Population Structure of Three Species of Anisakis Nematodes Recovered From Pacific Sardines (Sardinops sagax) Distributed Throughout the California Current System

Population Structure of Three Species of Anisakis Nematodes Recovered From Pacific Sardines (Sardinops sagax) Distributed Throughout the California Current System Author(s) :Rebecca E. Baldwin, Mary Beth Rew, Mattias L. Johansson, Michael A. Banks, and Kym C. Jacobson Source: Journal of Parasitology, 97(4):545-554. 2011. Published By: American Society of Parasitologists DOI: 10.1645/GE-2690.1 URL: http://www.bioone.org/doi/full/10.1645/GE-2690.1 BioOne (www.bioone.org) is a a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. J. Parasitol., 97(4), 2011, pp. 545–554 F American Society of Parasitologists 2011 POPULATION STRUCTURE OF THREE SPECIES OF ANISAKIS NEMATODES RECOVERED FROM PACIFIC SARDINES (SARDINOPS SAGAX) DISTRIBUTED THROUGHOUT THE CALIFORNIA CURRENT SYSTEM Rebecca E. Baldwin*, Mary Beth Rew, Mattias L. JohanssonÀ, Michael A. BanksÀ, and Kym C. Jacobson` Cooperative Institute for Marine Resources Studies, Hatfield Marine Science Center, Oregon State University, Newport, Oregon 97365. e-mail: rebaldwi@ualberta.ca ABSTRACT: Members of the Anisakidae are known to infect over 200 pelagic fish species and have been frequently used as biological tags to identify fish populations. Despite information on the global distribution of Anisakis species, there is little information on the genetic diversity and population structure of this genus, which could be useful in assessing the stock structure of their fish hosts. From 2005 through 2008, 148 larval anisakids were recovered from Pacific sardine (Sardinops sagax) in the California Current upwelling zone and were genetically sequenced. Sardines were captured off Vancouver Island, British Columbia in the north to San Diego, California in the south. Three species, Anisakis pegreffii, Anisakis simplex ‘C’, and Anisakis simplex s.s., were identified with the use of sequences from the internal transcribed spacers (ITS1 and ITS2) and the 5.8s subunit of the nuclear ribosomal DNA. The degree of nematode population structure was assessed with the use of the cytochrome c oxidase 2 (cox2) mitochondrial DNA gene. All 3 Anisakis species were distributed throughout the study region from 32uN to 50uN latitude. There was no association between sardine length and either nematode infection intensity or Anisakis species recovered. Larval Anisakis species and mitochondrial haplotype distributions from both parsimony networks and analyses of molecular variance revealed a panmictic distribution of these parasites, which infect sardines throughout the California Current ecosystem. Panmictic distribution of the larval Anisakis spp. populations may be a result of the presumed migratory pathways of the intermediate host (the Pacific sardine), moving into the northern portion of the California Current in summer and returning to the southern portion to overwinter and spawn in spring. However, the wider geographic range of paratenic (large piscine predators), and final hosts (cetaceans) can also explain the observed distribution pattern. As a result, the recovery of 3 Anisakis species and a panmictic distribution of their haplotypes could not be used to confirm or deny the presence of population subdivision of Pacific sardines in the California Current system. allozyme data from 5 Anisakis species (Mattiucci et al., 2008) were used to identify separate stocks of the Atlantic horse mackerel (Trachurus trachurus) within the Atlantic Ocean (western and southern) and North Sea. Atlantic horse mackerel were further separated into 3 different stocks within the Mediterranean Sea, with parasite data indicating the potential of fish exchange between the Atlantic southern stock and the west Mediterranean stock (Abaunza et al., 2008). In addition, Cross et al. (2007) suggested that Anisakis simplex s.s. may be a suitable biological tag for Atlantic herring (Clupea harengus), because this nematode species can be recovered throughout the year. The Pacific sardine (Sardinops sagax) is an economically and ecologically important forage fish that transfers energy resources from planktonic primary producers and secondary consumers to upper trophic predators (Cury et al., 2000). In the California Current, Pacific sardines are currently managed as 3 stocks: (1) Central California Offshore, (2) Baja California Sur Inshore, and (3) the Gulf of California (Smith, 2005). Pacific sardine allozyme (Hedgecock et al., 1989) and mitochondrial DNA (mtDNA) data (Grant et al., 1998; Lecomte et al., 2004) suggest a panmictic population with a shallow genetic structure; however, there is some evidence of more than 1 subpopulation within the Central California Offshore management unit based on the recovery of larger individuals at higher latitudes (Clark and Janssen, 1945; Hill, 1999; Emmett et al., 2005; McFarlane et al., 2005) and a temporal difference in sardine spawning off the Pacific Northwest (PNW) versus southern California (Emmett et al., 