1

INTRODUCTION

Background of the Study

Parasites are organisms that live on or in an organism of another species known

as the host from where nutrients are obtained and gain benefits at the expense of the host

(http://www.encyclopedia.com). Some parasites are responsible for the outbreaks of

diseases in fish populations and few (e.g., anisakid nematodes) are capable of infecting

humans (Barber and Poulin, 2002). Parasites can economically devaluate fish market

value as it can cause spoilage and it cause human health risks (Adams et al., 1997). Also,

parasites influence individual host survival and reproduction, alter fish behavior and

migration patterns, and even regulate fish populations and affect fish community

structure (Garnick and Margolis, 1990; Barber and Poulin, 2002). On the other hand,

prevalence of parasites in one habitat is indicative of the health of an ecosystem (Adams

et al., 1997; Hudson et al., 2006) and determinant on the status of fish stocks (Barber and

Poulin, 2002).

Nematode larvae of the genus Anisakis (Nematoda: Anisakidae) are common

parasites of marine fish with a worldwide distribution (Setyobudi et al., 2011). These

nematodes have a complex life cycle involving organisms at various levels of a trophic

web in the marine ecosystem (Costa et al., 2003). Akbar and Ghosh, (2005) added that

fish and squid are the intermediate or paratenic hosts of the parasites, whereas marine

mammals mainly cetaceans are the definitive ones. In the neighboring countries of the

Philippines such as in Indonesia and Thailand, there have been reports of A. simplex

sensu stricto (s.s.), A. typica, and possible local variation or sibling species of A. typica
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from marine fishes (Moravec et al., 2006; Chen et al., 2008; Palm et al., 2008). In the

Philippines alone, two (2) Anisakis species (i.e., A. typica, A. brevispiculata) and two (2)

unknown Anisakis genetically close to A. paggiae and A. ziphidarum, have been reported

from its final host Dwarf Sperm Whale stranded in southern Philippines (Quiazon et al.,

2013).

The information on the food habits of marine fishes, such as the predator-prey

relationship, which is useful in order to assess the role of marine fishes in the ecosystem

(Duffu and Hay, 2001), has encouraged the author to perform parasitological studies.

Additions to this are the limited studies on Anisakis infection in Philippine waters,

particularly in the southern Luzon island.

The aim of this research was to determine the prevalence, abundance and intensity

of third-stage larva (L3) of Anisakis worms infecting marine fishes at the southern part of

Luzon, specifically in Tayabas Bay. The collected data provided some information on the

infection levels on each fish species that can be used as guide during fish handling and

processing, as well as possibility for use as biological tag for fish stocks identification.

Thus, understanding of the parasites distribution and specific site of infection may be

used as biological indicators for several ecological studies.

Statement of the Problem

Marine fishes constitute an important food source for human consumption. It has a

high demand in the market because it is affordable to consumers (Roberts et al., 2001).

However, these fishes are susceptible to damages caused by parasites; thus, securing its

quality is very important (https://en.wikipedia.org/).

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Anisakis is usually present in the final host species including cetaceans (dolphins

and whales) and pinnipeds (walrus and sea lion). However, they can also be found in

several marine fishes serving as their intermediate hosts such as splendid ponyfish

(Eubleekeria splenders), longfin grouper (Epinephelus quoyanus), one-spot snapper

(Lutjanus monostigma), purple-spotted big eye (Priacanthus tayenus) and jarbua terapon

(Terapon jarbua), all of which are consumed by many Filipinos in raw or processed

forms. There are actually no known reports on the common parasites present in marine

fishes caught within Tayabas Bay, Quezon province. Thus, no scientific basis can be

made available to quantify the present quality or condition of fishes landed in this area.

Significance of the Study

The identification of parasitic nematodes in fishes, particularly that of the

zoonotic anisakid nematodes, will be a great help in assessing the present quality of these

marine fish species in Philippine waters, which also concerns possible human health

risks. Moreover, the use of these parasites as biological indicators for fish stock

assessment will be important for the possible management of this fish stocks in Tayabas

Bay benefiting not only the Bureau of Fisheries and Aquatic Resources (BFAR) for the

promotion of proper and sustainable marine capture fisheries, but mainly our local fisher

folks. With the increasing interests in fish production, parasitic infestations are becoming

threats for fish health management. Thus, the outcome of this study could also be a basis

of future researchers.
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Objectives of the Study

The main objective of the study was to identify the parasitic nematodes,

specifically focusing on anisakid nematodes infecting ten different marine species bought

at the Dalahican Fish Port Complex, Lucena City, Quezon province.

Specifically, the study aims to:

a) identify at the genus level (if possible up to species level), the anisakid

nematodes infecting ten different marine species bought at the Dalahican Fish

Port Complex, Lucena City, Quezon,

b) determine the prevalence, abundance and intensity of Anisakis infection, and;

c) correlate the length and weight of the fishes with the number of anisakid

nematode infection.

Scope and Limitation of the Study

Fishes, like all living organisms, can be infected with various parasites. Parasites

that live internally in the host are endoparasites, thus, it cannot be studied without

knowing the hosts in which they live (Roberts and Janovy, 2009). This study focused

mainly on the identification, counting and grouping of Anisakis spp. isolated in the

visceral organs of ten different marine species bought at Dalahican Fish Port Complex,

Lucena City, Quezon Province. To ensure that fishes bought at the Dalahican Fish Port

Complex in Lucena City was strictly caught within Tayabas Bay, the fish samples were

collected by the fishermen residing nearby and assisted by the local workers from BFAR

in Quezon province. In the identification of the anisakid nematodes, taxonomical keys

 5

previously reported (Koyama et al., 1969; Davey, 1971; Quiazon et al., 2008; Murata et

al., 2011; Quiazon et al., 2013) was used through morphological examinations only.

