Veterinary Parasitology 197 (2013) 221–230
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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
Anisakis species (Nematoda: Anisakidae) of Dwarf Sperm
Whale Kogia sima (Owen, 1866) stranded off the Pacific coast
of southern Philippine archipelago夽
Karl Marx A. Quiazon a,b,∗ , Mudjekeewis D. Santos c , Tomoyoshi Yoshinaga b
a
Freshwater Aquaculture Center and College of Fisheries, Central Luzon State University, Science City of Muñoz, Nueva Ecija 3120,
Philippines
b
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-8657, Japan
c
Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, 101 Mother Ignacia Street, Quezon City
1103, Philippines
a r t i c l e
i n f o
Article history:
Received 7 January 2013
Received in revised form 21 May 2013
Accepted 23 May 2013
Keywords:
Dwarf Sperm Whale
Philippine archipelago
Anisakis species
ITS region
mtDNA cox2 region
a b s t r a c t
Anisakid nematodes in the Pacific region of the Philippine archipelago still remain unexplored. This study was carried out to identify anisakid species from one of their final
hosts, the Kogiid whale (Dwarf Sperm Whale, Kogia sima) stranded off the southern part
(Davao Gulf) of the Philippine archipelago. Anisakid worms were initially identified morphologically using light and scanning electron microscopy, whereas identification to species
level was carried out molecularly using PCR-RFLP and sequencing of the ITS (ITS1–5.8s
rRNA–ITS2) and mtDNA cox2 regions. Parasitological study revealed new geographical
records for the presence of two Anisakis species (A. brevispiculata and A. typica) and two
unknown Anisakis species that are genetically close, at mtDNA cox2 region, to A. paggiae
and A. ziphidarum. Based on the molecular data on both genes, the current findings suggest
possible occurrence of local variations or sibling species of A. paggiae and A. ziphidarum in
the region. Given that Anisakis species have not been reported in the Philippine archipelago,
their presence in the Dwarf Sperm Whale inhabiting this region indicates high possibility of
Anisakis infections in the marine fishes, cephalopods and other intermediate hosts within
the Philippine waters.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
The Philippines, identified as the center of marine
fish biodiversity (Carpenter and Springer, 2005), is an
archipelagic country surrounded with vast marine fishery
夽 The nucleotide sequences of four Anisakis species have been deposited
in GenBank database with the accession numbers KC342886–KC342901,
KC821728–KC821738 and KC852163–KC852171.
∗ Corresponding author at: Freshwater Aquaculture Center and College
of Fisheries, Central Luzon State University, Science City of Muñoz, Nueva
Ecija 3120, Philippines. Tel.: +63 44 4560681; fax: +63 44 4560681.
E-mail address: karlmq@yahoo.com (K.M.A. Quiazon).
0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetpar.2013.05.019
resources. Aside from the marine fishes and cephalopods
serving as intermediate hosts of some zoonotic nematodes,
marine cetaceans (i.e., whales, dolphins and porpoises) represent part of the marine biodiversity. Cetaceans live in
distinct regions of the world oceans, wherein due to different habitat preferences, they inhabit particular depths,
temperature ranges (tropical, temperate, polar) or oceanographic regimes (Jefferson et al., 2008).
Whales, such as the baleen, sperm and some other
large toothed whales, have extensive and predictable seasonal migrations allowing maximum exploitation of food
resources (Jefferson et al., 2008). Sperm whales include
two families, i,e., Physeteridae (modern sperm whale) and
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K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
Kogiidae (Dwarf Sperm Whale, Kogia sima, and Pygmy
Sperm Whale, Kogia breviceps), which are known from typical deep-water habitats in both tropical and temperate
zones. Sperm whales mostly feed in deep waters (mesoand bathypelagial), primarily on cephalopods and less frequently on deep-sea fishes and crustaceans (i.e., decapods)
(Dos Santos and Haimovici, 2001; Beatson, 2007; West
et al., 2009). Marine mammals are known to be the final
host of zoonotic nematodes of the genus Anisakis Dujardin,
1845. Euphausiids (krills) serve as intermediate hosts of
Anisakis species, with cephalopods and marine fishes serving as paratenic hosts, whereas humans are regarded as
accidental hosts once infected paratenic hosts are consumed in raw, undercooked or even marinated form.