2005; Smith, 2005). Furthermore, the potential for subpopulations with connectivity poses questions to the long-standing paradigm of an annual migration of individuals to feeding grounds off the Pacific northwest in the summer with migrants returning to southern California in the fall to spawn the following spring (Clark, 1935). This accepted coastwide migration pattern was described with mark–recapture tagging studies prior to the fishery collapse in the 1940s (Janssen, 1938; Clark and Janssen, 1945). It Anisakid nematodes are known to infect more than 200 species of pelagic fish (Cross et al., 2007), and have been used as biological tags for fish population structure studies (see review by MacKenzie, 2002). These nematodes use euphausiids as their obligate first intermediate host, fish or squid as second intermediate or paratenic (transport hosts where no development occurs), and cetaceans as definitive hosts (Oshima, 1972; Smith and Wooten, 1978). With the availability of molecular markers, morphologically similar larvae of Anisakis species recovered globally have been separated into 9 genetically distinct species comprising 2 clades (Mattiucci et al., 2009). Despite the growing information on the global distribution of Anisakis species (see review by Mattiucci and Nascetti, 2008; Klimpel et al., 2010), there is little information on the genetic diversity and population structure of these nematodes, which could be useful in assessing the stock structure of their fish hosts. In the marine environment, there are few obvious physical barriers limiting gene flow between fish populations. Nevertheless, some fish species have been designated as distinctively and geographically separated stocks or subpopulations based on genetic identification of Anisakis species. For example, separate stocks of the European hake (Merluccius merluccius) were identified within the Mediterranean Sea and Atlantic Ocean based on the distribution of 7 species of Anisakis identified with the use of allozymes (Mattiucci et al., 2004). More recently, parasite community analysis (MacKenzie et al., 2008) and Received 2 November 2010; revised 2 March 2011; accepted 11 March 2011. * Present address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. { Coastal Oregon Marine Experimental Station, Hatfield Marine Science Center, Oregon State University, Newport, Oregon 97365. { Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 2030 South Marine Science Drive, Newport, Oregon 97365. DOI: 10.1645/GE-2690.1 545 546 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 4, AUGUST 2011 is unclear if this exact pattern of migration has been reestablished since the return of Pacific sardines to Pacific northwest in the 1990s, or if there are some sardine subpopulations along the West Coast with limited latitudinal migrations. The main goals of our study were to identify which Anisakis species infect Pacific sardine in the California Current, examine the genetic diversity and population structure of nematodes collected from sardines sampled off of Vancouver Island, British Columbia to San Diego, California, and assess the potential of using Anisakis species as a biological tag to help discriminate stocks of Pacific sardine. MATERIALS AND METHODS From 2005 through 2008, Pacific sardines (n 5 1,339) were opportunistically recovered in the California Current (Table I) between 32u and 50uN latitude, and 119u to 128uW longitude (Fig. 1). We divided the study area into 5 geographic regions: (1) Vancouver Island, British Columbia (part of PNW); (2) Washington and Oregon (part of PNW); (3) northern California; (4) central California; and (5) southern California. Sardines from Canadian waters were caught with the use of a modified Cantrawl 240 rope trawl (Cantrawl Nets Ltd., Richmond, British Columbia; see Morris et al., 2009, for details), and sardines from Washington to California were caught with the use of a 30-m-wide by 20-m-deep mouth-opening 264 rope trawl (Nor’Eastern Trawl Systems, Inc. Bainbridge Island, Washington; see Baldwin et al., 2008, for details). One tow of the net equaled 1 trawling event. Captured Pacific sardines were immediately frozen onboard and stored in the lab at 280 C until being processed for parasites. After being thawed, each fish was weighed to the nearest 0.1 g, and standard length (SL) measured to the nearest millimeter. Fresh SL of individual frozen sardine was estimated with the use of the following regression: Fresh SL 5 2.89 + 1.0286 (frozen and then thawed SL) (Lo et al., 2007). Anisakis spp. nematodes were recovered from stomachs, intestines, and body cavities according to standard necropsy procedures (Arthur and Albert, 1994). No nematodes were recovered in the flesh. A total of 191 anisakid nematodes was collected from these sardines and preserved in 95% ethanol. DNA was extracted from nematode tissue with the use of a glass fiber plate DNA extraction protocol (Ivanova et al., 2006). We used the polymerase chain reaction (PCR) to amplify a region including the internal transcribed spacers (ITS-1, ITS-2) and 