Time and Place of the Study

This study was conducted from July to August 2015. Ten marine fish species

were purchased at the Dalahican Fish Port Complex in Lucena City. A total of four

hundred fish samples were dissected and brought at Freshwater Aquaculture Center,

Central Luzon State University for the isolation and examination of fish endoparasites

from the visceral organs.

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REVIEW OF RELATED LITERATURE

Fish Parasitism

Parasitism is a symbiotic relationship between two organisms in which the

parasite, depends on the host, and either harms or lives on the host (Rohde, 2005).

Parasites may cause mechanical injury, such as boring a hole into the host or digging into

its skin or other tissues, stimulate a damaging inflammatory or immune response (Rohde,

2005). Most parasites inflict a combination of these conditions on their hosts (Taliaferro,

2009). This relationship also plays a significant role in fish biology and ecology (Hudson

et al., 2006; Luque and Poulin, 2008). Also, parasite infections on fish are a usual

ecological event because parasites are natural component of the aquatic environment and

fish serves as important part of their life cycle (Barber and Poulin, 2002).

Parasite is an organism that lives in or on another organism (its host) and benefits

by deriving nutrients at the host's expense (https://frankensteinfanclub.wordpress.com).

According to Yeh (2002), it is different from predators and parasitoids (which also derive

benefits from certain inter-specific interactions while harming the other participant) in

that the host of a parasite is not necessarily killed instead it only derives benefits from

their host. Though, it also often causes important disease to animals and humans which

may cause death to its host (Bogitsh et al., 2005). These authors shown the conspicuous

role in parasites on fish marine in reserves; Morand and Arias-Gonzalez (1997),

accompanied by Sasal et al. (1996) discussed parasitism and diseases are important

factors affecting the viability of natural populations and communities thus, in the marine
 7

environment, it has been demonstrated that individual fish may suffer from parasitic

attacks.

According to Alves and Luque (2001), the feeding habits and the wide diet

spectrum of fishes puts them into contact with several potential intermediate hosts of

parasites. This might be increasing the presence of endoparasites in these fishes (Alves

and Luque, 2001). The diet of the host species is the main factor affecting the parasite

community structure, especially for the trematodes, acanthocephalans and nematodes

transmitted to their host through a predator-prey relationship (Sasal et al., 1999). Parasite

species might accumulate along food chain (Aho and Bush, 1993).

Endoparasite

Endoparasites are parasites that are located within the flesh and cavities of the

host organism (Roberts and Janovy, 2005). These parasites can be found within the

muscles, organs, and membrane linings of the cavities of the host organism (Hoffman,

1998). Thus, the increased potential for infecting humans with a fish parasite is more

common for endoparasites since tapeworms and roundworms can be passed to humans

who eat undercooked or raw meat (Hoffman, 1998).

Dailey 2001 reported that endoparasites can cause various pathological disorders

in marine mammals. Thus digestive system parasites can cause internal bleeding,

ulceration, obstruction and malnutrition (Dailey, 2001).

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Nematodes

The most commonly observed parasites in marine food fishes are roundworms

called nematodes (Hilderbrand et al., 1985). According to Petersen et al. (1993), parasites

transferred by marine fish to human by consumption of poorly cooked infected fish are

found rarely among the acanthocephalans (Bolbosoma sp.), occasionally among the

digeneans (Diplogonoporus spp.) and more frequently among the nematodes (Anisakis

sp.).

Larval stages of anisakid nematodes (super family: Ascaridioidea, Family:

Anisakidae) of some genera such as Anisakis, Contracaecum, Pseudoterranova, and

Hysterothylacium are commonly found in the viscera and musculature of many species of

teleost fish (Costa et al., 2003). Since they are swallowed when fish eat their prey, which

are the intermediate hosts of these worms that are encapsulated in viscera or muscles

(Smith, 1983; Mattiucci et al., 1997) and can infect humans causing significant clinical

disease (i.e. anisakiasis) in a number of countries (Zhou et al., 2008).

Anisakis spp.

The nematodes of the genus Anisakis Dujardin, 1845 (Family Anisakidae) are

parasites of the alimentary tract of aquatic vertebrates with worldwide distribution

(Mattiucci and Nascetti, 2008) (Fig. 1). These parasites can also be found in cephalopods

and act as paratenic or transport hosts (Eissa, 2002; Choi et al., 2011; Tantanasi et al.,

2012).

There are currently nine species recognized under the genus Anisakis namely: A.

simplex s.s., A. pegreffii, A. berlandi, A. typica, A. ziphidarum, A. nascettii, A. physeteris,

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A. brevispiculata and A. paggiae. According to Mattiucci and Nascetti (2006), there are

two main clusters that exist separating these nine Anisakis species. The first clade (or

Anisakis type 1 group) includes A. simplex s.s., A. pegreffii, A. typica, A. ziphidarum, and

A. berlandi, while the second clade include A. physeteris, A. brevispiculata and A.

paggiae.

Figure 1. Anisakis spp. (Source: http://www.foodpoisoningtreatment.org)

Life Cycle of Anisakis

Adult stages of Anisakis simplex or Pseudoterranova decipiens reside in the

stomach of marine mammals, where they are embedded in the mucosa, in clusters

(http://www.cdc.gov). Unembryonated eggs produced by adult females are passed in the

feces of marine mammals (http://www.cdc.gov). The eggs become embryonated in water,

and first-stage larvae are formed in the eggs (http://www.cdc.gov). The larvae molt,
 10

becoming second-stage larvae (2a), and after the larvae hatch from the eggs, they become

free-swimming (2b). Larvae released from the eggs are ingested by crustaceans

(http://www.cdc.gov). The ingested larvae develop into third-stage larvae that are

infective to fish and squid (http://www.cdc.gov). The larvae migrate from the intestine to

the tissues in the peritoneal cavity and grow up to 3 cm in length (http://www.cdc.gov).

