Cochliomyia hominivorax (Coquerel), is one of the most damaging parasites of livestock, causing millions of dollars in annual losses to producers. The fly is an obligate parasite of warm-blooded animals, including humans. After a successful 50-year eradication campaign, C. hominivorax has been eradicated from the USA, Mexico and Central America by an area-wide integrated pest management approach. Recently, Caribbean and South American countries have expressed an interest in this approach. Aiming to support forthcoming projects in these countries, this review describes the main technical components of past and ongoing AW-IPM campaigns against C. hominivorax. The New World Screwworm,  Cochliomyia hominivorax  , is an obligate parasite of warm-blooded animals, causing primary myiasis in pre-existing wounds. The fly has a very high reproductive rate and females can oviposit up to 450 eggs at 3–4 days intervals on the dry edge of wounds or body orifices. The eggs hatch after 12–20 hours, the larvae migrate immediately to the wound and start feeding on the wound fluids. The second and third instar larvae burrow deeply into the host tissue for feeding. Mature larvae leave the animal and pupate in the soil and the entire life cycle can be a short as 20 days under optimal environmental conditions [The New World Screwworm (NWS), et al. [ca. US$ 20 million/year and US$ 50–100 million/year, respectively. In 1935, an infestation with 230,000 myiasis cases was reported in USA [et al. [The larvae from a single oviposition can kill smaller animals and multiple infections can kill mature cattle ,5,6. Othet al. , the estd in USA  and Parid in USA  reportedHumans are also hosts and the literature contains numerous reports of human infestations in Colombia, Suriname, Guyana, Ecuador, Paraguay, Peru, Bolivia and Uruguay. Leão  describeThe NWS was originally distributed throughout South and Central America, the southwestern States of USA and across the tropical belt to Florida ,12,13. HC. hominivorax. An IPM approach can be defined as “a decision support system for the selection and use of pest control tactics, singly or harmoniously, based on cost/benefit analyses that take into account the interests of and impacts on producers, society, and the environment” . The AW-Cochliomyia Towsend is comprised of four Calliphoridae species in the Neotropical region, Cochliomyia aldrichi , C. minima , C. hominivorax  and C. macellaria  [C. aldrichi and C. minima are restricted geographically to some Caribbean islands and Florida [C. minima myiasis has been reported in Puerto Rico [The genus C. macellaria, the secondary screwworm, is the species most commonly confused with C.hominivorax in myiasis. This fly is a sarcosaprophagous species, but can be found as a secondary invader in wounds. It is distributed from southern Canada to Argentina [C. macellaria and C. hominivorax can lead to misidentification by non-specialists, but some larvae and adult morphological characters are reliable to distinguish the two species [rgentina . The geo species . C. hominivorax third-instar larvae can be easily distinguished due to the presence of heavily pigmented tracheal trunks from posterior spiracles (up to segment 9 or 10) and 7–12 papillae in the anterior spiracle. In C. macellaria, the tracheal trunks are not heavily pigmented, and dorsal spines are not found in the posterior margin of segment eleven [t eleven . C. hominivorax adults, black setulae can be found in the parafacialia of the head. The central stripe on thorax extends just forward of mesonotal suture. The fifth segment of abdominal tergite is usually very slight dusting laterally. In females, the basicostal scale is dark brown to almost black [In st black .C. macellaria adults, only yellow hairs can be found (but their insertions appear as black dots). The central stripe on thorax extends well forward of mesonotal suture. The fifth abdominal tergite presents dense white spots laterally. In females, the basicostal scale is yellow [In the fronto-orbital plates of s yellow . C. hominivorax [Sex identification of adults can easily be carried out for both species as males are holoptic or subholoptic and females are dichoptic . The sexinivorax . et al. [C. hominivorax and C. macellaria through wing morphometrics, suggesting that the partial warp scores could be used as basis of a test to distinguish both species. The major disadvantage of a wing shape test is that only non-damaged wings can be used. Wing morphometry can be another tool for identification. Lyra et al.  describeC. hominivorax and C.macellaria have also been investigated with success [Different methods based on molecular markers for the identification of  success ,22,23. C. hominivorax.Egg masses collected from sentinel animals, submission of larval samples from wounds by the public and animal health authorities, and collection of adults using trapping systems are essential in surveillance for NWS populations and for monitoring the progress of any AW-IPM campaign against Larvae can be obtained through regular inspection of livestock and wild animals. After being pulled from the wound with forceps, larvae can be dropped directly into 100% ethanol (for molecular or genetic studies) or put in a sealed container for later processing. To extend and to prevent them from turning black, larvae can be killed by immersion in hot water (> 80 °C) for 15–30 s and then stored in formalin or 80%–100% ethanol at ambient or lower temperatures. Alternatively, live larvae can be fixed in Kahle’s fixative for up to 24 h, rinsing in water and stored in 80%–100% ethanol .e.g., animals exposed to ticks, horn and stable flies, animals undergoing husbandry procedures, neonatal calves and lambs), consideration should be given to deliberate wounding of sentinels. The cost of sentinel animals, their intensive care, difficulty to get approval from animal welfare authorities, problems in transporting and keeping them in diverse habitats are factors that favor alternate methods of attracting adults. Parker & Welch [Wounded sentinel animals, especially sheep or goats, have been used in research and eradication campaigns to obtain egg masses. Female flies are attracted to infested wounds’ odors resultant from destruction and necrosis of tissues by larvae and bacteria ,24,25. I & Welch  suggeste2 in central Texas [et al. [A few trapping systems are available for collecting adults. Bishopp  was the al Texas  to captual Texas  isolated [et al.  and thatet al. [p-cresol, indole, iso-butyl alcohol, sec-butyl alcohol, and acetone). The new chemical bait (the first “Swormlure”) was as attractive as liver to NWS adults and much less attractive to other blowfly species, reducing significantly the time required to identify trap catches [Under field conditions, Jones et al.  continue catches . Other aet al. [Swormlure by mixing the compounds in different proportions, adding methyl disulfide and eliminating acetone. The new attractant, called Swormlure-2, proved to be more attractive to NWS than the original mixture in Bishopp traps and as attractive as decomposing liver. The capture of non-target flies also remained low. Swormlure-2 has a strong odor which permeates almost any material . Trials conducted at the Moore Air Base, Texas, indicated that the instantaneous area of influence of Swormlure-2 extended 125–150 m downwind of the attractant source [Coppedge et al.  refined t source .Swormlure-2 has a shelf life of more than three months [Swormlure-2 in trapping systems in the field. Dimethyl disulfide, butanol, butyric acid, and acetic acid appeared to evaporate the most rapidly. On the other hand, phenol, valeric acid, p-cresol, benzoic acid, and indole exhibited low but constant rates of evaporation. Swormlure-2 was more attractive during the first 7 days of exposure. Loss of attractancy appeared to result from changes in relative composition with time of exposure, but sex ratios of fly collections were not affected. Temperature and wind were the main weather-related factors affecting evaporation [Once mixed, e months . Brown &e months  providedporation .Swormlure-2 [Swormlure-2 released by 10-fold without reducing the number of flies captured.Polyester fiber wicks coated with a propylene skin proved to be superior to cotton dental wicks as a slow release mechanism for the rmlure-2 . Their uSwormlure-2 became increasingly erratic, particularly in humid, tropical areas [Swormlure-2. The most attractive mixture (Swormlure-4) contained higher amounts of dimethyl disulfide, butanol, and acetic acid, becoming almost twice as volatile as Swormlure-2. Traps baited with the new mixture captured 81% more wild C. hominivorax than traps baited with Swormlure-2 during 7 days of exposure, but sex ratios for captured flies were similar [In the 1980s, as the eradication campaign moved southward from USA into Mexico, the performance of al areas . The casal areas  then eva similar . Swormlure-2 had been used more often in a collapsible trap, which was a modification of the Bishopp blowfly trap [Since its formulation , the Swofly trap , by placfly trap .Swormlure-2, Broce et al. [Swormlures could be deployed in two bottles with dental cotton wicks as described by Coppedge et al. [C. macellaria than the collapsible trap [A few months after the development of e et al.  developee et al.  and place et al. . After fe et al. ,41. WOT ble trap ,41. Peterson II  evaluateSwormlure-4 was rapidly incorporated into the Screwworm Eradication Program as a replacement for liver baited traps and served as the standard detection system. For many years, this system was used extensively by the United States Department of Agriculture (USDA) to determine the first appearance of NWS in new areas and to serve as a quality control  for the released sterile insects. The WOT baited with et al. [Coppedge et al.  reportedet al. , coupledSwormlure. Solar power can keep the batteries of portable, electrocuter grid traps charged, but this alternative is more costly [In certain situations , sticky traps or electrocuter grid traps can be much more efficient than WOT ,44,45 an2 [Swormlure-4 in tropical dense forests [Wild NWS populations are not considered to exceed 200 female adults/km2 . Adults 2 ,48,49,50 forests , which m forests . The exa forests ,53. Furt forests . Following IPM principles , a set oThe NWS requires soft tissue of living warm-blooded animals for larval development . UsuallyParish  investigKrafsur & Lindquist  observedChanges in management can virtually eliminate several predisposing causes of screwworm infestations  from theThe incidence of myiasis resulting from breeding and birth  can be reduced by limiting them to periods of the year when NWS is less prevalent . i.e., times of the year with low prevalence of NWS). The same may be applied for other practices such as branding, castrating, dehorning, docking, earmarking, and registration tagging [Some man-made wounds can be prevented by following proper practices and more careful handling of livestock. Shear cuts can be greatly reduced by applying suitable wound protectors and proper timing of shearing  or beef (in the case of cattle), being marketed as soon as their condition warrants . More recently, many farmers and livestock companies have adopted stricter animal welfare standards in order to meet requirements of customers and consumers worldwide. Welfare practices dictate that farm animals must be respected by producers, transporters, and operators during breeding, handling and slaughter, besides being provided with an appropriate environment. Fear, discomfort, stress, hunger, thirst, excess of heat or cold, injuries, and diseases must be avoided at every single step . Ultimatet al. [et al. [Pediculus humanus L.) and mosquitoes (Aedes aegypti L.). These findings, made at the Orlando, Florida, laboratory of the USDA in conjunction with wartime research on the development of military insecticides, justified the beginning of a project on animal insecticides at the Kerrville, Texas, Livestock Insects Laboratory.Protection from screwworm infestation and parasite control of livestock can be achieved by spraying, dipping, or injecting larvicidal insecticides. The first success administration of an insecticide to control a bloodsucking insect was attained by Knipling , who renet al.  found th [et al.  found the.g., easy rearing, short life cycle, and diversity of hosts), C. hominivorax was selected as a screening insect [As testing against NWS larvae offered many advantages  wound dressing or Smear-62 (based on 35% benzol and 10% turkey red oil). Other commonly used smears were based on 5% toxaphene, 2% coumaphos or 2% dioxathion . e.g., coumaphos, chlorfenvinphos, diclorvos, diazinon, dichlofenthion, fenchlorvos, fenthionethyl, and trichlorfon, among others) are commonly applied as aerosol sprays  or as dusts that are puffed into the wound from plastic squeeze bottles. Individual treatment sachets  can also be either sprinkled directly on to a wound or brushed into the wound as a paste after mixing with cooking oil [et al. [Spraying livestock with insecticides or to smear wounds with insecticides formulated in viscous carriers became widely recommended methods of treatment and are still very used by farmers especially in developing countries. Organophosphorus compounds  are effective against NWS [e.g., cypermethrin, cyhalothrin, deltamethrin) have potential for control of NWS myiasis and are currently being applied in combination with organophosphorus compounds [Saccharopolyspora spinosa, was effective in treating and preventing NWS myiasis when applied as an aerosol spray [Chlorinated hydrocarbons  and C.hominivorax for at least 7 weeks after one application of a single ear tag. Ahrens et al. [Controlled-release formulations and devices can provide season-long protection for livestock with a single application of the insecticide. Gladney  showed ts et al.  verifiedStreptomyces avermitilis, has been effective against NWS in preventing navel strike of newborn calves and scrotal strike of castrated calves [et al. [A single subcutaneous injection of ivermectin (200 µg/kg), a macrocyclic lactone produced by d calves . Studiesd calves ,83. Anzi [et al.  reportedThere have also been an increasing number of publications reporting that a subcutaneous injection of doramectin (200 µg/kg) can be up to 100% effective as a NWS prophylactic, preventing infestation of artificial wounds, umbilical or castration wounds of calves, and infestation of post-parturient cows for up to 12–15 days after application ,86,87. Cetc.) are allowed to be scheduled to maximize productivity rather than being limited to times of the year when the NWS is less prevalent.Effectiveness also depends on factors such as cattle breed and degree of fly strike. In general, the larvicidal activity of these endectocides remains as one of the most effective approaches to control screwworms, as routine management practices  [When these units were evaluated in the area of the sterile insect mass-rearing facility (in which vicinity thousands of sterile flies were available weekly), most units gave kills of sterile flies in excess of 100/h/SWASS unit . In a prs ratio) .2) Caribbean island in the Netherlands Antilles, 6.5 km from Venezuela coast [C. hominivorax had become firmly reestablished, presenting a serious human and animal health problem [ca. 100 traps, each baited with 60 mL of Swormlure-2). The preliminary trapping and data collection provided a reliable base-line data for about 6 months prior to the release of the suppression units [2) over the rural part of the island by airplane by using a system developed by Goodenough et al. [2) in the city of Willemstadt for a period of 15 weeks from February 26 to May 13, 1977 [etc. were reported. In mid-May, the distribution of SWASS units was discontinued, and releases of sterile flies  were initiated. The low residual NWS population was eradicated in 3-4 weeks, and Curaçao was declared screwworm free on October 25, 1977 [The first opportunity to test the new suppression system in large-scale and against an entire screwworm population occurred in Curaçao, a relatively small  [Swormlure)  [The SWASS pellets  were prepared from 30.5% sugar, 30.5% dried blood, 7.5% corn cob grits, 2% dichlorvos, and 29.5% of a hardened wax preparation of 18% wax) . When st 7 days) . 2 area of the Davis Mountains, Jeff Davis county, Texas, during 1978 [2) twice weekly  on lanes 1.6 km apart, with the second application flown between these lanes [C. hominivorax and C. macellaria by ca. 72%–95% and 90%, respectively, and proved to be about three times more effective than the original SWASS cylinder during a 7 day period  [The new SWASS units were field tested for the first time on a large-scale basis against a non-isolated NWS population in a 518 kming 1978 . An outbing 1978 . The SWAse lanes . The pel 3 days) . et al. [2/application) twice weekly over an area of 2,625 km2 in a period of 3 weeks. After the 6 applications, the number of NWS adults captured in traps was reduced 72% and the number of egg masses collected was reduced 81%, proving the effectiveness of the new system even in a tropical rainy region. Tannahill et al.  performe2 or the capture of 0.5 wild flies/trap in a similar area) [With the success of SWASS evidenced in semi-arid and tropical areas, special-use registration for this suppression system in Texas, Arizona, and New Mexico was accomplished in March 1979, and the pellets started being used in outbreaks  , areas war area) . 2 twice per week on a 0.8 km swath width [The pelletized formulation of SWASS started being used operationally in the joint U.S.-Mexico Screwworm Eradication Program in the following years. The standard treatment became an air dispersal of pellets over infested areas at the rate of 60 pellets/kmth width . It was th width . Until tth width . Swormlure was attractive. Peterson II et al. [Dichlorvos could also be replaced by alternative insecticides that would not be repellent and could remain effective for at least as long as the I et al.  reportedSwormlure-4 evaporated and more dichlorvos remained as pellet size increased. In many cases, the amounts of dichlorvos fell below acceptable levels after only 48 h of exposure, but the standard and 9.5 mm pellets contained similar percentages of the insecticide after exposure up to 4 days. Exposed pellets lost more Swormlure-4 and dichlorvos. The 6.4 mm pellet was considered ineffective and, therefore, unsuitable for application in the eradication campaign. Mackley & Brown  comparedet al. [C. macellaria represented 48.2% of the total number of specimens collected. None of the species killed were noted as important beneficial species, and immigration from outside the SWASS treated area would quickly repopulate it if any non-target species had been drastically reduced [Considering the environmental impact of SWASS applications, Peterson II et al.  determin reduced . Mammalset al. [Swormlure-2 (9.6 g of wax for 30 mL of Swormlure). In field tests, bait stations were equal or superior in effectiveness to WOTs for killing or capturing NWS for 45 to 60 days. Their results also indicated that a target population could be reduced at least 25 to 30% with bait stations at a density of 1 trap/2.6 km2, and that a random distribution of the baits over an area could be more effective than clumping them around watering sites. A constraint for the SWASS pellets was that they could not be dropped from air in certain areas, such as near lakes and other major accumulations of water, rivers, certain areas supporting endangered wildlife, and where the system was not registered for use. Coppedge et al.  then decet al.  suspendeFrom its first design until the bait station approach, SWASS proved to be highly effective in high population situations . Thus, SThe Sterile Insect Technique (SIT) can be defined as “a method of pest control that uses area-wide inundative releases of sexually sterile insects to reduce fertility of a field population of the same species” . Before e.g., Chaudhury et al. [et al. [The mass rearing of the target insect, in sufficient number and of adequate quality, is one of the main requirements for the implementation of the technique. In the 1930s, Melvin & Bushland  developey et al.  developey et al.  and Baum [et al. .Sterility in screwworms can be induced by chemosterilants ,119 whenDrosophila melanogaster. Some radiations can make insects reproductively sterile by specifically causing germ-cell chromosome fragmentation  that leads to the production of imbalanced gametes and, subsequently, the inhibition of mitosis and death of fertilized eggs and embryos [Muller  demonstr embryos . In 1946ca. 50 Gy of X-rays were applied to pupae during their last two days before emergence, the adults that emerged were sterile but could compete equally with non-irradiated flies. The development of gamma ray sources by the Atomic Energy Commission [During 1950, Bushland & Hopkins  conductemmission  offered ca. 108 Gy) was required to sterilize the females. Two decades later, Crystal [Bushland & Hopkins  then com Crystal  re-examiIrradiation presented many advantages over other sterilization methods as insects could be irradiated inside packaging materials, the sterile insects could be released immediately after irradiation, residues that could be harmful to humans or the environment were nonexistence, and temperature rise during the process is usually insignificant . The ind2), 4 km off the coast of Florida. The results corroborated the previous laboratory studies, and just after 8 weeks of releases , 100% of the egg masses collected from wounded sentinel goats were sterile. Nevertheless, eradication was not achieved because wild fertile flies continued reinfesting the island from the mainland [The first published field evaluation of sterile screwworm flies was performed during 1951-1953 at Sanibel Island  on August 1954 and eradication was accomplished within just 14 weeks [To prove the SIT effectiveness once for all, an eradication trial was initiated in Curaçao Island in 1954. The NWS flies were reared in the USDA Entomology Research Division Laboratory at Orlando, Florida, and irradiated pupae were packaged in paper bags, air shipped to Curaçao, and released by air twice a week. The sterile males started being released (14 weeks . 