5.8S subunit of the nuclear ribosomal DNA (rDNA) (hereafter referred to as ITS markers) to identify larval Anisakis nematodes to species genetically at 2 diagnostic nucleotide sites (Abollo et al., 2003; Nadler et al., 2005; Abe, 2008) with the use of the forward primer 93 and the reverse primer 94 (Nadler et al., 2005). All PCR reactions had a final volume of 20 ml comprised of 2 ml genomic DNA, 0.25 mM each forward and reverse primer, 0.25 mM deoxynucleoside triphosphates (dNTPs), 1.5 mM MgCl2, 13 PCR buffer, and 1 unit Taq DNA polymerase (Promega, Madison, Wisconsin). The temperature and cycling parameters included denaturation at 94 C for 2 min, followed by 30 cycles at 94 C for 30 sec, 53 C for 30 sec, 72 C for 45 sec, followed by postamplification extension at 72 C for 10 min. To examine the population structure of anisakid nematodes, we amplified the mitochondrial DNA (mtDNA) cox2 gene with the forward primer 210 and the reverse primer 211 (Nadler and Hudspeth, 2000). Modified from Valentini et al. (2006), all PCR reactions had a final volume of 20 ml comprised of 2 ml genomic DNA, 0.3 mM of each forward and reverse primer, 0.4 mM dNTPs, 2.5 mM MgCl2, 13 PCR buffer, and 1 unit Taq DNA polymerase (Promega). The PCR temperature and cycling parameters included denaturation at 94 C for 3 min, followed by 34 cycles at 94 C for 30 sec, 46 C for 1 min, 72 C for 1 min and 30 sec, and postamplification extension at 72 C for 10 min. All ITS and cox2 PCR products were cleaned for direct nucleotide sequencing with the use of an ExoSap-IT clean-up protocol (GE Healthcare, Piscataway, New Jersey). Cycle sequencing was conducted with the use of ABI-PRISM Big Dye terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, California), and DNA sequences were cleaned with a Sephadex protocol (GE Healthcare). Sequences were analyzed with an ABI 3730xl DNA automated sequencer (Applied Biosystems). DNA sequences were edited using BioEdit 7.0.1 (Hall, 1999) and aligned with ClustalW (Thompson et al., 1994) following the default parameters. For each nematode species, all unique sequences FIGURE 1. Geographic location of stations (solid circles) where Pacific sardine (Sardinops sagax) were caught in 5 regions of the California Current, and the relative proportions of the Anisakis species are indicated for each region (pie charts: solid white, Anisakis pegreffii; solid black, Anisakis simplex ‘C’; gray dotted, Anisakis simplex s.s.). For each region, the number of Anisakis nematodes was included. Region 1: off Vancouver Island, British Columbia (n 5 19), Region 2: Washington and Oregon (n 5 55), Region 3: Northern California (n 5 32), Region 4: Central California (n 5 13), and Region 5: Southern California (n 5 29). The 200m isobath is depicted by the solid black line oriented approximately north to south, west of the provincial and state coastlines. were deposited into GenBank under the following accession numbers: Anisakis simplex s.s. (JF423200–JF423247), Anisakis pegreffii (JF423248– JF423280), and Anisakis simplex ‘C’ (JF423281–JF423297). Data analysis Estimated sardine standard lengths (SL, in millimeters) among regions were not normally distributed (Kolmogorov-Smirnov test, P value 0.001), and variances were uneven among regions (Levene’s test, P-value 5 0.002; SPSS PASW Statistics 18). Thus for each year, Mann–Whitney U-tests were used to compare estimated sardine SL of uninfected and infected sardine between regions and between near-shore and offshore samples. Fish collected east of the 200-m isobath (approximating the continental shelf break) were classified as inshore samples, and fish collected west of this line were considered offshore samples (Fig. 1). A Mann–Whitney Utest was also used to examine parasite species abundance between regions for fish caught near shore versus offshore. We tested whether there was an effect of host size on parasite accumulation for each nematode species with the use of Spearman’s correlations (Ambrose and Ambrose, 1987). All Mann–Whitney U-tests and Spearman’s correlations were calculated in StatviewH (SAS, 1998). For each geographic region designated in the California Current, we calculated prevalence and intensity for each Anisakis species according to Bush et al. (1997). For each nematode species we used DNAsp (v.5.00.07) (Librado and Rozas, 2009) to calculate standard statistics: (1) haplotype diversity (h), BALDWIN ET AL.—POPULATION STRUCTURE OF ANISAKIS NEMATODES 547 TABLE I. Samples of Pacific sardine (Sardinops sagax) collected by year, region and latitude. The estimated mean standard length (SL) and range in millimeters (mm) are provided for each fish collection. Estimated mean fresh Year Location Region* Latitude (uN) No. fish 2005 .Vancouver Island, British Columbia, Canada .Willapa Bay, Washington, USA .Columbia River, Oregon, USA .Santa Cruz, California, USA .Point Arguello, California, USA .Vancouver Island, British Columbia, Canada .Willapa Bay, Washington, USA .Columbia River, Oregon, USA .Newport, Oregon, USA .Point Delgada, California, USA .Manchester, California, USA .Point Arguello, California, USA .San Nicolas Island, California, USA .East of San Nicolas Island, California, USA .Willapa Bay, Washington, USA .Astoria, Oregon, USA .Columbia River, Oregon, USA .Cape Blanco, Oregon, USA .Chetco River, California, USA .Patrick’s Point/Klamath River, California, USA .Point Delgada, California, USA .Point Arena/Point Reyes, California, USA .Golden Gate Inner, California, USA .Salmon Cone, California, USA .Point Arguello, California, USA .San Nicolas Island, California, USA .Ventura, California, USA .San Diego, California, USA . 