Upon the host's death, larvae migrate to the muscle tissues, and through predation, the

larvae are transferred from fish to fish (http://www.cdc.gov). Fish and squid maintain

third-stage larvae that are infective to humans and marine mammals (5)

(http://www.cdc.gov). When fish or squid containing third-stage larvae are ingested by

marine mammals, the larvae molt twice and develop into adult worms

(http://www.cdc.gov) The adult females produce eggs that are shed by marine mammals

(6) (http://www.cdc.gov). Humans become infected by eating raw or undercooked

infected marine fish (7) (http://www.cdc.gov). After ingestion, the anisakid larvae

penetrate the gastric and intestinal mucosa, causing the symptoms of anisakiasis

(http://www.cdc.gov).

The life cycle of these parasites involves various hosts at different levels in food

webs (Mattiucci and Nascetti, 2008) (Figure 2). According to Audicana and Kennedy

(2008), commercially important species which serves as hosts of Anisakis spp. include

herring (Clupea spp.), cod (Gadus spp.), salmon (Oncorhynchus spp.), mackerel

(Scomber spp.) and anchovy (Engraulis spp.). The infective L3 of Anisakis are common

in commercially important marine fishes and its presence is of great concern for both

public health and economic reasons (Choi et al., 2011; Tantanasi et al., 2012). The
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infective larval stage of A. simplex is the most important etiologic agent of anisakidiosis

in humans (Choi et al., 2011).

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Figure 2. Life cycle of Anisakis including accidental human hosts

(Source: Audicana and Kennedy, 2008).

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Anisakis as Biological Indicators

Parasites have been successfully used as tools to understand several aspects of

their hosts biology, namely; identify and discriminate fish stocks (Timi, 2007; Santos et

al., 2009), study population structures (Pozdnyakov and Vasilenko, 1994) and clarify

taxonomic relations (Luque and Poulin, 2008; Oliva et al., 2008). The use of parasites

with these purposes is based on the fact that the host can only become infected with a

particular parasite species within the endemic area of that parasite (MacKenzie and

Abaunza, 1998). However, not all parasites can be useful as biological indicators. In

order to be a good one, there are certain criteria that should be met according to

MacKenzie and Abaunza (1998) and Rohde (2005) and these are: (a) the parasite should

have significantly different levels of infection in different parts of the study area. (b) the

parasite should have a long life span in the target host, at least long enough to cover the

time scale of the investigation (Williams et al., 1992); (c) parasites with direct single-host

life cycles are easier to use as tags, but those with two or more hosts can be useful, since

there is information on biotic and abiotic factors influencing parasites transmission

(MacKenzie and Abaunza, 1998; Rohde, 2005); (d) the parasite should have constant

levels of infection from year to year (Rohde, 2005); (e) the parasite should be easily

detected and identified, and the examination of the host should involve the minimum of

dissection (MacKenzie and Abaunza, 1998; Rohde, 2005); and (f) the parasite should not

cause high pathogenicity in the host nor changes in host behaviour (Williams et al., 1992,

MacKenzie and Abaunza, 1998).

Several authors have consistently highlighted the utility of parasitological surveys

in understanding and differentiating fish populations, and have successfully demonstrated

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that indeed parasites are good biological indicators for fish populations (MacKenzie,

2002; Hemmingsen et al., 1991; George-Nascimento and Arancibia, 1992; Williams et

al., 1992; Santos et al., 2009).

Scientists are studying Anisakis because of its potential as a biological indicator of

fish stocks. In previous decades, marine parasites (anisakids) have been broadly used in

biological and ecological surveys of marine ecosystems as biological indicators (Table 1).

Table 1. Uses of Anisakis spp. as biological indicator

Concerns Reference

Food Chain Structure Thompson et al., 2005

Pollution Sures, 2004

Global Climate Changes Brooks and Hoberg, 2007

Poulin, 2006

Anthropogenic Impacts and Environmental Stresses Marcogliese, 2005

Fish Stock Assessment MacKenzie, 2002

Lloret et al., 2012

General Ecosystem Health Marcogliese, 2005

Biological Indicators for Fish Stock Assessments

One of the major goals from the last decade in the study of the parasite fauna of

aquatic organisms has been, among the others, the assessment of fish stocks, their

movement, and recruitment (Quiazon, 2015).

A parasite can be used as suitable biological tag for fish stock identification when

its geographical distribution and life-cycle are known, and when the parasites residence
 15

time in the host is long enough compared to the life span of the fish host (Quiazon, 2015).

In this sense, the parasite as biological indicator reflects the geographic origin of the fish

population on a spatial scale (Quiazon, 2015). Indeed, the genetic or molecular markers

define the stock on the evolutionary temporal scale, while the parasite taxa characterize

the stock on a spatial scale (Mattiucci et al., in press).

Anisakid Nematode (Anisakis) for Stock Assessment

Infection by anisakids can affect the commercial value of fish, particularly when

larvae are located in the musculature and thus represent some economical loss for the

fisheries industry (Angot and Brasseur, 1995). The worms in flesh reduce the market

value of fish, and thus represent some economical loss of the fisheries industry (Angot

and Brasseur, 1995). The Anisakis found in fish may cause the most severe problems for

human health causing human anisakiasis. Humans can acquire this disease by eating fish

subject to little heat treatment or fish that is smoked, soaked in vinegar, pickled with

spices and other raw fish specialties (Petersen et al., 1993; Beran and Steel, 1994).

Moller (1991) stated that the fish inhabiting nematode stages show a remarkable

resistance to low temperature, but are destroyed readily by heating to 70C or freezing at

-20C for more than 24 hours. The time to kill the nematodes by marinating depends on

the degree of acidity, temperature and salt addition (Moller, 1991). It produces severe

lesions in human stomach and is associated with gastric neoplasia (Mattiucci et al.,

1997). If larvae are loose or attached to digestive tract, they may produce irritation,

inflammation and ulceration (Mattiucci et al., 1997).