2) southeast of Orlando were begun on May 1957, at the rate of 193 sterile males/km2. Despite the initial outbreak proportions of the infestation, sterility of egg masses reached 70% during July. The test was considered successful, and realistic planning for eradication was initiated [The successful eradication of the NWS from Curaçao increased interest in the SIT for eradication of this pest from southeastern USA. During 1955, additional rearing facilities were constructed near Bithlo, Florida, with the initial goal of producing 1 million flies/wk (or 6 times the Curaçao production). This goal was rapidly achieved by expanding operations based on procedural modifications adopted for Curaçao . In the nitiated . ca. 78 flies/km2/wk throughout peninsular Florida. During the campaign, special releases were made in several areas in which screwworms persisted and, because sporadic outbreaks of wild flies occurred, sterile flies were released as far north as southern Georgia to maintain a 63 km barrier. Releases were discontinued on 3,861 km2 in southern Florida in late July 1959 and in entire Southeast on November 1959. After this two-year campaign (1957–1959), the southeastern USA was declared NWS free. Campaign cost was US$ 10 million, or half the annual loss to livestock owners from NWS infestations [The Florida State legislature then appropriated US$ 3 million to share the cost of a joint federal campaign, which led to the implementation of the eradication effort in the southeastern USA. In early 1958, a new mass rearing plant was planned to operate in a former US Air Force base near Sebring, Florida. The campaign got off a good start due to the winter of 1957–1958, which was one of the coldest in the history of Florida. To take advantage of this severe winter, sterile fly production at the Bithlo facility was increased from 2 million/wk to 14 million/wk. The new facility near Sebring was officially opened on July 1958, and within three months it was producing over 50 million/wk . During stations . Livestock producers in Southwest watched that eradication campaign with much interest, and some representatives of the livestock industry in Texas visited the Sebring plant. Soon afterwards, the Southwest Animal Health Research Foundation (SWAHRF) was formed to obtain support for an eradication campaign in the southwestern USA , and froca. 770 sterile flies per linear km. During the winter 1963–1964, releases were extended 125 km into strategic areas throughout northern Mexico, and only 233 cases were recorded in Texas during 1964. In 1965, Arizona and California joined the campaign. After 1963, with the southwestern campaign in operation, none of the southeastern States reported a single case of NWS infestation. In 1966, the entire USA was declared screwworm free [In February 1962, the southwestern campaign was inaugurated in a state of urgency to take advantage of the weather that was unfavorable to the NWS (as occurred in the Southeast in 1958), and the Animal Disease Eradication Division of the USDA-ARS expanded a rearing facility in Kerrville, Texas. From February to June 1962, SWAHRF constructed a new mass rearing facility at Mission, Texas, with a capacity to produce 200 million sterile flies per week. Isolated infestations  and along major watercourses in and beyond the primary grid zone were treated at orm free . The souDue to continued outbreaks in USA between 1966 and 1972, and interest of Mexican livestock producers in extending the eradication campaign into Mexico, it was decided to move the sterile fly biological barrier south to the Isthmus of Tehuantepec in Mexico  . Comisión México-Estados Unidos para la Erradicación del Gusano Barrenador del Ganado (COMEXA). The goals of the COMEXA were to construct a new mass rearing facility and to eradicate NWS southwards [An agreement was signed on August 1972, creating the uthwards . The camuthwards . The camuthwards .ca. 115 million sterile flies/wk were dispersed to cover the entire Guatemala. Dispersals ceased at the end of December 1993, and an official declaration of freedom from NWS was held on May 1994 [Following indications of interest from practically all the Central American countries and Panama, a plan was traced in 1985 to extend the eradication campaign through Central America and establish the biological barrier in Panama. After the enlargement of the campaign in Mexico to cover the entire country, in February 1991 Mexico was declared screwworm free . The COMMay 1994 .Dispersal of sterile flies began over Belize on August 1989. At the peak of dispersal, 24 million sterile flies/wk were released. Belize was officially declared NWS free on May 1994 . Aerial dispersal of sterile flies over El Salvador started in October 1991. An average of 24 million sterile flies/wk were dispersed. In June 1995, El Slvador was declared screwworm free. Also in late 1991, sterile fly dispersal over Honduras was begun. By June 1993, up to 120 million sterile flies/wk were dispersed. In August 1996, Honduras was free from NWS .In July 1993, releases of sterile flies were initiated in Nicaragua. Approximately 120 million sterile flies/wk were required to disperse over the entire country. The last myiasis case by NWS in Nicaragua was reported in June 1997. Flights operating out of Nicaragua were also used to disperse sterile flies in Costa Rica on early 1996. About 60 million sterile flies/wk were needed to cover Costa Rica, and the last reported case occurred in March 1999 .2 dispersed in 3.2 km swath widths [Comisión Panamá-Estados Unidos para la Erradicación y Prevención del Gusano Barredor del Ganado (COPEG) at Pacora. Dispersal of sterile flies over Panama began in July 1998, also using over-flights out of Nicaragua. In October 1998, 50% of the country was dispersed from a new dispersal center located at Tocumen International Airport near Panama City. Approximately 80 million sterile flies/wk were needed to cover the entire country at the programmed rate of 3,000 sterile flies/kmh widths . Panama 2 at the Darien Gap is being maintained by the weekly release of 25–50 million sterile males [Currently, only the mass rearing facility at Pacora is capable of providing millions of sterile screwworm flies per week, and the buffer zone of 30,000 kmle males ,130. The2/year of the SIT operation are the lowest for NWS (US$ 182.00/km2/year) when compared to other pests target of AW-IPM programs that release sterile insects, such as the Mediterranean fruit fly  [Although the production and release costs of sterile NWS flies are not low , the costs/kmm2/year) . The permanent sterile fly biological barrier between Panama and Colombia continues to protect the NWS free countries from reinvasion from South America, and the 50-year AW-IPM campaign ultimately resulted in estimated annual producer benefits in the USA, Mexico, and Central America of greater than one billion dollars .2 in northwestern Libya [2 were made over 40,000 km2 and this continued until October 1991. From January to August 1991, almost 17 million animals were inspected at quarantine stations and by 70 teams of mobile surveillance teams. The last confirmed screwworm infestation in Libya was in April 1991 [The NWS was historically restricted to the Americas, but in March 1988, the NWS was discovered in Tripoli, Libya, infesting domestic animals, and during 1990 an explosive outbreak of screwworms occurred in a region of 26,500 kmrn Libya . Shipmenril 1991 . 2) with sterile flies imported from COMEXA and, after just 13 weeks of air releasing ca. 1,545 sterile flies/km2, 21.5% of the egg masses collected were sterile [Efforts and isolated projects to suppress NWS populations continue in the Caribbean and South America. In 1996, an eradication campaign began in Jamaica, but it suffered many constraints and eventually failed . Between sterile . Cochliomyia hominivorax, causes obligatory myiasis in warm-blooded animals and is a major pest of livestock in the Caribbean and South America. Before the adoption of control measures, AW-IPM campaigns against this pest require active detection and surveillance of NWS populations through inspection of animals and trapping systems.The New World Screwworm (NWS), e.g., prevention procedures, larvicides, SWASS, and SIT) has strengthened previous AW-IPM campaigns against this pest. A campaign that depends solely on one component of control is much more vulnerable to failure since no backup method exists if the first system fails. Despite the appearance of lineages resistant to some insecticides, the chemical control and protection of livestock against screwworm strikes are still essential tools for improvement of animal health and management in tropical and sub-tropical areas.The availability of different methods of control (Sterile flies were essential in eradicating the residual NWS populations in North of Africa and the Americas through to Colombia, and the SIT would be strategic in forthcoming projects against the NWS in developing countries.