1 2 2 4 4 1 2 2 2 3 3 4 5 5 2 2 2 2 3 3 3 3 4 4 4 5 5 5 . 48.7 46.67 46.17 36.98 34.54 50.58 46.67 46.17 44.67 40.24 39.12 35.29 33.2 32.97 46.67 46.04 46.17 43 42 41.21 40 38.29 37.48 35.8 35.37 33.28 34.28 32.48 . 56 86 30 50 50 45 89 46 39 50 68 48 48 26 92 21 102 3 10 32 23 95 1 39 45 45 50 50 1,339 2006 2007 2008 Total SL (range SL) 185.69 197.42 254.88 171.63 172.63 223.88 202.16 213.28 185.24 205.11 196.95 181.40 174.45 191.52 220.16 220.56 203.66 211.70 217.87 205.43 199.89 195.57 209.74 207.95 178.80 208.30 222.26 (148.08–256.61) (143.28–258.53) (241.24–279.66) (106.78–198.98) (147.12–196.10) (194.21–285.76) (156.15–265.18) (192.15–268.27) (100.61–213.75) (184.58–247.00) (120.23–260.45) (163.35–200.38) (157.18–204.50) (158.21–206.55) (179.81–276.50) (207.58–255.93) (189.07–217.87) (142.78–247.70) (209.64–229.18) (190.10–219.93) (189.07–207.58) (170.55–212.72) .183.92 (185.98–267.24) (120.15–235.35) (161.29–205.52) (193.18–264.15) (207.00–257.00) . * Region 1 5 Vancouver Island, British Columbia; Region 2 5 Washington and Oregon; Region 3 5 northern California; Region 4 5 central California; and Region 5 5 southern California. the proportion of unique haplotypes recovered; (2) the number of polymorphic sites; (3) nucleotide diversity (p), the species-wide average number of nucleotide differences per site between 2 sequences; and (4) Tajima’s D (Tajima, 1989) to test for evidence of selection or demographic processes among all molecular mutations. The number of unique haplotypes and mean pairwise differences among cox2 sequences within and among species were determined with the use of Arlequin 3.1 (Excoffier et al., 1992). With the use of an analysis of molecular variance (AMOVA) for each Anisakis species, genetic variation was partitioned into variance components (within, and among, variation by either year or region), and PhiST (WST) values (analogous to Fst values) were calculated with the use of permutational estimates of significance in Arlequin 3.1 (Excoffier et al., 1992). PhiST (WST) values have a maximum value of 1, where 0 indicates no differentiation among sequences from predefined regions and 1 indicates complete differentiation among sequences from predefined regions. The program TCS 1.13 (Clement et al., 2000) was used to create statistical parsimony networks of cox2 haplotypes for each Anisakis species by region only. Parsimony networks were recalculated to assess the regional similarity among cox2 sequences from our study and cox2 sequences previously deposited in GenBank for A. simplex s.s., A. simplex ‘C’, and A. pegreffii (Table II). For sequences that were not included in a network at a 95% confidence level, we used a maximum of 100 steps to force sequences into the network to determine the number of steps from which these sequences differed from the main network. An AMOVA was used to compare the genetic subdivision between A. simplex s.s. sequences from Pacific sardine from the California Current (this study) to A. simplex s.s. recovered off Japan from walleye pollock (Theragra chalcogramma) (Quiazon et al., 2009) and chub mackerel (Scomber japonicus) (Suzuki et al., 2009). RESULTS General Pacific sardine information A total of 1,339 sardines were processed during this study. These sardines had an estimated fresh standard length (SL) ranging from 100.61 to 285.76 mm (Table I). The 809 fish caught near shore were larger (mean 205.31 ± 21.76 mm, with a mean rank of 747.84) than the 530 fish caught offshore (mean 193.23 ± 21.54 mm, with a mean rank of 551.18; z value 5 29.10, P-value , 0.0001). Except for 1 sardine off Newport, Oregon (estimated SL 5 100.61 mm), the smallest sardines were recovered in 2005 from region 4 (Central California; minimum estimated SL 5 106.78 mm), and the largest were caught in 2006 from region 1 (Vancouver Island, British Columbia; maximum estimated SL 5 285.76 mm). A total of 9.1% of sampled sardines was infected with anisakid nematodes. There was no difference in estimated SL for infected fish caught near shore versus offshore when all years were combined (z value 5 20.92, P 5 0.36) or for each year (2006: z value 5 20.84, P 5 0.40; 2007: z value 5 20.82, P 5 0.41). No near shore versus offshore comparison could be made for 2005 (sampled only near shore) or 2008 (sampled only offshore). Regionally, infected fish were smaller in region 1 (Vancouver Island, British Columbia; 198.02 ± 9.50 mm, mean rank 5 2.25) 548 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 4, AUGUST 2011 TABLE