Morphological identification has been used to identify adults and some third-stage larvae of Anisakis to
type or species levels (Koyama et al., 1969; Davey, 1971;
Fagerholm, 1988; Mattiucci et al., 2005; Quiazon et al.,
2008; Murata et al., 2011). Recently, the use of molecular markers revealed at least nine Anisakis species: A.
simplex sensu stricto (s.s.), A. pegreffii, A. simplex C (all
three species comprise the A. simplex complex), A. typica, A. ziphidarum, A. nascettii, A. paggiae, A. brevispiculata,
and A. physeteris (the latter three species comprise the A.
physeteris complex). In spite of morphological similarities
among some Anisakis species, they are found to be genetically different having distinct host preferences, life cycles
and zoogeographical distributions (Klimpel et al., 2008,
2011; Mattiucci and Nascetti, 2006, 2007, 2008; Mattiucci
et al., 2009).
A recent study conducted by Kuhn et al. (2011) on
the zoogeographical modeling of the zoonotic parasite
Anisakis clearly demonstrates that the distribution patterns
of Anisakis species can be narrowed down to certain areas
within climatic zones and oceans and are mainly influenced by the species ranges and feeding behaviors of their
respective intermediate and mammalian final hosts. Based
on their model, the Anisakis species inhabiting the tropical
region in the South China Sea, which also includes the West
Philippine Sea, is A. typica. In the Philippines, only Petersen
et al. (1993) have reported third-stage larvae of unknown
Anisakis species from the body cavity and muscle of Daggertooth pike conger (Muraenesox cinereus) caught from
central Philippine waters. In the neighboring countries of
the Philippines such as in Indonesia and Thailand, there
have been reports of A. simplex s.s., A. typica, and possible
local variation or sibling species of A. typica from marine
fishes (Moravec et al., 2006; Chen et al., 2008; Palm et al.,
2008).
Anisakid nematodes have also been reported in Kogiid
whales. Anisakis brevispiculata, A. paggiae, A. physeteris, A.
simplex s.s, A. typica, and Pseudoterranova ceticola have been
reported from the definitive host Pygmy Sperm Whale,
which is closely related to the Dwarf Sperm Whale (Abollo
et al., 1998; Mattiucci et al., 2001; Mattiucci and Nascetti,
2007; Cavallero et al., 2011). Although Oliveira et al. (2011)
did not find any anisakid nematodes in the Dwarf Sperm
Whale from the Pacific coast of Costa Rica, there are
reported infections of A. brevispiculata, A. paggiae, A. physeteris, A. simplex complex, A. typica, and P. ceticola in the
gastro-intestinal tract of this cetacean species (Deardorff
and Overstreet, 1981; Mattiucci et al., 2005; Colón-Llavina
et al., 2009; Cavallero et al., 2011).
Despite the frequent whale strandings within the Philippine’s long coastline, no parasitological studies have been
carried out, similar to those on zoonotic nematodes on
marine fishes and cephalopods causing human anisakiasis (or anisakidosis) and allergic reactions (Audicana and
Kennedy, 2008). Given that the Philippine archipelago
relies mainly on marine fisheries, both for local and export
consumption, this study was carried out to determine
presence of zoonotic anisakid nematodes in the region
by morphological and molecular identification of anisakid
worms isolated from the stomach of a stranded Dwarf
Sperm Whale in the Pacific coast of southern Philippine
archipelago. As local dish called kilawin (fresh fish products
marinated in vinegar and spices) is commonly consumed
in the country, the risk of transmitting these zoonotic parasites from the final host to fishes and cephalopods, and
accidentally to humans, is likely possible. Also, there are
some high valued marine fishes such as the bigeye (Thunnus obesus (Lowe, 1839)) and yellowfin tunas (Thunnus
albacares (Bonnaterre, 1788)) that are exported abroad for
sushi and sashimi consumption. Hence, a broad knowledge on the distribution of anisakid nematodes is very
important in understanding and forecasting possible future
infections, thereby serving as pro-active measure in reducing the risk of human anisakidosis and allergies to target
consumers locally and internationally.
