Chanos Chanos (FORSSKAL, 1775), FRY SEASONALITY in VANUATU: THEIR AVAILABILITY and ABUNDANCE in the COASTAL SHORELINE of EFATE ISLAND

MILKFISH, Chanos chanos (FORSSKAL, 1775), FRY SEASONALITY IN VANUATU: THEIR AVAILABILITY AND ABUNDANCE IN THE COASTAL SHORELINE OF EFATE ISLAND

Ronnick Spenly Shedrack

A Thesis submitted in fulfillment of the requirements for the Degree of Master of Science in Marine Science

School of Marine Studies Faculty of Science, Technology and Environment The University of the South Pacific

November, 2017

Declaration of Originality

Statement by Authoor

I, Ronick Spenly Shedrack, declare that this thesis is my own workk and that, to the best of my knowledgee, it contains no material previously published, or substantially overlapping with matterial submitted for the award of any other degree at any institution, except whhere due acknowledgement is made in the textt.

Signature: Date: 10 November 2017

Name: Ronick Spenlly Shedrack Student ID No: S11062153

Statement by Supervvisor

The research in this thhesis was performed under my supervision annd to my knowledge is the solee work of Mr. Ronick Spenly Shedrack.

Signature: Date: 20/11/22017

Name: Dr. Marta Feerriera Designation: Princippal Supervi

Dedication

This thesis is dedicated to my dear parents Belinda Toa Spenly and Spenly Shedrack Salemomo.

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Acknowledgements

I express my sincere thanks and appreciation to the Australian Centre of Agricultural Research (ACIAR) for funding me for the Master of Science degree under the ACIAR-USP scholarship Scheme and to USP for facilitating the sponsorship and funding another 6-month for the write up until completion of the program.

The completion of my thesis was made possible through the invaluable support from my principal supervisor, Dr. Marta Ferreira and co-supervisor, Dr. Susanna Piovano who provided constructive criticism, suggestion, comments and encouragement during the course of the study.

Dr. Marta is also immeasurably valued for providing me with support to Otolith analysis technique that is necessary to complete the thesis. I also acknowledge Dr. Susanna for various constructive criticisms polishing this study.

I express my appreciation to Mr. Sompert Gereva for his invaluable support, advice and assistance throughout the project with the field data collection, sampling materials and providing me space at the Vanuatu fisheries department to complete the thesis write up. Thanks are also extended to Dr. Timonthy Pickering for his various advices on the field data collection techniques.

I also express my gratitude to the Vanuatu Meteorological and Geo-hazard Department staff particular, a special thanks to Melinda Natapei, David Gibson and Philip Mansale for supporting with the data collection until the completion of the fieldwork.

I would like to thank Martinez and his colleagues (2006) for allowing me to reproduce their figure of milkfish life stages in Figure 1.2.

My heartfelt gratitude is extended to my family members, in particular Christian Shedrack, Semion Shedrack, Ajay Shedrack, Susila Salemomo and Welpi Tane for assisting me throughout the field data collection.

Lastly, I am thankful to my then girlfriend and now my wife, Rose Charley for her love, support and assistance with lab work, food preparation and for everything, she did for me.

Abstract

This study describes the seasonality and abundance of milkfish Chanos chanos fry in the coastal shorelines of Efate Island in order to assess the feasibility of milkfish aquaculture in Vanuatu. The study will enable the knowledge of fry collection so that this species can be cultured for food. Four sampling sites have been assessed in February 2016 for the presence of milkfish fry namely, Erakor, Kawenu, Mele and Teouma. The site with the highest abundance was selected for further seasonality sampling over a one-year period from February 2016 to February 2017.

Milkfish fry were collected from February to May and October to December 2016 and, again from January to February 2017. Milkfish fry was absent in other months of the year. Two peaks of fry occurrence were observed, the 1st peak was in April and the 2nd peak was in November. The fry abundance in Teouma was high (relative to other locations such as Erakor, Kawenu and Mele) over the period of seasonality sampling. The abundance between Moon phases showed that more fry were caught in New Moon and 3rd quarter Moon whereby a non-significant difference (p > 0.05) was observed. Milkfish fry collected on Teouma coast measured between 8 mm to 15 mm total length (TL) and the weight range was between 3 mg to 9 mg. The fry were aged 13 to 25 days old, which mean that they spend 2 to 4 weeks in the surf zone before disappearing into other habitats. Environmental variables assessed showed cloud cover is positively correlated with fry abundance, similarly, wind speed and current speed with length, and rainfall and turbidity with weight. Other variable such as temperature in situ do not significantly correlated with fry abundance, weight and length.

The prolonged fry seasonality in Vanuatu is advantageous for milkfish fry aquaculture, however the very low abundance documented in Teouma are not. Before a final decision on the viability of milkfish culture in Vanuatu is made, an assessment of multiple sites is recommended. To be cost-effective such a study could be informed by this research with sampling limited to the months when fry abundances peaks. If milkfish culture does proof to be viable, then the landowners of fry collection ground may need to manage activities along the coast to reduce impacts on fry recruitment habitat. Furthermore, fishers may want to consider

managing fishing of milkfish during spawning periods in order to protect spawning stocks.