II. GenBank cox2 sequences available for 3 species of Anisakis nematodes. The unique worm numbers designated by Valentini et al. (2006) are provided for those sequences with the same accession number. GenBank accession no. Unique worm no. Host species Common name Geographic location AE01 AE02 Delphinus delphis D. delphis Seriola dumerili Scomber japonicus S. japonicus S. japonicus S. japonicus S. japonicus Theragra chalcogramma T. chalcogramma Common dolphin Common dolphin Greater Amberjack Chub mackerel Chub mackerel Chub mackerel Chub mackerel Chub mackerel Alaska pollock Alaska pollock Spanish Atlantic coast Spanish Atlantic coast China Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Iwate Prefecture, Japan Iwate Prefecture, Japan AS09 AS10 AS11 AS12 AS13 AS14 Unknown Conger myriaster S. japonicus S. japonicus S. japonicus S. japonicus S. japonicus S. japonicus Phocoena phocoena P. phocoena Pseudorca crassidens P. crassidens P. crassidens P. crassidens Unknown Conger eel Chub mackerel Chub mackerel Chub mackerel Chub mackerel Chub mackerel Chub mackerel Harbor porpoise Harbor porpoise False killer whale False killer whale False killer whale False killer whale Unknown Korea Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Vancouver Island, British Columbia Vancouver Island, British Columbia Canadian coast Canadian coast Canadian coast Canadian coast AC02 AC07 AC10 Unknown P. crassidens P. crassidens P. crassidens Lissodelphis borealis Pacific coast rockfish False killer whale False killer whale False killer whale Northern right whale dolphin Californian coast Vancouver Island, British Columbia Vancouver Island, British Columbia Vancouver Island, British Columbia Californian coast Anisakis pegreffii DQ116432 DQ116432 EU933994 AB517565 AB517564 AB517563 AB517562 AB517561 EU560911 EU560907 A. simplex s.s. AJ132189 AY994157 or NC_007934 AB517570 AB517569 AB517568 AB517567 AB517566 AB517560 DQ116426 DQ116426 DQ116426 DQ116426 DQ116426 DQ116426 Anisakis simplex ‘C’ AF179905 AF179906 DQ116429 DQ116429 DQ116429 than in region 2 (Washington and Oregon, 248.60 ± 20.77 mm; z value 5 22.01, P 5 0.04; mean rank 5 10.41) in 2005. However, in 2006 infected fish were larger in region 1 than region 2 (z value 5 23.63, P 5 0.0003; region 1 mean rank 5 28.29; region 2 mean rank 5 14.74), and region 3 (northern California) (z value 5 23.29, P 5 0.001, region 1 mean rank 5 20.12, region 3 mean rank 5 9.46). In 2007, infected fish from region 3 were smaller than infected fish in region 2 (z value 5 21.93, P 5 0.05; region 2 mean rank 5 14.58; region 3 mean rank 5 21.20). No comparison between regions was possible for 2008 because all fish used in this study from that year were caught in region 5 (southern California). There were no correlations between the estimated fresh SL of infected fish and intensity of any Anisakis species for any region (Rho 5 20.59–0.50, P 5 0.10–0.90) or by year (Rho 5 20.011–0.51, P 5 0.06–0.96). Anisakis species recovery and genetics summary Nematode intensity ranged from 1 to 4 worms per host, with most infected fish harboring a single worm (94 of 122 fish). All nematodes were recovered from the body cavity. Of the 191 nematodes collected from 5 geographic regions, DNA was obtained from 148 nematodes. To identify to species, we used an 848–base pair (bp) portion of ITS rDNA, which spanned 2 diagnostic sites. Three genetically distinct species from the Anisakis simplex complex, i.e., A. pegreffii (n 5 76), A. simplex s.s. (n 5 51), and A. simplex ‘C’ (n 5 21, Table III, Fig. 1) were recovered in single- and mixed-species infections throughout the study area. Nine fish (7.3%) were infected with more than 1 species of Anisakis. Six of these fish were caught in regions 4 and 5 (California), 2 in region 2 (Columbia River, Oregon), and 1 in region 1 (Vancouver Island, British Columbia). Two sardines caught in regions 4 and 5 (California) were infected with all 3 Anisakis species. The population structure of each species of the Anisakis simplex complex was assessed with the use of a 524-bp portion of the cox2 mtDNA gene (Table III). Shared haplotypes were observed for all 3 nematode species: (1) A. pegreffii (n 5 10), (2) A. simplex s.s. (n 5 3), and (3) A. simplex ‘C’ (n 5 2). However, more shared haplotypes may become evident with increased sample sizes for each Anisakis species. Anisakis simplex s.s. had the most unique haplotypes (n 5 48), followed by A. pegreffii (n 5 33) and A. simplex ‘C’ (n 5 17) (Table III, Fig. 2A–C). Among the 3 Anisakis species, haplotype diversity ranged from 0.942 to 0.998, BALDWIN ET AL.