Economic constraints may lead to poor health among people from the developing world. Microfinance has proven to be useful in improving health outcomes elsewhere, but it still remains a neglected issue in Nepal. This study aims to assess perceived usefulness of the microfinance on health awareness and practices among different ethnic groups of Nepal.A community-based cross-sectional study was conducted in four districts of western Nepal. A total of 500 microfinance clients representing different ethnic groups  were selected by using systemic random sampling. Health awareness and practices among different ethnic groups were compared by logistic regression after adjustment for age, level of education, sex of household heads, occupation, and place of residence. Since participants were asked about their health awareness and practices, before and after microfinance intervention, during a single interview, there was a strong possibility of recall bias with respect to their preintervention awareness and other measures.p < 0.001). Similarly, the practices of eating nutritious food/balanced diet, drinking safe water, using toilet, immunizing the children, and regularly visiting the healthcare facility were improved from 4.2 to 63.8, 12.6 to 66.8, 15.2 to 70.4, 15.8 to 73.8, and 3.6 to 61.4%, respectively (p < 0.001).Microfinance intervention positively influenced self-reported health awareness and practices among different ethnic groups in Nepal, which was highest among the upper caste group , followed by the aadibasi/janajati  and dalit group . Self-reported awareness about environment and sanitation, family planning methods, and available health services at local level improved from 11.2 to 69.2, 9.2 to 65.0, and 3.8 to 59.8%, respectively, among the clients after microfinance intervention (Despite lack of control group and potential recall bias, this study reports positive effect of microfinance on self-reported health awareness and practices among different ethnic groups of Nepal. This finding supports further implementation and evaluation of equity-based microfinance to improve health and economic conditions of Nepalese people. Every year, 25 million households (more than 100 million people) are pushed into poverty due to recurrent illnesses and expensive healthcare. Access to the good health services and health protection are key components for fighting against poverty . In AsiaMicrofinance organizations can implement health programs that increase knowledge, change health-related behaviors, and improve access to health services . MicrofiNepal is a landlocked country with per capita income of just US$ 703 in 2013, and 23.8% of its population still live below the poverty line , 15. SavThe survey included 500 microfinance clients from four districts  of western development region, which consist of 300 government microfinance institutions (GMFIs) clients and 200 private MFIs (PMFIs) clients who were involved regularly in microfinance program for last 5 years. This research includes the “D class” MFIs working in western development region of Nepal , 15. DatA cross-sectional survey was used in this study. The multistage cluster sampling method was applied by covering a wide geographical area to ensure the representation from each zone of the western development region of Nepal in sample as shown in Figure The survey included the sample size of 500 microfinance clients from four districts  of western development region of Nepal, which was estimated using formula for estimating the optimal sample size .n = optimal sample size, N = size of population, Z = value of standard variant at 5% level if confidence interval = 1.96, p = variability (variability has been assumed 0.5), q = 1 − p, and e = level of precision or acceptable sampling error tolerated (assumed 5.0%).Then,N) for the clients’ survey. The sample size before adjustment of design effect was 317. The sample size was adjusted to account for intracluster correlation. The adjusted n = n × design effect. Here, the design effect 1.5 was taken as recommended for social rating. Thus, adjusted sample size after incorporating design effect was 476. Also, by allowing the 5% potential non-response, we inflated the final sample size to 500. All the participants were users of microfinance services with membership of MIF. Out of 565 such membership-based microfinance clients visited, 65 did not consent for an interview with 88.5% of response rate.The clients, who are involved in different clusters of MIFs for five or more years, are the total population  respondent’s general characteristics and (ii) health awareness and practices of respondents before and after a microfinance intervention. Seven research assistants, who were trained on questionnaires, conducted the interview and they were constantly supervised.The research proposal was approved by the Institutional Review Board, Banaras Hindu University, India. Respondents provided consent before the interview. Personal identifiers were removed before data analysis.The outcome variables of the study were health awareness and health practices. Health awareness included three variables: awareness of environment and sanitation, family planning methods, and available health services at their local levels. Health practices included use of nutritious foods/balanced diet, use of safe drinking water, use of toilet, use of traditional healers when they get sick, immunization of children, and visits to health facility for heath checkup. All these variables had Yes or No responses.Five confounding variables were defined as follows. Respondents’ ages were categorized into four groups . Education status was recorded as illiterate, literate only, primary, secondary, and above secondary levels. Respondents’ household heads were male or female. Occupation was coded as livestock, agriculture, business, and others . Place of residence was categorized as rural and urban.Ethnicity was based on the caste system in Nepal and was divided into three major groups based on available literature and similarities between the caste/ethnic groups: (i) advantaged upper caste, (ii) disadvantaged aadibasi/janajati (J&A), and (iii) disadvantaged dalit. The advantaged group included Brahmin, Chhetri, Thakuri, and Sanyasi. The disadvantaged J&A included indigenous groups of Nepal. Disadvantaged dalit included the castes that were considered lower in the caste-based stratification within the Nepalese society .p value ≤0.05 was considered statistically significant. Data were analyzed using Statistical Package for Social Sciences (SPSS version 17 for windows).The data were analyzed with the help of descriptive and inferential statistics mainly chi-square test and logistic regression methods to analyze the health awareness and practices of the clients by Nepalese ethnicities. Odds ratios were converted into rate ratios with adjustment of confounding variables . A p valTable p < 0.001). Similarly, the use of nutritious food/balanced diet, safe drinking water, toilet, immunization of children, and regular visits to health facility were found to increase from 4.2 to 63.8, 12.6 to 66.8, 15.2 to 70.4, 15.8 to 73.8, and 3.6 to 61.4%, respectively (p < 0.001). However, use of traditional healers seemed to decrease from 15.6 to 1.4% (p < 0.001). Among the three ethnic groups, the highest rates of health awareness and practices were observed among the upper caste group (77–92%), followed by the aadibasi/janajati group (60–76%) and the dalit group (33–52%).These results altogether indicate toward a positive effect of microfinance intervention in the study population.Self-reported health awareness and practices of the respondents after 5 years of membership-based microfinance intervention is shown in Table Multivariable analysis of self-reported health awareness and practices in relation to ethnicity of the respondents after microfinance intervention has been demonstrated in Table Previously, health benefits, in addition to financial and other non-financial benefits of microfinance, have been reported in sub-Saharan Africa and South Asia , 12. OurInterestingly, use of traditional healers to manage health problems in this study was significantly lowered after microfinance interventions. Moreover, the highest level of health awareness and practices were observed among microfinance clients of upper caste group, followed by aadibasi/janajati group and the dalit group. This could be due to the increased uptake of health information, higher education level, and better communication and economic benefits among upper caste. Based on these observations, it is presumed that an equity-based microfinance services with more emphasis to the lower caste groups would be more effective in Nepal.Our study indicates that awareness of environmental health and sanitation, different family planning methods, and availability of health services at local level were improved after microfinance intervention among upper caste group more commonly than others. The findings of current study are in line with other studies from India and Bangladesh , 24. ImpThis study indicates a significant improvement in the use of nutritious diet, safe drinking water, toilet, health checkup at local health facility, immunization of their children after the microfinance membership-based intervention. Some previous literatures reported that access to financial services improved health conditions, such as nutrition, higher immunization rates, and child health , 26. In Our results suggest that microfinance has been useful to improve health awareness and practices. To the best of our knowledge, no such previous study has been conducted on effect of microfinance on health awareness and practices in Nepal. Nevertheless, despite our best effort, this study also has some limitations. First, due to the lack of a control group, the methods do not allow to assess whether the improvements in health awareness and practices are indeed due to the intervention. This also holds true for the differences between the three subpopulations. In particular, it is not clear how the intervention has affected equity. Although the study explored an important area and the current observations are encouraging, better-controlled experimental designs would provide stronger evidence for the effectiveness of microfinance. Second, there was potential for a recall bias in data collection, particularly for the data related to outcome measures before the intervention as one set of interviews was conducted after the intervention had taken place.This study found positive effects of microfinance on health awareness and practices in different ethnic groups of Nepal. The positive health awareness and practice were more commonly observed among upper caste group. Therefore, the study indicates toward a need for an equity-based microfinance intervention for improving the health and economic status of the Nepalese people. Additional follow-up interventional studies are recommended in this regard.JS and BD participated in the design of the study. DA and JS performed statistical analysis. DA and JS wrote manuscript with significant contribution of BD and SG. BD, SG, and PP contributed in the analysis, interpretation of the results, and literature review. All of the authors contributed in revision and agreed on the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Flavivirus in the family Flaviviridae. JEV can cause febrile illness and encephalitis mainly in humans and horses, and occasionally in cattle.Japanese encephalitis virus (JEV) is classified into the genus In late September 2010, a 114-month-old cow showed neurological symptoms similar to the symptoms observed in previous bovine cases of Japanese encephalitis (JE); therefore, we conducted virological and pathological tests on the cow. As a result, JEV was isolated from the cerebrum of the affected cow. We determined the complete genome sequence of the JEV isolate, which we named JEV/Bo/Aichi/1/2010, including the envelope (E) gene region and 3’ untranslated region (3’UTR). Our phylogenetic analyses of the E region and complete genome showed that the isolate belongs to JEV genotype 1 (G1). The isolate, JEV/Bo/Aichi/1/2010, was most closely related to several JEV G1 isolates in Toyama Prefecture, Japan in 2007–2009 by the phylogenetic analysis of the E region. In addition, the nucleotide alignment revealed that the deletion in the 3’UTR was the same between JEV/Bo/Aichi/1/2010 and several other JEV G1 isolates identified in Toyama Prefecture in 2008–2009. A hemagglutination inhibition (HI) test was conducted for the detection of anti-JEV antibodies in the affected cow, and the test detected 2-mercaptoethanol (2-ME)-sensitive HI antibodies against JEV in the serum of the affected cow. The histopathological investigation revealed nonsuppurative encephalomyelitis in the affected cow, and the immunohistochemical assay detected JEV antigen in the cerebrum.We diagnosed the case as JE of a cow based on the findings of nonsuppurative encephalomyelitis observed in the central nervous system, JEV antigen detected in the cerebrum, JEV isolated from the cerebrum, and 2-ME-sensitive HI antibodies against JEV detected in the serum. This is the first reported case of JE in a cow over 24 months old. Flavivirus in the family Flaviviridae, and it is distributed throughout eastern, southern and southeastern Asia, Papua New Guinea, and the Torres Strait of northern Australia[Japanese encephalitis virus (JEV) is a member of the genus ustralia. JEV is ustralia,3. Althoustralia,4,5, JEVustralia-3,6, andustralia-9. A casustralia. In lateIn late September 2010, a 114-month-old Japanese Black cow on a beef farm in Aichi Prefecture, Japan showed decreased appetite and fever. Four days after the onset of these symptoms, the cow showed astasia. Despite palliative treatments, no improvement was seen, and the cow then became recumbent and was euthanized. A