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Anisakid parasites are an integral part of aquatic ecosystems as they play key

roles in population dynamics and community structure and can provide important

information about the general biodiversity of the ecosystem (Quiazon, 2015). Recent data

on the possible use of anisakid nematodes have been presented as biological indicators

of: a) the definition of fish stocks within a multidisciplinary approach, b) integrity and

stability of trophic webs, and c) habitat disturbance (Mattiucci et al., 2008). Among the

parasite species that have been used in fish stock definition is the larval anisakid

nematodes of the genus Anisakis which represent one of the best biological indicators

(MacKenzie, 2002).
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MATERIALS AND METHODS

Description of the Dalahican Fish Port Complex

One of the important fish landing sites in Quezon province is in Dalahican, a

small town located at the north western part of Tayabas Bay (Figure 3). The Dalahican

Fish Port Complex located in Dalahican was built along the fishing village of Brgy.

Talao-Talao, (Figure 4) (http://www.pdosoluz.com.ph). The Port of Lucena is about 140

kilometres southeast of Manila located on coordinates of 135413 north latitude and

1213736 east longitude (http://www.skyscrapercity.com).

The strategic location of the Dalahican Fish Port Complex and its proximity to

some of the major fishing grounds in the Philippines such the Tayabas Bay, Ragay Gulf

and other fishing grounds contiguous to the southern Tagalog region, have made the port

a major fish landing center of the province (http://www.pfda.da.gov.ph).

According to Trinidad et al. (1993), fish landing and trading usually start at 4:00

a.m. and last for four to five hours. Selling of fish is usually carried out by brokers who

are stationed in the area through whisper bidding. Moreover, brokers who sell fish in

behalf of fish producers get a commission in the gross sales (Trinidad et al., 1993).
 18

Figure 3. The map of Tayabas Bay (Source: https: //www.hubmaps.com)

Figure 4. Dalahican Fish Port Complex (Source: http://farm8.staticflickr.com)

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Research Design

This study was a descriptive type of a research. It was conducted from July to

August, 2015. The researcher randomly collected at least 10 samples of marine fishes for

single sampling. Sample collection started from 3:00-5:00 AM at the landing-places at

Dalahican Fish Port, the major fishing harbour in Tayabas Bay. The 400 number of

purchased fish samples were examined for the prevalence, abundance and mean intensity

of infection.

Collection of Fish Samples

A total of 400 fish individuals were randomly collected as samples during July

2015. BFAR personnel from Region 4-A identified the ten different marine species

(http://www.fishbase.org/) as follows; one-spot snapper (Lutjanus monostigma), striped

ponyfish (Eubleekeria splendens), splendid ponyfish (Aurigequula fasciata), longfin

grouper (Epinephelus quoyanus), redspine threadfin bream (Nemipterus nemurus),

purple-spotted bigeye (Priacanthus tayenus), spangled emperor (Lethrinus nebulosus),

jarbua terapon (Terapon jarbua), checkered snapper (Lutjanus decussates) and chocolate

hind (Cephalopholis boenak).

Dissection of Fish Samples

The collected fish samples were immediately documented via digital images. All

fish samples were stored in an ice box prior to travel at FAC, CLSU, Munoz, Nueva Ecija

for dissection of fish, and collection and identification of parasites. Fish samples were

dissected at the Fish Pathology laboratory. Each fish sample was weighed and total body

length measured prior to dissection.

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The fish samples were then is measured in centimetres (cm) for their total length

from the tip of the snout to the longest caudal ray and the wet weight to the nearest gram

(g). The sex of the fish (if possible) was also recorded. A compound microscope was

used in the identification of parasites up to genera level (if possible, up to species level).

Parasite Collection and Fixation

Parasite collections were conducted on the body cavity of each fish. The whole

body cavity and the viscera of each sample were carefully dissected and thoroughly

examined for presence of anisakids. The anisakid nematodes were collected from the

tissue outside the stomach and visceral organ of the fish samples. After opening, the

viscera were removed by cutting the oesophagus at the anterior end until the intestine at

the vent. The small intestines were placed immediately in a cylindrical tubes containing

Natural Saline Solution then transferred in petri plate for the collection of parasites. Most

larvae collected were examined directly under a light microscope but some anisakids

were fixed in 70% ethanol for morphological analysis under microscope.

For identification of species, each (ethanol-fixed) 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 cleared in glycerin solution for 24-48 hours

prior to mounting in slides and examined under light microscope at different

magnifications for the initial parasite identification using morphological keys (Koyama et

al., 1969; Davey, 1971; Quiazon et al., 2008; Murata et al., 2011; Quiazon et al., 2013).

Anisakid nematodes were identified up to genus level based on morphological

 21

characteristics as previously reported (Hurst, 1984; Navone et al., 1998; Hugot et al.,

1991) (Fig. 5).

Figure 5. Morphological features of anisakid nematodes (Sources: Hurst

(1984); Navone et al. (1998) and Hugot et al. (1991))

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Light Microscope Examination

A total of 178 worms were examined by the aide of light microscope. The parasites

were examined at Fish Pathology Laboratory, College of Fisheries, Central Luzon State

University. Characterization of different Anisakis species using morphological cues were

assessed through ventriculus length (i.e., Anisakis type I group has 0.5-1.5 mm

ventricular length, while Anisakis type II group has ventricular length of below 0.5 mm.).

A schematic drawing was made using Corel Draw Version 12 software on the anterior

and posterior ends of the worms containing the vital keys for morphological examination

(Fig. 6).

Figure 6. Diagram of morphology of Anisakis sp. (A) and Pseudoterranova sp.

(B). Anterior portion of the worms showing the boring tooth (BT),

esophagus (E), ventriculus (V), cecum (C), and intestine (I).