2. Materials and methods
2.1. Sample collection and fixation
Anisakid worms (around 800–900 worms) were collected from the stomach of a stranded Dwarf Sperm Whale
from Davao Gulf in southern Philippines (6◦ 35 55 N; 125◦
47 17 E) on December 2011. Each individual worm was
cut into three portions, the anterior end, middle portion,
and posterior end. The anterior and posterior ends of the
isolated worms were fixed in 70% ethyl alcohol, followed by
clearing in glycerin prior to mounting in slides for the initial
anisakid identification using morphological keys (Koyama
et al., 1969; Davey, 1971; Quiazon et al., 2008; Murata
et al., 2011). Following light microscopic examination, the
samples were further processed for SEM examination as
previously described (Quiazon et al., 2008). On the other
hand, the remaining middle portions were fixed in 100%
ethyl alcohol for molecular analyses.
2.2. DNA extraction and PCR
The genomic DNA was extracted from 100% ethanolfixed middle portion of every individual worm using
DNeasyTM Tissue Kit (Qiagen, Hilden, Germany). Initial
species identification was carried out through PCRRFLP and sequencing of the ITS (ITS1–5.8s rRNA–ITS2)
region. Final confirmation of species identity was
performed by sequencing the mitochondrial DNA
cytochrome oxidase subunit 2 (mtDNA cox2) region.
The ITS region was amplified using forward primer
NC5f (5 -GTAGGTGAACCTGCGGAAGGATCATT-3 ) and
K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
reverse primer NC2r (5 -TTAGTTTCTTTTCCTCCGCT-3 )
with the following PCR protocol: initial denaturization
at 94 ◦ C for 4 min, followed by 30 cycles consisting of
denaturization at 94 ◦ C for 30 s, annealing at 55 ◦ C for
30 s, and extension at 72 ◦ C for 30 s, with final extension at 72 ◦ C for 7 min (Zhu et al., 1998). On the other
hand, the mtDNA cox2 region was amplified using
the primers 210 (5 -CACCAACTCTTAAAATTATC-3 ) and
(5 -TTTCTAGTTATATAGATTGRTTYAT-3 )
(Nadler
211
and Hudspeth, 2000) with the following PCR protocol:
denaturization at 94 ◦ C for 3 min, followed by 34 cycles
consisting of 94 ◦ C for 30 s, 46 ◦ C for 1 min, 72 ◦ C for 1 min
and 30 s, with final extension at 72 ◦ C for 10 min (Valentini
et al., 2006). For both DNA regions, the PCR assays were
performed with 1 ␮L sample DNA as template in a total
volume of 20 ␮L: containing 0.6 ␮L forward and reverse
primers, 14.1 ␮L DDW and 3.7 ␮L Taq mix (containing
0.1 ␮L TAKARA Ex TaqTM HS; 2 ␮L [10×] Ex Taq Buffer; and
48 ␮L dNTP mixture).
2.3. PCR-RFLP and DNA sequencing
Among the morphologically identified anisakid samples, digestion of PCR products with restriction enzymes
(Alu I, Hae III, Hha I, Hinf I, Mbo I, Pvu II, Dra I, EcoR V,
Sca I and Pst I (Takara Bio Inc., Otsu, Japan)) was carried
out on the ITS region to initially determine species differences using reported molecular keys (D’Amelio et al.,
2000; Pontes et al., 2005; Farjallah et al., 2008; Quiazon
et al., 2009; Murata et al., 2011). The digested products
stained with GR Green Loading Buffer (Bio-craft, Tokyo,
Japan) were electrophoresed in 2.0% agarose gel and visualized by illumination with shortwave ultraviolet light.
For further confirmation on the taxonomical identity of the PCR-RFLP-identified Anisakis species, the ITS
and mtDNA cox2 regions of a minimum of three representative specimens from each species were individually
amplified and sequenced. After amplification, the PCR
products having the expected molecular weight were purified using Qiaquick gel extraction kit (Qiagen, Hilden,
Germany) and sequenced in both directions with the same
primers as used in PCR. The sequencing reactions were
carried out using BigDye terminator kit version 3.1 and
resolved with ABI 3730XL genetic analyzer (Applied Biosystems, Japan). For the mtDNA cox2 region, the nucleotide
sequences were translated into amino acid sequences,
followed by comparison and alignment with the previously reported mtDNA cox2 sequences from GenBank.