serum sample was collected after the onset of the initial symptoms, and organs were collected at necropsy for virological and pathological investigations including the brain, spinal cord, heart, lung, liver, kidney, spleen, rumen, reticulum, omasum, abomasum, intestines, right axillary lymph node, and right popliteal lymph node. Serum samples were collected from the other 14 cows on the farm for anti-JEV hemagglutination inhibition (HI) antibody.Homogenate was prepared from the cerebrum of the affected cow, and RNA was extracted from the homogenate and the cow’s serum sample with a QIAamp Viral RNA Mini Kit . The RNA was tested for the presence of JEV by reverse transcription-polymerase chain reaction (RT-PCR). For virn = 1,000)[To analyze the phylogenetic relationships among virus isolates, we aligned sequences using the Clustal W program, and we = 1,000). The nucA hemagglutination inhibition (HI) test was conducted to determine the anti-JEV HI antibody titers of the affected cow and all other cows at the farm. Prior tWe examined the affected cow for gross and histological lesions. The collected organs were fixed in 10% neutral buffered formalin. Fixed organs were then embedded in paraffin wax, sectioned at 3 μm, and stained with hematoxylin and eosin. An immunohistochemical (IHC) assay was performed using a Histofine Simple Stain MAX-PO (MULTI) Kit . Anti-JEV (AS-6 strain) polycloThe cerebrum of the affected cow tested positive for JEV, but the serum tested negative. We then subjected the cerebrum to virus isolation, and JEV was isolated after the second passage. From the JEV isolate, which we named JEV/Bo/Aichi/1/2010, cDNAs containing the E region and the 3’UTR were successfully amplified and sequenced. Our phylogenetic analysis of the E region showed that the isolate clustered with other isolates belonging to JEV G1 . Histological findings in other organs included  Figure .We diagnosed the case as JE of a cow based on the following findings: nonsuppurative encephalomyelitis observed in the CNS, JEV antigen detected in the cerebrum, JEV isolated from the cerebrum and 2-ME sensitive HI antibodies against JEV in the serum. The histopathological findings observed in the CNS of the affected cow were similar to those observed in the 141-day-old calf in the previous JE case, includiHowever, two other lesions observed in the present case were not observed in the 141-day-old calf but were seen in other bovine JE cases: pulmonary emphysema was observed in a 24-month-old milking cow in 1950, and congestion of the brain was seen in an 18-month-old Japanese Black cow in 1996,9. It isSarcocystis spp. in the myocardium, although some species of Sarcocystis protozoan parasites are highly pathogenic to ruminants[The affected cow’s wound and muscle necrosis of the right upper limb seemed to have resulted from the onset of astasia and recumbency, and the infiltration of neutrophils in the right axillary and right popliteal lymph nodes may have been immune responses to wound infections. However, it is unclear whether the other pathological findings in the affected cow had relevance to the onset of JE, such as the severe diffuse hemosiderosis in the spleen and the infection of uminants.Our genetic analyses revealed that JEV/Bo/Aichi/1/2010 was most closely related to several other JEV G1 isolates in Toyama Prefecture, which is located approx. 100 miles away from Aichi Prefecture. The JEV isolate JEV/Bo/Aichi/1/2010 was most closely related to several JEV G1 isolates in Toyama Prefecture identified in 2007–2009 by phylogenetic analysis of the E region. In addiThere were some differences in the phylogenetic characteristics between JEV/Bo/Aichi/1/2010 and another JEV isolate from an affected calf in Miyazaki Prefecture, a regioIn the bovine JE cases reported to date, the ages of onset were 141 days, 6 months, 18 months and 24 months-10; the A 114-month-old cow showed several clinical signs including decreased appetite, fever, astasia and recumbency in the summer of 2010 in Aichi Prefecture, Japan. The histopathological investigation revealed nonsuppurative encephalomyelitis in the affected cow, and JEV antigen was observed in the lesions. We isolated JEV from the cerebrum, and we thus diagnosed the case as JE of a cow. Our genetic analyses revealed that the isolate was closely related to other JEV G1 isolates from pigs and mosquitoes in Toyama Prefecture, which is approx. 100 miles away from Aichi Prefecture. Although our data clearly indicated that the causative agent was JEV in the cow’s case, the causes of the onset are still unclear, since JEV infection usually results in an asymptomatic infection in cattle. Further study is needed to clarify the actual causes of the onset after JEV infection in cattle.https://datadryad.org/resource/doi:10.5061/dryad.s0c34).Alignments and phylogenetic trees created in the present study are available from the Dryad Digital Repository (The authors declare that they have no competing interests.NK and SS performed the pathological and virological investigations, respectively. NK, SS and NS made the diagnosis of the cow’s case. TK, TY, MY and HS performed the genetic and phylogenetic analyses of the JEV isolate. NK and HS wrote the manuscript, and all authors read and approved the final manuscript.


Bison bonasus L. 1758) was the largest surviving herbivore of the post-glacial megafauna in Europe before it became extinct in the wild, surviving only in captivity since 1919. We investigated the exploration behavior of a herd of European bison reintroduced into the Rothaargebirge, a commercial forest in low range mountain intensively used and densely populated by humans, in the first six months after release. We focused on three questions: (1) how did the European bison move and utilize the habitat on a daily basis, (2) how did the animals explore the new environment, and (3) did their habitat preferences change over time. The European bison dispersed away from their previous enclosure at an average rate of 539 m/month, with their areas of daily use ranging from 70 to 173 ha, their movement ranging from 3.6 km to 5.2 km per day, and their day-to-day use of areas ranged between 389 and 900 m. We could identify three major exploration bouts, when the animals entered and explored areas previously unknown to them. During the birthing phase, the European bison reduced daily walking distances, and the adult bull segregated from the herd for 58 days. Around rut, roaming behavior of the herd increased slightly. The animals preferred spruce forest, wind thrown areas and grassland, all of which are food abundant habitat types, and they avoided beech forest. Habitat preference differed slightly between phases of the study period, probably due to phenological cycles. After six months, the complete summer home range was 42.5 km2. Our study shows that a small free-ranging herd of European bison can live in an area intensively used by humans and describes in detail the initial roaming behavior and habitat utilization of the animals.Biodiversity is rapidly declining globally. One strategy to help to conserve species is to breed species in captivity and release them into suitable habitats. The way that reintroduced animals explore new habitats and/or disperse from the release site is rarely studied in detail and represents key information for the success of reintroduction projects. The European bison ( Bison bonasus, L. 1758). Most species became endangered due to overexploitation, habitat loss, invasive species and the synergistic effects of these factors , , , , t, tex sitountries , 18], . I. I32]. IWe investigated the exploration behavior of the released herd for the first six months following reintroducing. We focused on three main questions to better understand the exploration behavior of a newly reintroduced large herbivore: (1) how did the European bison move and use habitat on a daily basis, (2) how did the animals explore the new environment, and (3) did their habitat preferences change over time.Since April 2010, a herd of European bison, obtained from different zoos in Germany and Belgium, were kept in an 89 ha enclosure within the Rothaargebirge  centrally located in the study area as described above. During a three-year period between 2010 and 2013 we intensively studied ethology and the habituation process of the animals and their impact on the area , 38], , , 40], , , 42], , , 44]. A. A44]. A2 and additional 1.5 million tourists visiting the area annually , , , , 53] an an53] anWe removed redundant data from the data set if temporal proximity between pooled GPS positions were too close (≤ 15 min). Average interval between GPS fixes was 24 ± 13.8 min (mean ± SD) with minimal/maximal intervals of 0:17 h/16:40 h. Data were subdivided into single days, counting from midnight to midnight, as European bison usually do not move during the night , 54]. B. B54]. Bref) to determine the total area and 25-%-isopleths to determine the core area of areas of daily use. Börger et al. , , , , 66], , . HabitatThe animals were housed in an 89 ha enclosure under semi-natural conditions prior to reintroduction, and were under control of the local district veterinarian. The animals were owned by the NGO “Wisent-Welt Wittgenstein e.V.”. The enclosure and project area were in private ownership of Richard, Prince of Sayn-Wittgenstein-Berleburg. No protected animals were sampled. Animal handling were conducted by the NGO “Wisent-Welt Wittgenstein”, under supervision of the district veterinarian. The permit for handling and releasing the animals was granted by a contract between the NGO “Wisent-Welt Wittgenstein e.V.”, the county government of North-Rhine Westphalia, the Bezirksregierung Arnsberg, the Landesbetrieb Wald und Holz NRW and the Wittgenstein-Berleburg'sche Rentkammer.2 = 58.13, df = 17, p < 0.001; total area: χ2 = 58.19, df = 17, p < 0.001, The herd significantly altered the size of the areas of daily use during the observation period  and was analyzed separately. He left the herd at the day of the birth of the first calf on day 24 and successively increased his distance from the herd to a median of 2,002 m , at the days of the two births the adult bull entered the herd (by reducing the distance to less than 500 m) immediately after each birth and then left the herd for a few days again. During his entire roaming phase of 58 days he reduced the distances to the herd three times for short events, and finally returned to the herd on day 82. The highest values of more than 11 km were shown by the adult bull when he searched for the herd on day 72. The herd did not show an increase in roaming activity , Fig 2.2 = 101.18, df = 17, p < 0.001, 2, the core area covered 1.85 km2  started to explore the new habitat around day 10 (21st April) after removal of the fence on 11th April 2013. The exploration rate during the first six months was 539 m/month, calculated from average values . The dis2, 10,468 location data were obtained . In general the animals showed preferences for spruce forest, storm damaged areas and grasslands. They avoided beech forest and other structures like settlements, functional forestry areas and pine plantations. Other forest types were frequented in relation to their size .The distribution of the animals' locations within each habitat type was significantly different from a random distribution in all six phases  is described as “a series of [population] increases in local areas followed by pulses of dispersal and range expansion” , , , 93]. A. A93]. AOther studies find widely different patterns of habitat preferences for the European bison, each of which differs slightly from the patterns found in our study: European bison in Białowieża prefer deciduous forest types and avoid coniferous forests . In the In recent decades many reintroductions of European bison were conducted in Eastern Europe and Asia. In 2012, 35 populations in seven countries were introduced , 18], , , but theAlthough we described in detail the exploration behavior of the herd after release, this study can only put a spotlight on a single herd. Therefore our findings should be interpreted cautiously, since the behavior of other populations under different conditions might differ. Nevertheless, our study gives detailed insight into the exploration behavior of this large herbivore in a human-dominated landscape back into the wild.S1 Tableref 85%); (c) areas of daily use of the herd, complete area (kernel href 25%); (d) day-to-day shift of daily used areas; (e) minimal daily walking routes.Pairwise Wilcoxon comparison with sequential Bonferroni correction. Measurements of the different 10-day periods are compared. Significant results are indicated by light gray background. Calculations are given for (a) expansion rate; (b) areas of daily use of the herd, complete area (kernel h(PDF)Click here for additional data file.S2 TableFor each period of higher ranking behaviour Jacobs' preference indicies for each habitat type and Bailey's confidence intervals were calculated. The area of each habitat type is given. According to the number of location counts the expected and observed location data  and the respective proportions (p(exp), p(obs)) are given. Bailey's confidence intervals  are calculated and Jacobs preference index (Jacobs). The preferences are calculated according to these borders; p(obs) lies between Bailey–and Bailey +: (=) p(exp) lies within the range of the confidence intervals, the habitat type is used according to its size. (-) p(exp) lies above the range of the confidence intervals, the habitat type is avoided. (+) p(exp) lies below the range of the confidence intervals, the habitat type is preferred.(PDF)Click here for additional data file.S3 TableFor each record the timestamp (UTC), Gauss-Krueger coordinates (EPSG 31467), the animal ID, the dilution and the day after release is given.(CSV)Click here for additional data file.