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Anisakis sp. (sensu lato) has a simple digestive tube, esophagus-ventriculus-

intestine (Fig. 6a), while Pseudoterranova sp. (sensu lato) has esophagus-ventriculus-

intestine with anteriorly directed cecum (Fig. 6b); (vi) total body length of Anisakis

ranges from 10 to 29 mm and 0.44 to 0.54 mm in width, while Pseudoterranova has 25 to

50 mm in body length and 0.3 to 1.2 mm in body width.

Identification of Anisakis Type Groupings

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).

Morphologically, Anisakis spp. were identified based on the following morphological

characters: (1) the shape and the presence of the boring tooth, (2) the shape of the tail, (3)

the presence of mucro, and (4) the shape of ventriculus (Chai et al., 1986; Sohn et al.,

1983). Berland (1961) stated that Anisakis spp. could only be categorized

morphologically into Anisakis type I and type II groups, in which the former has a longer

ventriculus and a mucro, while the latter has short ventriculus and no mucro.

Taxonomic keys for nematodes were used to confirm the morphological

classification, as well as to determine the larval stages of Anisakis spp. Recent studies

showed that molecular diagnostic techniques could be used to identify Anisakis to a

species level (Abe et al., 2006). PCR-RFLP has been widely used to

identify Anisakis spp. in different fish species (Abe et al., 2006; Pontes et al., 2005;

Quiazon et al., 2009; Setyobudi et al., 2011; Umehara et al., 2010). However, this study
 24

only focused on the morphological identification of anisakid nematode, particularly on

the type group of those identified in the genus Anisakis.

Molecularly, the Anisakis type I group is known to consist of 6 species (A.

ziphidarum, A. nascettii, A. typica, and 3 sibling species of A. simplex complex,

namely A. simplex s.s., A. pegreffii, and A. berlandi, whereas Anisakis type II group

consists of 3 species (A. paggiae, A. brevispiculata, and A. physeteris) (Mattiucci et al.,

2008; Mattiucci et al., 2009).Recent studies in Japan showed that L3, L4, and adult of A.

simplex (s. s) and A. pegreffii, both belonging to type I group, could be distinguished

morphologically based on the ventriculus length, wherein the former has a longer

ventriculus length (i.e., 0.90-1.50 mm) than the latter (0.50-0.78 mm) (Quiazon et al.,

2008).

Statistical Analysis

The prevalence, mean intensity and abundance of parasites present in different

marine fishes was calculated using the following formula proposed by Margolis et al.

(1982) and Bush et al. (1997):

umber of infected fish

Prevalence = x 100
otal fish e amined

umber of parasites
Intensity = nfected fish

number of lar ae detected

Abundance = umber of fish e amined

umber of lar ae detected

Infection rate x 100
umber of fish e amined
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Relationship between the length and weight of the fish with the number of

parasites observed were computed using Paleontological Statistics Version 7.0 and was

correlated using Sigma plot Version 10.0.

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RESULTS AND DISCUSSION

Anisakis spp. were categorized into Anisakis type I and type II based on the size

of its ventriculus and the presence of the mucro. A total of 101 out of 178 worms from

the fish samples were morphologically identified as Anisakis type I. Three species of

anisakids were identified; Anisakis simplex s.s, Anisakis typica and Pseudoterranova sp.

(Figure 7). Philometrid nematode was also classified.

A B

Figure 7. Microscopic images of the parasites: a) Anisakis typica.,b) Pseudoterranova

sp. Ventriculus c) Anisakis sp. Posterior (V=ventriculus; M=mucro; A=anus)

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In terms of parasitic worm infection, Table 1 shows the highest prevalence of

infection was found in jarbua terapon (100%), followed by longfin grouper (58.33%),

striped ponyfish (50%), purple-spotted bigeye (33.33%), spot snapper (27.27%) and

splendid ponyfish (15.94%).

Table 1. Prevalence of larval anisakid parasites in the examined fish species

Fish species No. of fish No. of infected Prevalence

examined fish %
Splendid ponyfish 69 11 15.94

Striped ponyfish 4 2 50

Longfin grouper 12 7 58.33

One-spot snapper 11 3 27.27

Redspine threadfin bream 9 0 0

Purple-spotted bigeye 3 1 33.33

Spangled emperor 5 0 0

Chocolate hind 1 0 0

Checkered snapper 1 0 0

Jarbua terapon 1 1 100

Sardinella fimbriata 137 0 0

Sardinella fimbriata 146 0 0

Total 399 25 47.48

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The mean intensity and highest rate of abundance were 37.71 and 11 larvae,

respectively, per fish (Table 2). Results showed the highest intensity rate was found in

longfin grouper (14.57 %), followed by jarbua terapon with 12 %. Low intensity rate

were found in splendid and striped ponyfish by (4.64 %) and (2.5 %), respectively.

Table 2. Intensity and abundance of larval anisakid parasites in the examined fish species

Fish species No. of fish No. of infected Intensity Abundance

examined fish (%) (%)
Splendid ponyfish 69 11 4.64 0.072
Striped ponyfish 4 2 2.5 0
Longfin grouper 12 7 14.57 6.67
One-spot snapper 11 3 2 0.36
Red spine threadfin bream 9 0 0 0
Purple-spotted bigeye 3 1 2 0.33
Spangled emperor 5 0 0 0
Chocolate hind 1 0 0 0
Checkered snapper 1 0 0 0
Jarbua terapon 1 1 12 11
Sardinella fimbriata 137 0 0 0
Sardinella fimbriata 146 0 0 0
Total 399 25 37.71 18.44
MEAN 6.28 3.69
 29

Infection status of parasitic worms in fish purchased from the Cooperative fish

market in Dalahican Fish Port Complex, Lucena City, Quezon is shown in Table 3,

whereas the infection status of the identified Anisakis spp. in fish examined is shown in

Table 4.