—POPULATION STRUCTURE OF ANISAKIS NEMATODES 549 TABLE III. Summary information of the genetic variability among 524 nucleotide sites of cox2 mitochondrial DNA for 3 Anisakis species. Anisakis pegreffii Anisakis simplex s.s. Anisakis simplex ‘C’ No. of sequences No. shared haplotypes No. unique haplotypes h diversity 76 51 21 10 3 2 33 48 17 0.942 0.998 0.967 with a total of 98 unique sequences recovered from 148 individual worms. Nucleotide diversity ranged from 0.007 to 0.018, and the number of polymorphic sites ranged from 23 to 79. All 3 Anisakis species had negative Tajima’s D values, but these values were significant only for A. simplex s.s. and A. simplex ‘C’ (Table III). The mean pairwise sequence differences within species were 0.70% p diversity No. polymorphic sites Tajima’s D Tajima’s D P value 0.014 0.018 0.007 40 79 23 20.321 21.61 21.718 0.47 0.03 0.03 (A. simplex ‘C’), 1.4% (A. simplex s.s.), and 1.8% (A. pegreffii). Corrected mean pairwise differences among the 3 species were higher than within each species, i.e., 3.1% (A. pegreffii vs. A. simplex s.s.), 4.7% (A. pegreffii vs. A. simplex ‘C’), and 5.4%. (A. simplex s.s. vs. A. simplex ‘C’), providing further evidence that 3 separate Anisakis species were recovered. FIGURE 2. Regions in the California Current System where 3 species of Anisakis nematodes were recovered from Pacific sardine (Sardinops sagax): off Vancouver Island, British Columbia (blue), Washington and Oregon (green), northern California (maroon), central California (purple), and southern California (yellow). In total, 29 GenBank cox2 mitochondrial DNA sequences (white) that are described in Table III were compared to our cox2 data. Statistical parsimony networks of our cox2 mitochondrial DNA sequences for each Anisakis species either without GenBank sequences: (A) Anisakis pegreffii, (B) Anisakis simplex s.s., (C) Anisakis simplex ‘C’; or with GenBank sequences: (D) A. pegreffii, (E) A. simplex s.s., and (F) A. simplex ‘C’. Each connection is a single base difference; solid black circles are inferred haplotypes, and colored circles are observed haplotypes. The colored circles indicate the geographic region where each haplotype was collected. The number of worms with identical sequences is represented by the size of the colored circles. Unless indicated by a number inside or next to the circle, each haplotype represents an individual nematode. Homoplasies among the sequences are indicated by reticulations within the networks. 550 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 4, AUGUST 2011 TABLE IV. Analyses of molecular variance (AMOVA) results for cox2 mitochondrial DNA sequences from 3 Anisakis species grouped either by year or by geographic region: By year (Y), Vancouver Island and Southern California (VI vs. SC); near shore versus offshore (N vs. O), where offshore includes stations west of the 200-m isobath lines indicated in Figure 1; Japan versus California Current (J vs. CC) only for Anisakis simplex s.s. The number of worms compared per species, region, and year are indicated for each AMOVA test. AMOVA test Source Variance components Percentage of variation P value DF Sum of squares .Among years .Within years .Total . 3 72 75 12.99 262.59 275.58 . 0.04 3.65 3.69 . 1.06 98.94 . . 0.01 . . . 0.26 . . . .Among regions .Within regions .Total 1 16 17 4.44 48.50 52.94 0.23 3.03 3.26 6.97 93.03 . 0.07 . . 0.24 . . .Among regions .Within regions .Total 1 74 75 4.99 270.59 275.59 0.04 3.66 3.70 0.98 99.02 . 0.01 . . 0.21 . . .Among years .Within years .Total . 3 17 20 5.38 29.67 35.05 . 0.01 1.75 1.76 . 0.54 99.46 . . 0.01 . . . 0.42 . . . WST Anisakis pegreffii Y 2005 2006 2007 2008 5 5 5 5 8 25 31 12 VI vs. SC VI 5 4 SC 5 14 N vs. O N 5 45 O 5 31 . Anisakis simplex ‘C’ Y 2005 2006 2007 2008 5 5 5 5 3 6 6 6 VI vs. SC VI 5 3 SC 5 6 N vs. O N 5 10 O 5 11 . .Among regions .Within regions .Total 1 7 8 1.44 13.00 14.44 20.10 1.86 1.75 25.88 105.88 . 20.06 . . 0.72 . . .Among regions .Within regions .Total 1 19 20 1.59 33.46 35.05 20.02 1.76 1.74 20.92 100.92 . 20.01 . . 0.52 . . .Among years .Within years .Total . 3 47 51 15.37 222.71 238.08 . 0.03 4.74 4.77 . 0.70 99.30 . . 0.01 . . . 0.30 . . . .Among regions .Within regions .Total 1 20 21 5.49 81.10 86.59 0.13 4.06 4.19 3.14 96.86 . 0.03 . . 0.20 . . .Among regions .Within regions .Total 1 49 50 7.47 230.61 238.08 0.12 4.71 4.82 2.46 97.54 . 0.02 . . 0.10 . . .Among regions .Within regions .Total 1 57 58 67.11 296.14 363.25 4.48 5.20 9.67 46.28 53.72 . 0.46 . . ,0.0001 . . A. simplex s.s. Y 2005 2006 2007 2008 5 5 5 5 7 24 10 10 VI vs. SC VI 5 12 SC 5 10 N vs. O N 5 33 O 5 18 J vs. CC J58 CC 5 51 . Temporal variation among cox2 sequences was not significant when individual worms were compared by collection year (Table IV). Geographic separation among cox2 sequences was minimal for each Anisakis species when the individual worms were compared from the northern and southern ends of the study region (Table IV). Genetic variation was associated with differences within regions, as opposed to differences among regions resulting in non-significant AMOVA WST values ranging from 20.06 to 0.07. The remaining regional comparisons that were geographically closer were also nonsignificant (data not shown). Additionally, WST values (20.01 to 0.02) were nonsignificant (P values ranged from 0.10 to 0.52) for near shore versus offshore nematode sequences compared throughout the study region. In contrast, a WST 5 0.46 (Table IV) was significantly different BALDWIN ET AL.