To verify whether body mass index (BMI) classification proposed by the Institute of Medicine (IOM) is valid in Japanese women.A study was conducted in 97,157 women with singleton pregnancies registered in the Japan Society of Obstetrics and Gynecology (JSOG) Successive Pregnancy Birth Registry System between January 2013 and December 2013, to examine pregnancy outcomes in four groups stratified by pre-pregnancy BMI category according to the 2009 criteria recommended by the Institute of Medicine (IOM). The groups comprised 17,724 underweight women with BMI <18.5, 69,126 normal weight women with BMI 18.5–24.9, 7,502 overweight women with BMI 25–29.9, and 2,805 obese women with BMI ≥30. The pregnancy outcomes were also compared among subgroups stratified by a gestational weight gain below, within, and above the optimal weight gain.The higher the pre-pregnancy BMI, the higher the incidences of pregnancy-induced hypertension, gestational diabetes mellitus, macrosomia, cesarean delivery, postpartum hemorrhage, and post-term birth, but the lower the incidence of small for gestational age (SGA). In all pre-pregnancy BMI category groups, excess gestational weight gain was associated with a higher frequency of large for gestational age and macrosomia; poor weight gain correlated with a higher frequency of SGA, preterm birth, preterm premature rupture of membranes, and spontaneous preterm birth; and optimal weight gain within the recommended range was associated with a better outcome.The BMI classification by the IOM was demonstrated to be valid in Japanese women. The patient records was anonymized and de-identified prior to analysis. The present study was a retrospective investigation of women with singleton pregnancies who were included in the Japan Society of Obstetrics and Gynecology (JSOG) registry system. Approximately 280 secondary and tertiary hospitals participated in the JSOG Successive Pregnancy Birth Registry System, which collected information on successive deliveries occurring at gestational week 22 or later. A total of 186,235 women were registered in the system between January 1, 2013 and December 31, 2013. Women with concomitant hypertension or diabetes as the underlying disease, with a history of cervical conization, who delivered a newborn with congenital anomalies, or whose data were unknown, were excluded. After the exclusion, 97,157 women were included in the study .2), the pre-pregnancy overweight group (BMI = 25–29.9 kg/m2), the pre-pregnancy normal weight group (BMI = 18.5–25 kg/m2), or the pre-pregnancy underweight group (BMI <18.5 kg/m2).Based on the 2009 IOM guidelines, 97,157 women were classified into the pre-pregnancy obese group , gestational diabetes mellitus (GDM), macrosomia, small for gestational age (SGA), large for gestational age (LGA), preterm birth, spontaneous preterm birth, preterm premature rupture of membranes (PPROM), induced preterm birth, cesarean delivery, severe postpartum hemorrhage, and post-term birth; the incidence of these outcomes was compared among the groups and analyzed. PIH was defined as a case in which hypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg) developed after 20 weeks of gestation. GDM  was diagnosed when at least one of the following was found: fasting blood glucose level of ≥92 mg/dL, blood glucose level at 1 h of ≥180 mg/dL, blood glucose level at 2 h ≥153 mg/dL. Macrosomia was defined as a neonatal birthweight 4,000 g or more. SGA was defined as a neonatal birthweight below the 10th percentile of the reference curves of birthweight for gestational week. LGA was defined as a neonatal birth weight above the 90th percentile of the reference curves of birth weight for gestational week. Induced preterm birth was defined as preterm delivery by cesarean section or induction of labor due to maternal complications, such as preeclampsia or non-reassuring fetal status. Spontaneous preterm birth was defined as other than preterm birth medically indicated by cesarean section or labor induction. Postpartum hemorrhage with vaginal delivery (PPH with VD) was defined as an estimated blood loss of 500 mL or more; postpartum hemorrhage with cesarean delivery (PPH with C/S) was defined as an estimated blood loss of 1,000 mL or more. Post-term birth was defined as a delivery at ≥42 and 0/7ths weeks of gestation. Gestational age was determined based on the last menstrual period. If gestational age according to the last menstrual period differed by more than 7 days from that based on ultrasonography at less than 11 weeks, the latter was used to assign a gestational age [Next, in order to investigate the relationship between weight gain during pregnancy and the pregnancy-delivery outcome, a stratified analysis was performed in three subgroups of gestational weight gain below, within, and above the optimal range, based on the IOM-recommended 2009 guidelines, to compare the frequency of PIH, GDM, SGA, high-fat diet, preterm birth, spontaneous preterm birth, PPROM, induced preterm birth, PPH with VD, PPH with C/S, macrosomia, and post-term birth in each group by pre-pregnancy BMI categories of underweight, normal weight, overweight, and obese.Data were expressed as means ± standard deviation or frequencies (percentages). SPSS Statistics software version 23  was used for the statistical analyses. One way analysis of variance (ANOVA) for continuous and a chi-square test for categorical data were used to compare the maternal characteristics and pregnancy outcomes among the four groups, with Bonferroni correction for multiple comparisons. A p value of <0.05 was considered statistically significant. Logistic regression analysis was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for confounding variables, including maternal age, maternal height and parity. Generalized estimating equations (GEE) for logistic regression was used to adjust for clustering of deliveries by hospitals.Of 97,157 pregnancies, the pre-pregnancy underweight group accounted for 18.2% , the pre-pregnancy normal weight group for 71.1% , the pre-pregnancy overweight group for 7.7% , and the pre-pregnancy obese group for 2.9% . Tables In the underweight and normal weight groups, the incidences of PIH, LGA, and macrosomia were significantly higher with increasing gestational weight gain (p < 0.001), and the incidences of SGA, preterm birth, PPROM, spontaneous preterm birth, and induced preterm birth were significantly higher in the subgroup with gestational weight gain below the optimal range (p < 0.001). In both groups, the frequency of cesarean delivery was lowest in the subgroup within the recommended weight gain.In the overweight group, the incidences of PIH and LGA were significantly higher with increasing gestational weight gain, and the incidence of macrosomia was significantly higher in the subgroup above the optimal weight gain (p < 0.001). In addition, the incidences of SGA, preterm birth, PPROM, and spontaneous preterm birth were significantly higher in the subgroup below the optimal weight gain (p < 0.001). The incidence of cesarean delivery was lowest in the subgroup within the recommended weight gain.In the obese group, the incidences of LGA, and macrosomia were significantly higher with increasing gestational weight gain (p < 0.001), and the incidences of SGA, preterm birth, PPROM and spontaneous preterm birth were significantly higher in the subgroup with gestational weight gain below the optimal range (p < 0.05); the incidence of PIH and cesarean delivery were not different by weight gain during pregnancy.The four pre-pregnancy BMI category groups demonstrated marked differences in the pregnancy outcome profile, indicating that the IOM-BMI classification is valid in Japanese women as well. In all groups, the gestational weight gain above the optimal range was associated with a higher incidence of LGA; the weight gain below the optimal range correlated with a higher incidence of SGA, preterm birth, PPROM, and spontaneous preterm birth; and the weight gain within the recommended range was associated with a better outcome. However, only the obese group did not show a difference in the incidence of PIH by gestational weight gain or a reduction in the incidence of cesarean delivery, even within the recommended weight gain.The BMI classification by the IOM was found to be valid in Japanese women.There were differences in the incidence of adverse pregnancy outcomes according to IOM recommended BMI categories. Due to a trend toward a higher proportion of leaner women and a lower proportion of obese individuals in the Japanese pregnant population, we defined those with BMI ≥25.0 as obese, in contrast to the IOM classification. However, the present study results indicated that Japanese pregnant women should also be categorized into an overweight group of pre-pregnant BMI 25–29.9 and an obese group with BMI ≥30. In addition, the group with BMI ≥30 did not show a difference in the incidence of PIH by weight control status during pregnancy or a reduction in cesarean delivery rate, even when the body weight was kept within the recommended range. These findings also indicated that weight reduction prior to pregnancy was important in improving pregnancy outcomes in obese women with BMI ≥30 in pregnancy. Poston et al.  reportedThe incidence rates of LGA and macrosomia were higher in women who had excess weight gain during pregnancy. Johnson et al.  reportedThe incidences of SGA, preterm birth, PPROM, and spontaneous preterm birth increased when the weight gain was below optimal. We previously reported that below optimal weight gain during pregnancy was associated with an increased risk of spontaneous preterm birth in underweight women ; howeverThe present study has limitations. First, our data set included only Japanese women, and it is unclear whether the results can be extrapolated to women of other ethnic groups. Second, we used the database of the JSOG Successive Pregnancy Birth Registry in tertiary centers. This may have introduced selective bias in the patient background characteristics. Third, we calculated the gestational weight gain as the change over the entire pregnancy period, and did not assess weekly weight gain.In Japan, pre-pregnancy BMI is currently classified into three categories: BMI <18.5, 18.5 to <25.0, and ≥25.0. On the basis of the present study results, we propose that the Japanese category of BMI ≥25.0 should also be further divided into overweight (BMI 25.0–29.9) and obese (BMI ≥30), in keeping with the IOM-recommended BMI classification. Our results also demonstrated that obese women with BMI ≥30 had a higher risk of adverse pregnancy outcome, and that gestational weight control did not have an impact on the cesarean delivery rate, supporting the importance of pre-pregnancy weight control within the normal range.