Sequence alignment was performed using BioEdit 7 (Hall,
1999), and a square matrix based on Kimura 2-parameter
(K2P) mode was made using MEGA5 (Tamura et al.,
2011). Partition homogeneity test and phylogenetic analysis using Maximum Parsimony (MP) was performed
by PAUP* version 4.0 (Swofford, 2003). Also, phylogenetic analysis was performed using Maximum Likelihood
(ML) using jModelTest 2.1.1 (Darriba et al., 2012). Estimation of genetic distance and Neighbor-Joining (NJ)
analyses from the ITS region was carried out using Kimura
2-parameters (K2P) mode (complete deletion, MEGA5),
whereas that of the mtDNA cox2 region was carried out
using p-distance (pairwise deletion, MEGA5). Nucleotide
223
sequences were deposited and made available in the
GenBank under accession numbers KC342886–KC342901,
KC821728–KC821738, and KC852163–KC852171.
3. Results
3.1. Morphological examination
Only pre-adult stages of anisakid nematodes of the
genus Anisakis were morphologically identified from
stranded Dwarf Sperm Whale using previously reported
morphological keys (Koyama et al., 1969; Davey, 1971;
Murata et al., 2011). Since pre-adult stages (i.e., more
advanced larval stages) of Anisakis spp. were observed in
the current specimens, this cannot be compared with thirdstage larvae and adults from previous reports (Mattiucci
et al., 2005; Murata et al., 2011). Current specimens were
found to exhibit two major morphotypes based on the morphological differences on the tail end (i.e., Morphotype 1 –
has a blunt tail; Morphotype 2 – has a conical tail) (Table 1,
Fig. 1).
3.2. PCR-RFLP
The possible Anisakis species differences among the
two morphotypes were initially determined using PCRRFLP. Based on the ten restriction enzymes used, only six
enzymes (Alu I, Hae III, Hha I, Hinf I, Mbo I, and Pvu II) were
found to show clear species differences based on the generated fragment patterns. Three different fragment patterns
were generated wherein two of these fragment patterns
(i.e., fragment pattern 1 and 2) coincide with the previously reported genetic markers for A. brevispiculata and A.
typica after digestion with Hha I and Hinf I (D’Amelio et al.,
2000; Pontes et al., 2005; Farjallah et al., 2008; Quiazon
et al., 2009; Murata et al., 2011), whereas the identity of
the remaining fragment pattern (i.e., fragment pattern 3)
remains unknown (Table 1, Fig. 2).
3.3. ITS sequences
The two species initially identified using PCR-RFLP
analyses, as well as the unknown species generating fragment pattern 3, were further confirmed and identified,
respectively, by sequencing the ITS region. Nucleotide
sequence data analyses on the ITS region confirmed the
species identity of A. brevispiculata and A. typica. All
three A. brevispiculata specimens revealed low genetic
variation (genetic distance of 0.003; 2 nucleotide bases
difference) compared to the deposited A. brevispiculata
(EU624344) from the GenBank. Also, all four specimens
of A. typica revealed low genetic variation (0.006; 4) compared to the deposited A. typica (EU346093). Furthermore,
the remaining 11 specimens generating fragment pattern 3 were genetically close to the deposited Anisakis
sp. (EU718474, KC121370, and JN005761) revealing very
low genetic variation (0.000–0.001; 0–1) (Table 2). The
generated NJ and MP trees showed that Anisakis spp.
obtained in the present study join the second clade composed of the reported A. paggiae, A. brevispiculata, and A.
physeteris (Fig. 3). Although Anisakis spp. from the current
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K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
Fig. 1. SEM micrographs of pre-adult stages of Anisakis species showing the two different morphotypes on the tail end. (A) and (B), morphotype 1 (A.
brevispiculata and A. typica); (C) and (D), morphotype 2 (unknown Anisakis species that are genetically close to A. paggiae and A. ziphidarum). Species
identities in parentheses are based on the mtDNA cox2 region. Scale bar = 100.
study revealed closest identity with A. paggiae (0.097; 62
nucleotide bases difference) than to any other deposited
Anisakis species (0.100–0.175; 64–106), species confirmation of these Anisakis spp. requires further examination on
the mtDNA cox2 region.
3.4. mtDNA cox2 sequences
To further verify the species identity of A. brevispiculata, A. typica, and particularly that of Anisakis spp., the
mtDNA cox2 region was sequenced and translated into
amino acids. The generated NJ tree, based on the translated amino acid sequences, finally confirmed the identity
of A. brevispiculata and A. typica. Intra-species comparisons
within A. brevispiculata (genetic distance of 0.000–0.010;
1–2 amino acid differences) and A. typica (0.000–0.005;
0–1) specimens from the current study showed very low
genetic variations.