—POPULATION STRUCTURE OF ANISAKIS NEMATODES (P , 0.0001) with 46.28% of the variance explained by differences among regions of A. simplex s.s. from walleye pollock (data from Quiazon et al., 2009) and chub mackerel (data from Suzuki et al., 2009) collected off Japan, and Pacific sardine caught in the California Current (this study). For Anisakis species recovered in the California Current, the lack of distinct population structure associated with defined geographic regions was also evident in the cox2 parsimony networks based on 524 bp of the sequence data (Fig. 2A–C). There was no separation by region within the network for individual cox2 haplotypes, and shared cox2 haplotypes were observed from multiple regions with 95% confidence. An additional sequence, A. simplex s.s, from Region 2 was forced into the main network by 23 steps (Fig. 2C). A similar pattern was observed in the parsimony networks based on 507 bp when 29 GenBank cox2 sequences of the 3 Anisakis species were compared to our sequences (Fig. 2D–F, Table II). Seventeen base pairs were removed from our sequences to enable an alignment with the GenBank sequences. Eight A. pegreffii sequences from GenBank fit into our network with 95% confidence, 5 of which were identical to sequences in our study (Fig. 2D). Only 4 of the 5 A. simplex ‘C’ sequences fit into our network with 95% confidence (Fig. 2E). When forced, the remaining A. simplex ‘C’ sequence was 7 steps away from the main network. Ten A. simplex s.s. GenBank sequences fit into the network with 95% confidence, 5 of which were identical to our sequences. An additional sequence A. simplex s.s. from our data and 6 A. simplex s.s. GenBank sequences were forced into the main network by either 21 or 23 steps (Fig. 2F). DISCUSSION We recovered larval A. pegreffii, A. simplex s.s., and A. simplex ‘C’ throughout the California Current in Pacific sardines. Analyses of the cox2 haplotypes supported a panmictic distribution of larval Anisakis species in the California Current for all 3 Anisakis species. For each, haplotype diversity was high, and related haplotypes among each species were distributed throughout our study area. Nucleotide diversity was low to medium compared to other nematode studies (Derycke et al., 2005; review by Höglund et al., 2006). This overall pattern of molecular variation across a large geographical region was similar to previous studies examining mtDNA of A. simplex s.s. infecting Atlantic herring (Cross et al., 2007), and parasitic nematodes infecting livestock (Blouin et al., 1995). Population subdivision was described among different river basins using AMOVA analyses for parasitic nematodes infecting freshwater fish. For example, Mejı́a-Madrid et al. (2007) observed 19.3% variation among 7 river basins in Central Mexico for cytochrome c oxidase subunit 1 (COI) sequences of Rhabdochona lichtenfelsi. In addition, Wu et al. (2009) observed 46.6% variation among COI sequences of Camallanus cottis among 3 river basins in China, identifying haplotypes unique to the Pearl River compared to the Yangtze and Minjiang Rivers. Regional differences in our study were only detected by AMOVA when sequences of A. simplex s.s. from the California Current were compared to A. simplex s.s. sequences collected off Japan (Quiazon et al., 2009; Suzuki et al., 2009). If different anisakid populations are observable in the Pacific Ocean only at the basin scale, then it is unlikely the population structure of Pacific 551 sardines in the California Current can be determined with the use of the recovery or population genetics of Anisakis nematodes. Our cox2 data support the hypothesis that host movement strongly influences the population structure of parasites (Blouin et al., 1995; Jarne and Theron, 2001; Criscione and Blouin, 2004; Criscione et al., 2005). The Pacific sardines in this study are considered part of the Central California Offshore subpopulation and thought to be capable of migrating between Vancouver Island, British Columbia, and San Diego, California (Dahlgren, 1936; Hart, 1943; Ahlstrom, 1957; Smith, 2005). In the Anisakis spp. nematode life cycle, cetacean definitive hosts likely comprise the most mobile hosts, traveling thousands of kilometers during their annual migrations, while dispersing nematode eggs. For example, humpback whales (Megaptera novaeangliae) are capable of traveling latitudinally between Mexico and Alaska (Lagerquist et al., 2008), and longitudinally between Japan and British Columbia (Perrin et al., 2009). With approximately 200 pelagic fish species known to be paratenic hosts for Anisakis species (Sabater and Sabeter, 2000), opportunities for gene flow between geographically distant and potentially distinct populations of Anisakis species likely result from both migrating fish species and cetacean hosts (Nadler, 1995; Cross et al., 2007; Mattiucci and Nascetti, 2008). All 3 Anisakis species were distributed from 50uN to 32uN latitude, a larger geographical area than previously reported by Mattiucci and Nascetti (2008). Thus, the panmixia of haplotypes found in our study could be a reflection