Prospects list, Draft Tracker, a live interactive application that includes a searchable database of every Draft-eligible player, and Draft Caster. You can also keep up to date by following @MLBDraft on Twitter. And get into the Draft conversation by tagging your tweets with #mlbdraft.Dierker, who had his No. 49 retired by the Astros in 2002, is currently serving as a community outreach executive for the team. Fastaia was driving the fire engine carrying members of Brooklyn's Engine 246 early on Sept. 11, 2011, when they were dispatched to respond to the terrorist attacks at the World Trade Center.
Brian McTaggart is reporter for MLB.com and writes an MLBlog, Tag's Lines. Follow @brianmctaggart on Twitter. This story was not subject to the approval of Major League Baseball or its clubs.