On the other hand, generated NJ tree revealed the
grouping of four ITS-identified Anisakis spp. specimens
within the phylogenetic branch of the deposited sequences
of A. paggiae, whereas seven specimens grouped within
the branch of the deposited A. ziphidarum. Intra-species
comparison of the four specimens with the deposited
A. paggiae samples from the GenBank revealed very low
genetic variations (0.000–0.005; 0–1). Furthermore, those
seven specimens that grouped within the A. ziphidarum
branch generated further two sub-branches with very high
bootstrap value (99%); i.e., sub-branch 1 includes four specimens from the present study (KC821732–KC821734, and
KC821736) and two specimens from the deposited A. ziphidarum (DQ116430 and AB517573) (Valentini et al., 2006;
Suzuki et al., 2010), while the remaining three specimens
(KC821735, KC821737, and KC821738) are included in the
sub-branch 2. Intra-species differences among the four
specimens included in the sub-branch 1 revealed a slightly
higher genetic variation (0.000–0.011; 0–2) than the three
specimens included in the sub-branch 2 (0.000–0.006; 0–1)
(Table 3, Fig. 4).
4. Discussion
Despite the groupings of the ITS-identified Anisakis sp.
specimens with the A. paggiae and A. ziphidarum (based on
the mtDNA cox2 region), the exact identities of these specimens still remain unclear. Phylogenetic analyses on both
gene regions indicate that the current specimens seemed
to be not A. paggiae and A. ziphidarum. In this regard, we
report them as unknown Anisakis species that are genetically close to A. paggiae and A. ziphidarum, respectively.
For the specimens that are genetically close to A. paggiae,
the reported distinct morphological feature on the presence of violin-shaped ventriculus in A. paggiae (Mattiucci
et al., 2005) was not observed. On the other hand, the high
K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
225
Fig. 2. PCR-RFLP results showing three different fragment patterns after digestion with restriction enzymes Alu I (A), Hae III (B), Hha I (C), Hinf I (D), Mbo I
(E), and Pvu II (F). Lane 1 – fragment pattern 1 (A. brevispiculata); lane 2 – fragment pattern 2 (A. typica); lane 3 – fragment pattern 3 (Anisakis species that
are genetically close to A. paggiae and A. ziphidarum); ladder – 100 bp, 500 bp (with asterisks). Species identities in parentheses are based on the mtDNA
cox2 region.
genetic variation in the ITS-identified Anisakis sp. explains
why PCR-RFLP results did not conform to the fragment patterns of previously reported A. paggiae and A. ziphidarum
(D’Amelio et al., 2000; Quiazon et al., 2009; Sequeira et al.,
2010; Suzuki et al., 2010; Murata et al., 2011; Hermida et al.,
2012).
Although the species identity of the reported Anisakis
sp. HD (Sequeira et al., 2010) was not further examined
using the mtDNA cox2 region, such similar fragment pattern with the ITS-identified Anisakis sp. in the present study
was also observed after digestion with restriction enzymes
Hinf I and Hha I. Thus, its identity could possibly be similar to those specimens that are either genetically close
(at mtDNA cox2 region) to A. paggiae or A. ziphidarum.
Moreover, such conflicting species identity using the ITS
and mtDNA cox2 region had also been previously reported
(Quiazon et al., 2009), wherein the taxonomical identity
of the mtDNA cox2-identified A. paggiae (EU560910) was
found to be different on the ITS region. Based on the mtDNA
cox2 sequences, the 98% similarity of the current unknown
Anisakis specimens that are genetically close to A. paggiae with EU560910, and around 96–97% similarity with
the truly identified A. paggiae (Valentini et al., 2006) are
not considered high enough to confirm the similarities
on the identity of two individual Anisakis worms. Hence,
the unknown Anisakis species that are genetically close
to A. paggiae could possibly be a local variation or sibling species of this species. Such possible existence of local
variations or sibling species (Sequeira et al., 2010) could
also be applicable in the unknown Anisakis species that
are genetically close to A. ziphidarum. Based on the mtDNA
cox2 region, the presence of two sub-branches in the NJ
tree (with 99% bootstrap value) within the A. ziphidarum
branch (Fig. 4) indicates possible existence of separate sibling species of A. ziphidarum. Therefore, further studies
and examinations of adult male specimens included in this
group of Anisakis species that are genetically close to A. paggiae and A. ziphidarum are needed to confirm such existence
of local variations or sibling species.