With respect to Anisakis infection, the overall infection rate was 37.55%, with

mean intensity of 7.3. Anisakis infection was observed only on five fish species, namely;

Epinephelus quoyanus, Lutjanus monostigma, Priacanthus tayenus, Eubleekeria

splenders and Jarbua terapon.

 30

Table 3. Summary of infection status of parasites in different marine species purchased in Dalahican Fish Port Complex

Fish Samples Total No. Total No. of Parasites Total No. of Prevalence Mean Abundance
of Samples Infected Intensity
Anisakis Philometra Unidentified TOTAL (%)
Fish (%)
spp. spp.
Splendid ponyfish 69 5 46 0 51 11 15.94 4.64 0.072
Striped ponyfish 4 0 5 0 5 2 50 2.5 0
Longfin grouper 12 80 17 5 102 7 58.33 14.57 6.67
One-spot snapper 11 4 2 0 6 3 27.27 2 0.36
Redspine threadfin bream 9 0 0 0 0 0 0 0 0
Purple-spotted bigeye 3 1 1 0 2 1 33.33 2 0.33
Spangled emperor 5 0 0 0 0 0 0 0 0
Chocolate hind 1 0 0 0 0 0 0 0 0
Checkered snapper 1 0 0 0 0 0 0 0 0
Jarbua terapon 1 11 1 0 12 1 100 12 11
Sardinella fimbriata 137 0 0 0 0 0 0 0 0
Sardinella fimbriata 146 0 0 0 0 0 0 0 0
Total 399 101 72 5 178 25 47.48 37.71 0.25

MEAN 6.28
 31

Table 4. Infection status of Anisakis spp. in fish purchased in Dalahican Fish Port Complex, Lucena City, Quezon

Fish Samples Total No. No. of fish infected No. of Anisakis Infection Intensity
of Samples by Anisakis spp. spp. present Rate
Splendid ponyfish 69 2 5 2.90 2.5
Striped ponyfish 4 0 0 0 0
Longfin grouper 12 4 80 33.33 20
One-spot snapper 11 2 4 18.18 2
Redspine threadfin bream 9 0 0 0 0
Purple-spotted bigeye 3 1 1 33.33 1
Spangled emperor 5 0 0 0 0
Chocolate hind 1 0 0 0 0
Checkered snapper 1 0 0 0 0
Jarbua terapon 1 1 11 100 11
Sardinella fimbriata 137 0 0 0 0
Sardinella fimbriata 146 0 0 0 0
Mean 37.55 7.3
 32

Table 5. Parasite measurement in millimeters using ImageJ Software.

Stage Length and Anisakis spp. Philometra spp. Others

width of
parasite
Adult TBL 30.24 22.15
MBW 0.20 0.17
TBL 20.37
MBW 0.26
TBL 18.50
MBW 0.15
Third-Stage TBL 14.40 13.38 17.15
Larvae MBW 0.24 0.26 0.25
TBL 10.16 12.19 16.77
MBW 0.23 0.20 0.15
TBL 10.11 16.23
MBW 0.18 0.17
TBL 7.34 7.16 15.43
MBW 0.17 0.18 0.2282
TBL 15.20
MBW 0.19
TBL 11.69
MBW 0.20
TBL 3.62
MBW 0.08
TBL 3.04
MBW 0.08
TBL 2.84
MBW 0.04
(TBL= Total Body Length; MBW=Maximum Body Width)
 33

Correlation of Length and Weight to the Infection of Parasites

A significant positive correlation was found in one-spot snapper and longfin

grouper. However, this correlation was not apparent in fish which do not change greatly

in their length and weight after they are fully grown.

 34

80
r2 = 0.04 A
r2 = 0.51
60

40

20
Number of Parasites

16 18 20 22 24 26 28 30 32

Total Length (cm)

80
r2 < 0.01
r2 = 0.56 B
60

40

20

0 100 200 300 400 500

Body Weight (g)

Figure 8. Relationship of length (A), and weight (B) of Epinephelus quoyanus

(solid circles , n = 12) and Lutjanus monostigma (open circles , n =

11) to the number of observed parasites.

A higher prevalence of anisakid nematode infestation depends on the availability

of the final hosts in the region and the parasites ability to complete its life cycle (Palm,
 35

1999). It also may be related to the food ingested and to the layer of the water column

inhabited (bottom versus pelagic) (Palm et al., 2007). The infestation dynamic is strongly

fish species and area specific (Rokicki et al., 2009). The said fishing ground is a suitable

place for investigation of marine fish infection since the market is the biggest in fish sales

scale in Tayabas Bay. The high loads of Anisakis found in fish host, and the fact that

anisakid larvae were seen penetrating muscles, may point to a possible health problem for

consumers (Costa et al., 2003).

The infection rate, however, varied greatly depending on the species of fish. The

infection rate ranged from 0% to100, which means that some species of fish show higher

affinity to Anisakis spp. than others. Fish showing high infection rate exhibited flabby

flesh and high fat contents.

The presence of Anisakis sp. larvae in marine organisms have been investigated in

several studies and a great number of fish species have been found and reported to be

receptive to anisakid infection (Smith and Wotten, 1978; Abollo et al., 2001).
 36

SUMMARY, CONCLUSIONS AND RECOMMENDATION

Nematode larvae of the genus Anisakis are common parasites of marine fishes

serving as their intermediate hosts such as splendid ponyfish (Eubleekeria splenders),

longfin grouper (Epinephelus quoyanus), one-spot snapper (Lutjanus monostigma),

purple-spotted big eye (Priacanthus tayenus) and jarbua terapon (Terapon jarbua), all of

which are consumed by many Filipinos in raw or processed forms. The information on

the food habits of marine fishes, such as the predator-prey relationship, which is useful in

order to assess the role of marine fishes in the ecosystem. Additions to this are the limited

studies on Anisakis infection in Philippine waters, particularly in the southern Luzon

island.