Abbreviation and Acronyms

ACIAR Australian Center for International Agricultural research ANOVA Analysis of variance BOD Biological demand for oxygen CT Concentration test oC Degree Celsius DO Dissolve oxygen FAO Food and Agricultural Organization GSI Gonadosomatic Index IPCC Intergovernmental Panel on Climate Change VFD Vanuatu Fisheries Department PIC Pacific Island Country ppt Parts per thousand VMGD Vanuatu Meteorology and Geo-hazard Department COSPPac Climate and Ocean Support Program in the Pacific SST Sea surface temperature SPC Pacific Community USP The University of the South Pacific TL Total length

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Table of Contents

Acknowledgements ...... iv

Abstract ...... v

Abbreviation and Acronyms ...... vii

List of figures ...... x List of tables ...... xii Thesis organisation...... xiii Introduction ...... 1 1.1 Milkfish aquaculture ...... 1 1.2 Reviews on milkfish (Chanos chanos) ...... 1 1.2.1 Systematics ...... 2 1.2.2 Morphology ...... 3 1.3 Habitat and life history ...... 3 1.3.1 Life history ...... 4 1.4 Food, Growth and Feeding Habit ...... 7 1.5 Milkfish distribution ...... 7 1.6. Milkfish aging, spawning and fry seasonality ...... 8 1.6.1 Milkfish spawning ...... 9 1.6.2 Seasonality of fry ...... 10 1.7 Migration and movement of fry ...... 11 1.8 Predation on milkfish fry ...... 12 1.9 Overview of main methods of fry collection ...... 12 1.10 Food demand in the Pacific island countries ...... 14 1.11 Conclusion ...... 16 1.12 Research objectives ...... 16 Chapter 2: Methodology ...... 18 2.0 Site selection and description ...... 18 2.1 Sampling materials and methods ...... 20 2.1.1 Sampling materials ...... 20 2.1.2 Bulldozer net construction ...... 21 2.1.3 Sampling method ...... 23 2.2 Fry identification ...... 24 2.3 Fry length and weight measurement ...... 24 viii

2.4 Otolith analysis ...... 25 2.5 Data analysis ...... 26 Chapter 3: Results ...... 27 3.1 Site assessment and selection ...... 27 3.2 Milkfish fry occurrence in Teouma ...... 29 3.2.1 Monthly occurrence and abundance ...... 29 3.2.2 Milkfish fry length and weight ...... 30 3.3 Effect of Moon phases ...... 32 3.3.1 Abundance by Moon phase...... 32 3.3.2 Total length and weight in Moon phase over one year ...... 33 3.4 Comparison of fry abundance with other indices ...... 34 3.4.1 Environmental variables correlation with fry abundance ...... 34 3.4.2 Environmental variables correlation with length and weight of milkfish fry ...... 35 3.5 Otolith analysis and age of the fry per month and spawning estimates ...... 37 3.6 The age distribution ...... 38 3.7 Spawning time estimates for Teouma over one year period ...... 40 3.8 Comparison of fry abundance in the Melanesian PICs ...... 41 Chapter 4: Discussion ...... 42 Chapter 5: Conclusion ...... 50 References ...... 52 Appendix ...... 66 Appendix A. Table of p-values for the ANOVA test ...... 66 Appendix B. The daily SST from VMDG and monthly mean, maximum, minimum, median and standard deviation beginning from February 17th 2016 to February 17, 2017...... 69 Appendix C. Milkfish fry mean length and weight plus the descriptive statistic for Teouma over one year period ...... 70

List of figures

Figure 1.1 Picture of adult milkfish……………………………………………...…...2

Figure 1.2 Milkfish life cycles, 1-7 indicates the seven development stages in the milkfish life cycle (obtained with permission from Martinez, Tseng et al. (2006)…..4

Figure 1.3 A graphical representation of peak and lean season of milkfish fry occurrence in several countries with data retrieved from Kuo et al. (1979),Villaluz (1986) and Vuto et al. (2014)…..………………………………………...………….19 Figure 2.1 Map of Vanuatu where the study is based. Retrieved from http://www.nationsonline.org/oneworld/map/vanuatu-map .…….…………….……...19

Figure 2.2 Map of the four sites Erakor, Kawenu, Mele and Teouma where milkfish fry was assessed during the preliminary study, and the Vanuatu capital (marked with a star)…………………………………..………………………………………….…20

Figure 2.3 The bulldozer net built and used for this study. Picture shot at Teouma on March 2016. ……………..……………………………………….…………………22

Figure 2.4 The bamboo frame design for the bulldozer net (Pickering, Tanaka et al. 2012)…………………………………………………….………………………..…22

Figure 2.5 Net size and design modified from Pickering, Tanaka et al. (2012)..…...23