In the present study, a multiple infection of advanced
larval/pre-adult stages of different Anisakis species from
single Dwarf Sperm Whale is a clear evidence of their
sympatric occurrence. Such absence of adult worms could
possibly due to recent infection of the definitive host
through consumption of infected paratenic hosts. On the
other hand, A. typica is a common parasite of various
dolphin species belonging to the families Delphinidae,
Phocoenidae and Pontoporidae from warmer temperate,
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K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
Fig. 3. Phylogeny of Anisakis species identified from Dwarf Sperm Whale (with asterisks) based on the ITS region (A – NJ tree, K2P, complete deletion,
bootstrap method, MEGA5; B – MP tree, bootstrap method, PAUP*4.0.b10).
tropical, and subtropical waters, as well as from the South
West (Brazil) and North West (Florida) Atlantic and from
Mediterranean (North Africa) (Mattiucci et al., 2002, 2005;
Nadler et al., 2005; Farjallah et al., 2008; Palm et al.,
2008; Colón-Llavina et al., 2009; Kuhn et al., 2011). This
species had recently added Dwarf Sperm Whale and Pygmy
Sperm Whale as new definitive hosts (Cavallero et al.,
2011; Iñiguez et al., 2011). In addition, these two kogiid
whales from the Central and South-Eastern and West
Atlantic Ocean and Gulf of Mexico have been reportedly
infected with A. brevispiculata, while those from Puerto Rico
and Atlantic coast of Florida are infected with A. paggiae
(Mattiucci et al., 2001, 2005; Colón-Llavina et al., 2009;
Cavallero et al., 2011). Moreover, A. ziphidarum has only
K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
227
Fig. 4. Phylogeny of Anisakis species identified from Dwarf Sperm Whale (with asterisks) based on the translated amino acid of the mtDNA cox2 sequences
(NJ tree; p-distance; pairwise deletion; bootstrap method; MEGA5).
228
Table 1
Summary data on the identity of Anisakis species from the Dwarf Sperm Whale based on morphology, PCR-RFLP, and sequencing of the ITS and mtDNA cox2 regions.
Morphotypes a
PCR-RFLP
fragment
patterns
Estimated fragment size b
Alu I
Hae III
Hha I
Hinf I
Mbo I
Pvu II
ITS
mtDNA cox2
Morphotype 1
Fragment
pattern 1
Fragment
pattern 2
460–270–250
700–220
435–370–191
1000
500–370
650–600
320–250–220–200
750–450
310–210–180–150–100
700–400
550–450
700–600
Fragment
pattern 3
320–250–200–110
700–450
400–250–200–150
900
480–460
600–550
A. brevispiculata
(KC342886–KC342888)
A. typica
(KC342889–KC342891;
KC852163)
Anisakis sp.
(KC852164–KC852171)
A. brevispiculata
(KC342899–KC342901)
A. typica
(KC342897–KC342898;
KC821728–KC821729)
A. ziphidarum-like c
(KC821732–
KC821738)
A. paggiae-like d
(KC342895–KC342896;
KC821730–KC821731)
Morphotype 2
Species identity (GenBank Acc. Nos.)
a
b
c
d
Morphotype 1, blunt tail; Morphotype 2, conical tail.
The estimated fragment size is based on the band size after electrophoresis.
Anisakis species that are genetically close to A. ziphidarum based on mtDNA cox2 region.
Anisakis species that are genetically close to A. paggiae based on mtDNA cox2 region.
Table 2
Pairwise comparison on the genetic distances (Kimura 2-parameters (K2P) mode; complete deletion; MEGA5) and number of nucleotide base differences (in parentheses) in the ITS region between Anisakis
species from the current study with Anisakis species deposited from GenBank.
Species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
GenBank Acc. No.