The aim of this research was to determine the prevalence, abundance and intensity

of third-stage larva (L3) of Anisakis worms infecting marine fishes at the southern part of

Luzon, specifically in Tayabas Bay. The main objective of this study was to identify the

parasitic nematodes, focusing on anisakid nematodes infecting ten different marine

species and correlate the length and weight of the fishes with the number of anisakid

nematode infection. Since there are actually no known reports on the common parasites

present in marine fishes landed in the area, a broad knowledge on the identity and

distribution of anisakid nematodes is very important in understanding existing infection

and as well in forecasting possible future infections.

Morphological methods were used in this study for the identification of Anisakis

species infecting marine fishes marketed in Dalahican Fish Port Complex, Lucena City.

Totally 400 fish specimens were collected during July 2015. The prevalence, intensity,

abundance and distribution of Anisakis spp. larvae which infected some fishes at the
 37

Tayabas Bay were investigated. It was calculated following the formula proposed by

Margolis et al. (1982) and Bush et al. (1997).

Results showed a total of 101 out of 178 worms were collected and identified

under the genus Anisakis from different host fishes namely, splendid ponyfish

(Eubleekeria splenders), longfin grouper (Epinephelus quoyanus), one-spot snapper

(Lutjanus monostigma), purple-spotted big eye (Priacanthus tayenus) and jarbua terapon

(Terapon jarbua). The identified Anisakis belong to Anisakis type 1 group based on

morphological examination. Three species of anisakids were identified; Anisakis simplex

s.s, Anisakis typica and Pseudoterranova sp. Philometrid nematode was also classified. In

terms of parasitic worm infection, the highest prevalence of infection was found in jarbua

terapon (100%, followed by longfin grouper (58.33%), striped ponyfish (50%), purple-

spotted bigeye (33.33%), spot snapper (27.27%) and splendid ponyfish (15.94%). On the

other hand, with respect to Anisakis infection, the overall infection rate was 37.55%, with

mean intensity of 7.3. Anisakis infection was observed only on five fish species which

includes the Epinephelus quoyanus, Lutjanus monostigma, Priacanthus tayenus,

Eubleekeria splenders and Jarbua terapon.

Presence of this parasite may be harmful for consumer; however it can be used for

several ecological studies as biological tags. Consumption of raw or undercooked fishes

with high prevalence of Anisakis infection should be avoided. The collected data

provided some information on the infection levels on each fish species that can be used as

guide during fish handling and processing, as well as possibility for use as biological tag

for fish stocks identification. Thus, understanding of the parasites distribution and

specific site of infection may be used as biological indicators for several ecological
 38

studies. Hence, the result of this study can serve as pro-active measure in reducing the

risk of human anisakiasis and allergies to target consumers locally and internationally.

Anisakis simplex s. s. and A. pegreffi have been reported to cause human

anisakiasis and allergies to consumer. Given that A. typica is the dominant species, which

have not been reported to cause human anisakiasis and allergies, this could possibly be

one of the reason why incidence of such human anisakiasis is not known in the country

(i.e., apart from not consuming mostly of raw fish and limited knowledge of the

infection). With the absence of A. simplex s. s. and A. pegreffi from our marine fishes

specifically grouper, we can assure the international community of the safeness for

human consumption. On the other hand, importation of marine products may pose threat

like dissemination of Anisakis infection in Philippine waters. If this scenario materializes,

it may cause anisakiasis and allergies to Filipino consumers.

Following recommendations in using morphological examination, one must be

very careful in cutting and fixing the worm because even a slight deformation of the

sample can be problematic in the microscopic examination. On the other hand, the use of

the molecular examination for final species identification specifically in the identification

of Anisakis spp. is more accurate and highly recommended than in morphological

characterization.
 39

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 45

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http://www.foodpoisoningtreatment.org

https://www.hubmaps.com/en/a/google-earth

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 46

APPENDICES
 47

APPENDIX TABLE

Appendix Table 1. Longfin grouper, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis spp. Philometra Unidentified
Epinephelus 1 205.5 25.5 21 74 16
quoyanus 2 75.9 18.9 15.2
Longfin grouper 3 75.9 18.7 15.4 3
Lapu-lapu, 4 75.9 17 14.6 2
Sigapo 5 75.9 17.5 14.7 1
6 438.5 30.3 26.2 1
7 134.5 23.3 19.3 2 1
8 179.75 21.5 17.7
9 179.75 23.9 19.8
10 285.25 27.5 23.4 2
11 285.25 25.5 21.4
12 159.5 22.2 18.5

Appendix Table 2. One-spot snapper, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis spp. Philometra Unidentified
Lutjanus 1 117.3 23 18.4
monostigma 2 117.3 21 17.5 1
One-spot 3 391.8 30.3 25.5 3
snapper 4 231.25 25.4 21
Maya-maya, 5 231.25 25.8 21.9
Talingan 6 267.8 27 22.3 1 1
7 267.8 25.8 22.4
8 137.5 21.3 17.7
9 148.5 20.7 17.3
10 257.5 26.3 22.2
11 216.5 24.3 20.9
 48

Appendix Table 3. Chocolate hind, number examined, length and weight, and parasites
infection.