To eliminate this as far as possible, we focused on presence and impact of headache on the preceding day (“headache yesterday”).Surveys enquiring about burden of headache over a prior period of time (Adults (18-65 years) were surveyed from the general populations of Germany, Italy, Lithuania, Luxembourg and the Netherlands, from a work-force population in Spain and from mostly non-headache patient populations of Austria, France and UK. A study of non-responders in some countries allowed detection of potential participation bias where initial participation rates were low.Participation rates varied between 11% and 59% (mean 27%). Non-responder studies suggested that, because of participation bias, headache prevalence might be overestimated in initial responders by up to 2% (absolute). Across all countries, 1,422 of 8,271 participants  had headache yesterday lasting on average for 6 hours. It was bad or very bad in 56% of cases and caused absence from work or school in 6%. Among those who worked despite headache, 20% reported productivity reduced by >50%. Social activities were lost by 24%. Women (21%) were more likely than men (12%) to have headache yesterday, but impact was similar in the two genders.With recall biases avoided, our findings indicate that headache costs at least 0.7% of working capacity in Europe. This calculation takes into account that most of those who missed work could make up for this later, which, however, means that leisure and social activities are even more influenced by headache. The World Health Organization (WHO) has acknowledged headache disorders as of global public-health importance . This isIn fact there are large gaps in our knowledge of the prevalence and burden of headache worldwide ,6. In EuThe Eurolight project was a collaboration of 25 partners from 15 countries in Europe  supporteBurden-of-headache studies generally estimate 1-year prevalence and enquire into quantifiable components of burden  through questionnaires that depend upon recall over a preceding period (commonly 3 months). It is not known how reliable recall is for such matters, but at best it is likely to be variable and at worst highly misleading . Recent The full methodology ,16 and pEurolight was essentially an EU-wide study employing a modified and pragmatic form of cluster sampling to reach a large number of participants in many countries. Questionnaire-based surveys were conducted in 10 countries: Austria, France, Germany, Ireland, Italy, Lithuania, Luxembourg, Netherlands, Spain and United Kingdom (UK).The National Ethics Committee of Luxembourg gave overall approval of the protocol. Further approvals were obtained from national or local ethics committees wherever needed as the methods for recruitment of participants differed between countries. Similarly, data protection approvals were obtained centrally in Luxembourg and at country levels in compliance with national and European privacy laws.In each country, prospective participants received a written information sheet explaining the project and enquiry, and their purpose.The development, content and validation of the structured questionnaire, and its translation into all local languages, have been previously described . DemograMethods varied between countries according to what was feasible. Again, these are fully described elsewhere . Table 1Additional surveys in Spain and the Netherlands, and the only survey in Ireland, were performed among members of the national headache patients’ organizations. The samples generated from these were inevitably biased  and the http://www.cefalea.it). In the other countries, non-responders were selected randomly and called by telephone. All were asked whether headache had occurred ever (yes or no), during the last year (and how often), and yesterday (yes or no).In all countries, questionnaires were more likely to be completed and returned by those most affected by headache (participation bias), and thus would not be representative of the initial samples (particularly relevant because of the rather low participation rates in some countries). Therefore, studies of non-responders were performed in Luxembourg (n = 357), Italy (n = 202), Netherlands (n = 188) and Germany (n = 260). In Italy, non-responders were invited by means of advertisements in local newspapers to complete a short questionnaire on the website of Centro Italiano di Ricerche Neurologiche Applicate (CIRNA) , participated in the survey. A majority (58%) were female. For each country, the number of participants and their gender are shown in Table In nine countries, 8,271 participants not derived from specific headache-patient populations (Most participants (47%) reported a duration of headache yesterday of 1-4 hours, which might reflect effect of treatment; 24% reported 5-12 hours and 15% reported >12 hours. The estimate of the overall mean (taking the mid-point of each range) was 6 hours Table . In all Functional impairment yesterday because of headache showed that most people 72%) were adversely affected to some extent, while about a quarter  were severely affected  and a minority  could do nothing Table . Variati% were advs 12%). However, with regard to headache duration and intensity and the measures of functional impairments, men and women were quite similar  of cases it was, there was no diagnosis made in the remaining 31%. This is not important to our message, which is a public-health one: headache disorders, without focus on migraine, are a major cause of population ill-health, representing a need for which health services must make due provision.today: participants with headache on the day they encountered the questionnaire might have deferred responding until they became headache-free, and then been highly likely to report headache yesterday. Headache today is as probable as headache yesterday; therefore, it might seem that this factor could double the reported prevalence of headache yesterday. In reality this was not so, because about half of the 1-day prevalence of headache is attributable to daily headache . When headache occurs daily, it is of no consequence whether the enquiry is conducted today or tomorrow. The problem would not have occurred in Lithuania, where participants were visited without prior notice, and did not have the option of deferring their responses until tomorrow. The reported prevalence of headache yesterday was lowest in this country, but not outlying ; the non-responder study therefore represented the other 73%, of whom 64% (gender-adjusted) had headache last year . The weievery day and signals a huge level of population ill-health. This range should be internally consistent with the approximate estimate derived from the 1-year prevalence (78.6% [An estimated 15-17% (the lower limit = 17*68/79) of adults aged 18-65 years in Europe had headache yesterday, which implies a similar percentage e , we calculate that, at this and any moment, between 6.3 (300*0.56*0.15/4) and 7.1 (300*0.56*0.17/4) million people are suffering in Europe with bad or very bad headache.The purpose of reporting headache yesterday was, however, to focus not on prevalence but on impact. Duration and intensity are dimensions of symptom burden; whilst they may be misleading because they are reported subject to the effects of any treatments taken, they give rise to the following estimation. If 15-17% of a population had headache yesterday of average duration 6 hours, then one quarter of these (say 4.0%) had headache at any particular moment (which compares with 1.8% reported in China ). HeadacAs telling are the secondary effects – in particular, functional impairment and its consequences. Thus, an estimated 0.4 (0.5*68/79) to 0.5% of working-age adults in Europe lost the whole of yesterday at work because of headache, again implying a similar percentage every (working) day. A further 1.2-1.6%, working despite headache yesterday, reported a significant level of inefficiency . In line with the theory behind the MIDAS instrument  on whichThe evidently greater negative impact of functional impairment upon household chores and social activities Table  reflectsOur data show that the burden of headache is much larger in women than in men, mostly because women have headache more often. During headache, men and women appear to have similar symptom burdens and functional impairment.Headache is common, and its impact is high. Assessment in nine countries in Europe based on headache yesterday, and therefore free from recall bias, shows that headache affects 15-17% of adults aged 18-65 years on any day. It removes at least 0.7% (and up to three times this) from workforce capacity. Leisure time and social activities are also serious casualties.Lifting The Burden, a UK-registered non-governmental organization conducting the Global Campaign against Headache in official relations with the World Health Organization.None of the authors has a conflict of interest relevant to the content of this manuscript. TJS, ZK and LJS are directors and trustees of The Eurolight project was conceived by CA and designed by CA, TJS and LJS. The concept of “headache yesterday” was developed by TJS and LJS. Data were acquired by CA, ZK, JML, CL, ML-M, DR, ERdlT, CT and LJS, and analysed and interpreted by JB, TJS, LJS and CA. The manuscript was drafted by TJS and revised for intellectual content and approved in the final version by all authors.


