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
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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.
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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.
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