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Cephalopholis boenak 1 81.5 17.2 14
Chocolate hind
Lapu-lapu, Sigapo

Appendix Table 4. Redspine threadfin, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Nemipterus 1 130.2 20.9 17.4
nemurus 2 130.2 22.5 18.3
Redspine 3 130.2 20.2 17.1
threadfin bream 4 130.2 23 18.1
Bisugo 5 191.25 24.6 20.1
6 191.25 26.1 21.5
7 209.5 25.1 20.9
8 78.75 16.1 13.4
9 78.75 18.5 15.4

Appendix Table 5. Purple-spotted bigeye, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Priacanthus 1 224.25 28.5 21.3
tayenus 2 224.25 30.4 23.5 1 1
Purple-spotted 3 180.5 29.1 21.7
bigeye
Siga, Dilat
 49

Appendix Table 6. Spangled emperor, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Lethrinus 1 227.75 22.9 19
nebulosus 2 227.75 24.8 21.2
Spangled 3 303.5 26.8 23.8
emperor 4 94.75 17.8 14.7
Puting 5 94.75 17.9 15
kanuping

Appendix Table 7. Checkered snapper, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Lutjanus 1 191.5 23.4 19.7
decussates
Checkered snapper
Madrigal, Maya-
maya

Appendix Table 8. Jarbua terapon, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Terapon 1 233.5 25.2 21.1 11 1
jarbua
Jarbua terapon
Bagaong,
Gung-gong
 50

Appendix Table 9. Striped ponyfish, number examined, length and weight, and parasites
infection

Species Fish W TL SL Number of Parasites

No. (g) (cm) (cm)
Anisakis spp. Philometra Unidentified
Aurigequula 1 83.25 16.7 14
fasciata 2 83.25 17.2 14.3
Striped ponyfish 3 237.5 24.5 20.8
Sap-sap, Waling 4 106.5 17.4 15.4

Appendix Table 10. Splendid ponyfish, number examined, length and weight, and parasites
infection
Species Fish W TL SL Number of Parasites
No. (g) (cm) (cm)
Anisakis Philometra Unidentified
spp.
Eubleekeria 1 130.2 20.9 17.4
splenders 2 130.2 22.5 18.3
Splendid 3 130.2 20.2 17.1
ponyfish 4 130.2 23 18.1
Sapsap, Laway, 5 191.25 24.6 20.1
Kurag 6 191.25 26.1 21.5
7 209.5 25.1 20.9
8 78.75 16.1 13.4
9 78.75 18.5 15.4
10 110.1 18.6 15
11 110.1 17.5 14.3
12 110.1 18.5 14.9
13 110.1 18.4 14.8
14 110.1 17.5 14.2
15 63.3 15 11.8
16 63.3 15 12.7
17 63.3 16.2 13.5
18 63.3 14.7 12.3
19 63.3 14.4 11.8
20 63.3 14.5 11.8
21 63.3 14.4` 11.6
22 63.3 15.7 12.6
23 63.3 15.8 12.9
24 63.3 17.4 14.2 3
21 63.3 14.4 11.6
25 84.75 15.3 13.4
 51

26 84.75 15.5 14.6

27 84.75 18.1 14.8
28 84.75 15.4 12.7
29 84.75 17.1 14.4
30 83.4 15.5 13.8
31 83.4 17 13.9
32 83.4 15.3 13.3
33 83.4 15.4 13.4
34 83.4 16.6 13.5
35 83.4 15 12.9
36 72.1 15 12.8
37 72.1 16.2 13.4
38 72.1 16.3 13.2
39 72.1 14.5 12
40 72.1 17.6 14
41 72.1 16 13
42 72.1 17 14 5
43 147.8 12.7 17.4
44 147.8 20 16.2 21
45 147.8 17.5 14.7 2 2
46 137.9 19.4 15.1 1
47 137.9 19.3 16.9
48 137.9 19.7 17.3 7
49 137.9 20.2 16.2
50 82.5 15.3 12.4
51 82.5 15.5 12.3 1
52 82.5 17.2 14.7 2
53 82.5 18.3 14.5
54 82.5 17.5 15.1
55 99.7 18.9 15.4
56 99.7 18.6 14.9
57 99.7 18.8 15.3
58 99.7 15.3 13.2
59 99.7 18.6 13.4
60 180.8 21.2 17.1
61 180.8 21.2 17.2 1
62 180.8 21.3 17.5
63 120.9 17.7 13.5
64 120.9 18.5 15
65 120.9 18.3 14.9
66 120.9 19.5 15.4
67 86.5 16 13.5 2
68 86.5 16.1 13.3
69 86.5 18.7 15.5 4
 52

Appendix Table 11. Summary of Ventriculus Length (mm) of identified parasites using

ImageJ software.

Anisakis spp. Anisakis simplex s.s Anisakis typica

1.20 1.05 1.10
1.16 1.03 1.06
1.51 1.05 1.41
1.19 1.12 1.06
1.19 1.48 1.07
1.21 1.10 1.04
1.10 1.10 1.21
1.08 1.12 0.88
1.095 1.18 1.23
1.08 1.21 1.24
1.42 1.17 1.14
1.06 0.86 1.10
1.10 1.04 1.09
1.06 1.33 0.98
1.05 1.25 1.91
1.05 1.20 0.90
1.05 1.14 1.22
1.06 1.17 1.22
1.06 1.09 1.14
1.06 1.10
1.06 1.09
1.11 0.98
1.06 0.90
1.07 1.22
1.22
0.60
0.62
1.15
 53

APPENDIX FIGURES

Appendix Figure 1. Collected samples

Appendix Figure 2. Collected samples in Styrofore box

 54

A B
A

C D

E F

G H

I J

Appendix Figure 3. Different marine fishes collected: (A) Eubleekeria splenders, (B)
Aurigequula fasciata, (C) Epinephelus quoyanus, (D) Priacanthus tayenus, (E)
Epinephelus quoyanus,(F) Lutjanus monostigma, (G) Lethrinus nebulosus, (H) Terapon
jarbua,,(I) Lutjanus decussates, (J) Cephalopholis boenak
 55

Appendix Figure 4. Measuring the length and weight of the samples

Appendix Figure 5. Dissection of fish samples

 56

Appendix Figure 6.Visceral organs of fish samples

Appendix Figure 7. Visceral organs in petri plates containing Natural saline solution
 57

Appendix Figure 8. Pouring 70% ethyl alcohol in Petri dish

Appendix Figure 9. Collected parasite ready for preservation