Aix galericulata and Tadorna ferruginea are two Anatidae species representing different taxonomic groups of Anseriformes. We used a PCR-based method to determine the complete mtDNAs of both species, and estimated phylogenetic trees based on the complete mtDNA alignment of these and 14 other Anseriforme species, to clarify Anseriform phylogenetics. Phylogenetic trees were also estimated using a multiple sequence alignment of three mitochondrial genes  from 68 typical species in GenBank, to further clarify the phylogenetic relationships of several groups among the Anseriformes. The new mtDNAs are circular molecules, 16,651 bp  and 16,639 bp (Tadorna ferruginea) in length, containing the 37 typical genes, with an identical gene order and arrangement as those of other Anseriformes. Comparing the protein-coding genes among the mtDNAs of 16 Anseriforme species, ATG is generally the start codon, TAA is the most frequent stop codon, one of three, TAA, TAG, and T-, commonly observed. All tRNAs could be folded into canonical cloverleaf secondary structures except for tRNASer (AGY) and tRNALeu (CUN), which are missing the "DHU" arm.Phylogenetic relationships demonstrate that Aix galericula and Tadorna ferruginea are in the same group, the Tadorninae lineage, based on our analyses of complete mtDNAs and combined gene data. Molecular phylogenetic analysis suggests the 68 species of Anseriform birds be divided into three families: Anhimidae, Anatidae, and Anseranatidae. The results suggest Anatidae birds be divided into five subfamilies: Anatinae, Tadorninae, Anserinae, Oxyurinae, and Dendrocygninae. Oxyurinae and Dendrocygninae should not belong to Anserinae, but rather represent independent subfamilies. The Anatinae includes species from the tribes Mergini, Somaterini, Anatini, and Aythyini. The Anserinae includes species from the tribes Anserini and Cygnini. Anseriformes is a highly differentiated order of birds with worldwide distribution, containing more than 150 species Aix galericulata) and Ruddy shelduck (Tadorna ferruginea) are two typical Anseriform waterfowl, yet their phylogenetic status has remained controversial Aix galericulata belongs in Tadornini, and Tadorna ferruginea is a member of Cairinini; both of them placed inside Anatinae. The two birds have a moderately large body size, contrastingly pale dorsal wing-coverts, and blunt carpal (wing) spurs, along with other shared morphological characteristics; and both also feed through a combination of wading and dabbling Aix and Tadorna form an independent lineage together, the subfamily Tadorninae Cairina a so-called “perching duck”, and Aix clustering together as a sister group, belonging to Tadornini Aix and Cairina should to be placed in their own tribe, Cairinini, within Anatinae Aix and Cairina as forming the subtribe Cairinina, placed in the tribe Anatini Aix and Cairina) should not belong to Anatinae at all, but rather represents the independent subfamily Tadorninae, placed in Anatidae Aix and Tadorna together as a tribe in the Anatinae Tadorna) together with the sheldgeese , forming the tribe Tadornini, based on morphological and molecular data, but consider it a non-monophyletic grouping Anseranas, Dendrocygna, Sarkidiornis, Tadorna, Cairina, Aix, and ChenonettaMandarin duck  Anseranas semipalmataAnseranas semipalmata, that is, whether it should be considered a member of an independent family by itself, or whether it is contained within a subfamily of Anatidae Nonetheless, important groups in the Anseriformes remain controversial. Traditionally, the order Anseriformes has been considered to be composed of the families Anhimidae (two genera and three species) and Anatidae  , two rRNAs (12S rRNA and 16S rRNA), 22 tRNAs and a putative CR (D-loop) (The complete mtDNAs of (D-loop) . The hea(D-loop) .Aix galericulata, but ND4 starts with ATT, while COI, COII, and ND5 begin with the nonstandard start codon GTG. In Tadorna ferruginea ten PCGs start with ATG, and ATT is the start codon only in ND4, while COI and ND5 begin with GTG. The standard stop termination codon TAA occurs in most of the same genes in the two birds' mtDNAs, except ND2 stops with TAA in Aix galericulata and TAG in Tadorna ferruginea. Furthermore, AGG terminates the ND1 and COI genes, TAG terminates the ND3 and ND6 gene, and the incomplete termination codon T- occurs in the COIII and ND4 genes in both birds.Through the 13 protein-coding genes, ATG is the start codon in nine of the 13 PCGs in Aix galericulata and Tadorna ferruginea 12S rRNA  and 16S rRNA  genes are located between the tRNAPhe and tRNALeu genes, separated by the tRNAVal gene. The two complete mtDNAs contain 22 tRNAs genes, and except for tRNASer (AGY) and tRNALeu (CUN), which lack dihydrouridine (DHU) arms, all other tRNAs could be folded into the typical cloverleaf structure. The longest tRNAs are tRNAAsn (78 bp) and tRNAGlu (87 bp) in Aix galericulata and Tadorna ferruginea, respectively, and the shortest is tRNACys (65 bp in both).In Glu and tRNAPhe genes, which are 1,071 bp and 1,077 bp, respectively, in Aix galericulata and Tadorna ferruginea. Additionally, 11 gene junction regions spacer by a total of 31 bp, with the longest one being 10 bp between ND6 and tRNAGlu in Aix galericulata  and Bayesian inference (BI) analyses share identical topologies and high node support values . The resThe gene order and arrangement of the two new mtDNA sequences, including such features as gene length, base composition, and RNA structure, are extremely conservative, and similar to that of other Anseriform birds Anseranas semipalmata , and the shortest is Anas formosa . Homologous regions comprise 12,748 bp, representing 79.68% of the complete genome  to −0.327 (Dendrocygna javanica), with an average value of −0.353  and the shortest is Anser fabalis . The 13 PCGs have a total length of 11,331 bp in Aix galericulata and 11,385 bp in Tadorna ferruginea, which is 68.05% and 68.42% of each entire mtDNA genome, respectively. In both species, the longest gene is ND5, located between the tRNALeu (CUN) and Cyt b genes, and the shortest is ATP8, which is between the tRNALys and ATP6 genes. Most PCGs used ATG as start codons, only a few start with GTG, GTC, or ATA. Stop codons are also similar across species, with TAA, TAG, and T- occurring most frequently. In Tadorna ferruginea and Tadorna ferruginea, the start condons are ATG, GTG and ATT, and TAA, AGG, TAG and T- as stop termination codons occur in most of the same genes in the two birds' mtDNAs. Among the 13 PCGs, specific examples include the following: the COI initiation codon is GTG and the termination codon is AGG in all 16 species; Cyt b starts with ATG and ends with TAA; COII starts with ATG and ends with TAG, except in Branta canadensis, where it starts with GTC; ND6 starts with ATG and ends with TAG, except in Anser fabalis it ends with TAA; ND1 starts with ATG and ends with AGG, except in Anser albifrons where the stop codon is TAA; and ND2 starts with ATG and ends with TAG, except in Anseranas semipalmata, Cygnus atratus, and Tadorna ferruginea, where the stop codon is TAA  to 1,335 bp  with an average length of 1,127 bp. The high AT content (53.55%) is similar to other organisms 11 repeats in Anser albifrons and Anser anser, and (ATCAAACG)15 elements in Anseranas semipalmata. The average genetic distance (0.376) between the CR of Anseranas semipalmata and the other species in our analysis is higher than average (0.288)  .Anseranas semipalmata thus has the most divergent CR of the studied species .Our mtDNA analysis suggests that the genus Aix galericulata and Tadorna ferruginea were collected using non-invasive methods at the Hefei Wild Animal Park in May 2013. No animal was killed for the purpose of the experiment, the method will not affect the health of the animals, and it conforms to our animal ethics committee's guidline in this study. The Hefei Wild Animal Park is authorized to administer animal rescue and medical treatment by Anhui Provincial Conservation and Mangement Station for Wildlife (APCMSW), a provincial government agency for wildlife conservation in the Anhui Province of China. We were authorized to study the birds by the APCMSW. The samples were stored at −20°C at the Institute of Biodiversity and Wetland Ecology, School of Resources and Environmental Engineering, Anhui University .Trace blood samples from Whole genomic DNA was isolated from blood samples using the phenol/chloroform method. Extracted DNA was examined on a 1.0% agarose/TBE gel and stored at −20°C as templates for PCR.Cygnus atratus (NC_012843), Anas platyrhynchos (NC_009684), and Aythya americana (NC_000877), we designed three primer pairs  using Primer 5.0. We also used other primers developed from Anser fabalisAix galericulata and Tadorna ferruginea , and sterile doubly-distilled water to final volume. PCR amplification conditions follow: denaturation for 5 min at 94°C, followed by 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 49–55°C (depending on primer combinations), elongation for 2 min at 72°C, and a final extension step of 10 min at 72°C. PCR products were examined using electrophoresis on a 1% agarose/TBE gel , and their cloverleaf secondary structures and anticodon sequences were determined using DNASIS . Two rRNAs were identified by comparison with complete mtDNA sequences of other Anseriformes available in GenBank. The complete mtDNA sequences have been deposited in GenBank under accession numbers KF437906 and KF684946.Sequences were checked and assembled using the programs Seqman , BioEdit, and Chromas 2.22, and then adjusted manually. PCGs were identified by comparison with the known complete mtDNA sequences of Anseriform birds using Sequin 11.0. The 22 tRNA genes were identified using the software package tRNA Scan-SE 1.21  sequence was used as outgroup. Phylogenetic trees were estimated for two cases: one based on the complete mtDNA of 16 Anseriforme species  and a general time-reversible model with invariant sites and among-site variation (GTR+I+Γ) that was selected as the best fit model of evolution using Modeltest (version 3.06) based on the AIC criterion. Internal ML tree branch support was evaluated using a bootstrap test with 100 iterations. Bayesian phylogenetic inference was done using MrBayes 3.1.2, with the same best-fit substitution model as that selected for the ML analysis. MrBayes 3.1.2 simultaneously initiates two Markov Chain Monte Carlo (MCMC) runs to provide additional confirmation of convergence of posterior probability distributions. Analyses were run for one million generations until the average standard deviation of split frequencies was less than 0.01, which indicated that convergence was reached. Chains were sampled every 1,000 generations.Appendix S1Phylogenetic Classification of the Anseriformes.(DOC)Click here for additional data file.