1. Anisakis sp.
2. A. brevispiculata
3. A. typica
4. A. paggiae
5. A. brevispiculata
6. A. physeteris
7. A. typica
8. A. simplex s.s.
9. A. pegreffii
10. A. simplex C
11. A. ziphidarum
12. Anisakis sp.
13. Anisakis sp.
14. Anisakis sp.
–
0.104
0.171
0.097
0.100
0.120
0.175
0.148
0.150
0.148
0.145
0.000
0.000
0.001
(66)
–
0.194
0.050
0.003
0.033
0.198
0.142
0.144
0.147
0.148
0.104
0.104
0.105
(104)
(116)
–
0.190
0.194
0.198
0.006
0.157
0.159
0.155
0.139
0.171
0.171
0.171
(62)
(33)
(114)
–
0.047
0.058
0.193
0.144
0.142
0.147
0.148
0.097
0.097
0.098
(64)
(2)
(116)
(31)
–
0.036
0.198
0.146
0.147
0.151
0.151
0.100
0.100
0.102
(75)
(22)
(118)
(38)
(24)
–
0.202
0.148
0.146
0.153
0.157
0.120
0.120
0.122
(106)
(118)
(4)
(116)
(118)
(120)
–
0.161
0.163
0.159
0.143
0.175
0.175
0.175
(91)
(88)
(97)
(89)
(90)
(91)
(99)
–
0.001
0.004
0.048
0.148
0.148
0.150
(92)
(89)
(98)
(88)
(91)
(90)
(100)
(1)
–
0.006
0.047
0.150
0.150
0.151
(91)
(91)
(96)
(91)
(93)
(94)
(98)
(3)
(4)
–
0.048
0.148
0.148
0.149
(89)
(91)
(87)
(91)
(93)
(96)
(89)
(32)
(31)
(32)
–
0.145
0.145
0.145
(0)
(66)
(104)
(62)
(64)
(75)
(106)
(91)
(92)
(91)
(89)
–
0.000
0.001
(0)
(66)
(104)
(62)
(64)
(75)
(106)
(91)
(92)
(91)
(89)
(0)
–
0.001
(1)
(67)
(104)
(63)
(65)
(76)
(106)
(92)
(93)
(92)
(89)
(1)
(1)
–
This study
This study
This study
GU295976
EU624344
AB592792
EU346093
EU718471
GQ131688
AY821739
EU718473
EU718474
KC121370
JN005761
K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
Anisakis sp.
(KC342892–KC342894)
EU560910
DQ116430
DQ116433
JQ934884
DQ116432
EU560911
DQ116428
DQ116429
GQ118165
(11)
(3)
(18)
(6)
(13)
(10)
(10)
(11)
–
(15)
(10)
(21)
(15)
(15)
(3)
(3)
–
0.053
a
These are the specimens (KC821732–KC821734, KC821736) within the sub-branch 1 of A. ziphidarum branch in the NJ tree.
These are the specimens (KC821735, KC821737, KC821738) within the sub-branch 2 of A. ziphidarum branch in the NJ tree.
5. Conclusion
b
(12)
(9)
(14)
(14)
(13)
–
0.024
0.018
0.060
(10)
(11)
(9)
(13)
–
0.078
0.068
0.073
0.063
(11)
(7)
(18)
–
0.063
0.084
0.068
0.073
0.029
(13)
(18)
–
0.087
0.044
0.084
0.107
0.102
0.087
(9)