of the extensive movement of all of the potential hosts utilized by these nematodes. Our results suggest that the limited oceanographic barriers and complexity in the California Current are not preventing the mixing of anisakid species or populations. The major biogeographic break in the California Current at Point Conception, California, does not appear to limit the distribution of highly migratory fish or cetacean taxa (Checkley and Barth, 2009) that propagate Anisakis species. Further, the hydrography of the north–south-oriented California Current is considered less complex than the hydrography of the Atlantic Ocean from Europe to northwest Africa, where 5 major currents interact along a European coastline that alternates between an east–west and north–south orientation (Checkley et al., 2009). The phylogeographic breaks located in the Mediterranean Sea (Peloponnesian) and the Atlantic–Mediterranean transition zone (Gibraltar and Oran-Almerı́a) (Patarnello et al., 2007; SalaBozano et al., 2009) limit the movement of European sardines (Sardina pilchardus), as well as other fishes and cetaceans, resulting in the recovery of different Anisakis species in the Mediterranean Sea and Atlantic Ocean. For example, A. pegreffii was most common in the Mediterranean Sea, and A. simplex s.s. was most prevalent in the northeast Atlantic Ocean for both European hake (Mattiucci et al., 2004) and Atlantic horse mackerel (Mattiucci et al., 2008). Our observations differ from the disjointed geographical recovery of Anisakis species in European hake (Mattiucci et al., 2004), Atlantic horse mackerel (Mattiucci et al., 2008), and European sardines. To date, European sardines infected with Anisakis spp. have been observed only along the Adriatic coast off Italy (Fioravanti et al., 2006), and off western Portugal (Silva and Eiras, 2003). No Anisakis spp. were observed in sardines off northwest Spain in Galician waters (Abollo et al., 2001), southern and eastern Spain (Rello et al., 2008), or off western Africa by 552 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 4, AUGUST 2011 Morocco and Mauritania (Kijewska, Dzido, Shukhgalter and Rokicki, 2009). Larval Anisakis species in European sardines have not yet been genetically identified to species. However, the geographic distribution of genetically identified Anisakis in European hake (Mattiucci et al., 2004) and horse mackerel (Mattiucci et al., 2008) suggest that A. simplex s.s. and A. pegreffii could infect sardines off Portugal and A. pegreffii, Anisakis typica, and Anisakis physeteris could infect sardines off Italy. Once Anisakis species from European sardines are genetically identified, and a population genetic study is conducted for each Anisakis species, it can be determined if the panmictic distribution of the 3 Anisakis species in Pacific sardine is unique because of the hydrography of the California Current. In summary, the distribution and population structure of Anisakis species throughout the California Current could suggest a single population of Pacific sardine. Just as elevated gene flow in several marine fish species obscures geographic structuring of genetic variation (Waples, 1998), gene flow among marine parasites would also connect subpopulations from distant geographic locations. For instance Mattiucci et al. (1997) reported high gene flow for Atlantic region populations of A. pegreffii, A. simplex s.s., and A. simplex ‘C’ based on 24 allozymes, and Kijewska, Dzido, and Rokicki (2009) suggested a genetic division between the Atlantic and Pacific Oceans for individual A. simplex s.s. and A. simplex ‘C’ based on the AT-rich region of mtDNA. The diversity and availability of fish and cetacean species that undergo extensive migrations along the full length of the California Current system may enable large geographically distributed population sizes of anisakids. Thus, we cannot confirm or deny the existence of Pacific sardine subpopulations within the California Current by the distributional patterns of Anisakis species. Complex oceanographic conditions and host migrations may influence the genetic diversity and population structure of Anisakis species along other coastlines. Investigation of these influences could clarify whether high genetic diversity and connectivity among anisakid populations over large geographical distances is a common pattern, or whether hydrography can restrict gene flow of a widely dispersed generalist marine parasite. ACKNOWLEDGMENTS We would like to thank R. Emmett, M. Blouin, V. Lesser, J. Bolte, E. Casillas, J. Butzerin, and an anonymous reviewer for providing comments on earlier versions of this manuscript. We would also like to thank S. Mattiucci for providing Anisakis spp. sequences and reviewing the manuscript. LITERATURE CITED ABAUNZA, P., A. G. MURTA, N. CAMPBELL, AND R. CIMMARUTA. 2008. Stock identity of horse mackerel (Trachurus trachurus) in the Northeast Atlantic and Mediterranean Sea: Integrating the results from different stock identification approaches. 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