–
0.087
0.034
0.053
0.054
0.044
0.049
0.015
–
0.046
0.066
0.056
0.051
0.072
0.082
0.077
0.056
0.047–0.054
0.077–0.081
0.010–0.020
0.077–0.081
0.031–0.041
0.078–0.084
0.097–0.102
0.092–0.097
0.077–0.081
0.050–0.055
0.006–0.011
0.073–0.077
0.045–0.049
0.050–0.055
0.048–0.054
0.045–0.049
0.050–0.055
0.022–0.027
0.047–0.060
0.000–0.011
0.086–0.093
0.036–0.049
0.046–0.059
0.054
0.036–0.049
0.041–0.054
0.015–0.027
0.005
0.041
0.066–0.067
0.061–0.062
0.056–0.057
0.066
0.077
0.071–0.072
0.051–0.052
6. A. paggiae
7. A. ziphidarum
8. A. brevispiculata
9. A. typica
10. A. physeteris
11. A. simplex s.s.
12. A. pegreffii
13. A. simplex C
14. A. nascettii
229
been reported on definitive hosts under the family Ziphiidae (Mesoplodon densirostris, M. europaeus, M. layardii,
and Ziphius cavirostris) from South African coast, Carribean
Sea, Mediterranean Sea, Gulf of Mexico and New Zealand
(Mattiucci and Nascetti, 2008, 2007; Colón-Llavina et al.,
2009; Mattiucci et al., 2009; Cavallero et al., 2011). Up to
date, A. ziphidarum has never been reported in any kogiid
whales. Hence, the identification of Anisakis species that are
genetically close to A. ziphidarum from the current study
could represents a new definitive host record of the possible local variation or sibling species of this species. Finally,
in view of human food safety, it would be necessary to
look closely on the intensity and diversity of such Anisakis
infection to various marine fishes and cephalopods within
the Philippine waters to have a clear understanding on
what fish species and from what locality should be taken
in consideration particularly in preparing dishes that may
transfer such infection to consumers.
(16)
(9)
(22)
(14)
(14)
(4)
–
0.015
0.049
This study
(6–7)
(15–16)
(14–15)
(13–14)
(11–12)
0.036–0.055
0.062–0.068
5. A. typica
0.076–0.082
1. A. paggiae–like
2. A.
ziphidarum–like a
3. A.
ziphidarum–like b
4. A. brevispiculata
0.045–0.049
0.056–0.059
0.044–0.054
0.000–0.005
(0–1)
0.058–0.063
0.036–0.042
0.088–0.095
0.000–0.005
0.067–0.074
0.084–0.090
0.073–0.079
0.078–0.084
0.031–0.037
(7–8)
(17–18)
(0–1)
(14–15)
This study
(15)
(18)
(13–14)
(6–8)
(9–10)
(14–16)
0.000–0.010
(1–2)
0.073–0.086
(15)
(2–4)
(15)
(19)
This study
(4–5)
(9–10)
(8–9)
(9–10)
(9–10)
(8–10)
(12–14)
0.000–0.006
(0–1)
0.067–0.077
(1–2)
(13–14)
(8–9)
(8–9)
This study
This study
(10)
(3–5)
(14)
(8–10)
(11)
(9)
(11)
(9–11)
(8–10)
0.000–0.011
(0–2)
0.006–0.011
0
0.041–0.055
(1)
(9–11)
(12–13)
(7–10)
(11)
(14–15)
(8–9)
(1–2)
(8)
(0–2)
(13)
(17)
(12)
(7–9)
(15)
(7–9)
GenBank
Acc. No.
14
13
12
11
10
9
8
7
6
5
4
3
2
1
Species
Table 3
Pairwise comparison on the genetic distances (p-distance; pairwise deletion; MEGA5) and number of amino acid differences (in parentheses) in the mtDNA cox2 region between Anisakis species from the current
study with Anisakis species deposited from GenBank.
K.M.A. Quiazon et al. / Veterinary Parasitology 197 (2013) 221–230
We report a new geographical record for both A. brevispiculata and A. typica, including the two unknown
Anisakis species that are genetically close to A. paggiae and
A. ziphidarum, off the Pacific coast of the southern Philippine archipelago. Isolation of different Anisakis species
in the Philippine archipelago indicates Anisakis distribution among paratenic hosts (i.e., fishes and cephalopods).
Further extensive studies from fishes, cephalopods and
cetaceans within Philippine waters are needed in order to
clarify the possible occurrence of other Anisakis species, and
to determine if the observed genetic variations in the ITS
and mtDNA cox2 regions represent normal intra-species
variation or these are a characteristic of a possible existence
of local variation or sibling species, particularly on the two
Anisakis species that are genetically close to A. paggiae and
A. ziphidarum.
Acknowledgments
The authors wish to thank Darrell D. Blatchley, Director and Curator of the D’ Bone Collector Museum, Davao
City, Philippines for collecting the worm samples from the
stranded Dwarf Sperm Whale, and Joanne Krisha M. Lacsamana, Research Assistant, NFRDI for pre-processing and
sending the worm samples to Japan. The authors would
also like to thank Bureau of Fisheries and Aquatic Resources
– Regional Office 11 (Philippines) for facilitating transport and documentation of samples used in the study. This
study was partly supported by JSPS (Japan Society for the
Promotion of Science) Postdoctoral Fellowship for Foreign
Researchers and a Grant-in-Aid for JSPF fellows (23-01405).
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