Figure 2.6 Milkfish fry collected in a white plastic basin for visual identification. The black dot from left to right showed position of grey spot on caudal fin, dark spot on middle of body (air bladder) and two dark spot (eyes) on side each side of head…..24

Figure 3.1 Milkfish fry total abundance (number of fry captured during the exploratory trial done on Teouma, Erakor, Mele and Kawenu). The different letters indicate significant difference in abundance (p < 0.05) while the same letter indicate no significant difference (p > 0.05).…………………………………………….…...29

Figure 3.2 Milkfish fry abundance and occurrence with SST by month for Teouma. The different letters indicate significant difference in fry abundance (p < 0.05)..….30

Figure 3.3 The total length frequency of milkfish fry (N = 369) in the Teouma……31

Figure 3.4 The frequency distribution of milkfish fry (N = 369) weight in Teouma……………………………………………………………………………...31

Figure 3.5 Milkfish fry mean total length (A) and weight (B) by month of occurrence. The error bars represent the standard deviation of the mean. …….…...32

Figure 3.6 Milkfish fry cumulative abundance in all Moon phases for Teouma throughout the year. The different letters indicate significant difference is abundance between Moon phases (p < 0.05). ………………………………...... …………….33

Figure 3.7 Milkfish fry mean total length (A) and weight (B) in four Moon phases. The different letters show significant difference in mean total length or weight (p < 0.05) and the error bars represent the standard deviation of the mean. .……..……..34

Figure 3.8 The environmental variable correlation with milkfish fry abundance, rainfall, cloud cover, wind speed, temperature, turbidity (transparency) and current speed...……………………………………………………………………………....35

Figure 3.9 Environmental variable, temperature, cloud cover, rainfall, turbidity (transparency), wind speed and current speed correlation with milkfish fry total length ..…………………………………………………………..…………………..36

Figure 3.10 Environmental variables, temperature, cloud cover, rainfall, turbidity (transparency), wind speed and current speed, correlation to milkfish fry weight ...…………………………………………………………………………..…………37

Figure 3.11 Photographs a, sagittae otolith from 14 days old larvae, 11 mm total length (TL). (x400), b an otolith of a 15 day larva of 11 mm TL and, c a otolith of a 22 day larva of 14 mm TL. In photograph a, letters. A, anterior; P, posterior; D, dorsal; V, ventral. The dot (●) are the increment dark zone, the star (ღ) indicate the core and the arrow (↓) indicate the discontinuity zone. …………...………………..38

Figure 3. 12 The age frequency of milkfish fry in Teouma coastal shoreline ….…..39

Figure 3.13 The mean age of fry per month of occurrence in Teouma. The error bars represent the standard deviation of the mean. ..………...…...………………………39

Figure 3.14 Correlation between the age and length of milkfish fry ………….……40

Figure 3.15 The spawning season of milkfish fry (N =63) based on different days over a one year period with mean Monthly SST…………………...……………….41

List of tables

Table 3.1 The monthly environmental parameters (turbidity (transparency), current speed, salinity and SST) assessed and the landscape features for Mele, Kawenu, Erakor and Teouma. The monthly values are presented as mean ± standard deviation…………………………………………………………………………..…28

Table 3.2 the fry abundance between Fiji, Solomon Islands and Vanuatu……….....41

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Thesis organisation

There are six chapters in this thesis.

Chapter 1 presents a literature review about what is known and where the gaps of knowledge which require further research on milkfish (Chanos chanos) larval recruitment. Other parameters affecting milkfish larval development and fry presence along coastal areas were also reviewed, and seasonality of spawning and culture of milkfish in the Pacific Island Countries (PICs) were outlined. This chapter also presents the purpose and scope of the study, and the hypotheses and statement of problems to be addressed. The reason for a milkfish fry seasonality study in Vanuatu is also outlined and the advantages of having an additional fish species for the local aquaculture industry are discussed.

Chapter 2 describes the materials and methods used to undertake this study. The study sites and the methodologies applied to collect the larvae are shown and the data and statistical analyses highlighted.

Chapter 3 presents the results that include seasonality of fry occurrence and abundance, abundance in Moon phases, environmental variables affecting abundance and size of fry, and finally the spawning period estimation.

Chapter 4 contains the discussion of research findings and reflection within the literature context. The assessment for each single site was discussed with respect to the abundance of milkfish fry and the parameters assessed. The length, weight and age frequency of milkfish fry were also discussed with respect to seasonality and Moon phases.

Finally, chapter 5 contains the conclusion and recommendations for further research to address limitations and constraints in this study and to further engage researchers to develop milkfish farming in Vanuatu.

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Introduction

1.1 Milkfish aquaculture

Milkfish Chanos chanos is one of the many species cultivated to address food shortage and meet the food protein demand in the Asian-Pacific region (Agbayani et al. 1989; FAO 2014; Martinez et al. 2006). The knowledge about biology, spawning and culture of the species has aided the development of milkfish culture industry in many countries, in particular Asian nations (Bagarinao 1991; Martinez et al. 2006; Sulu et al. 2016). Milkfish aquaculture dated back about 4 to 6 centuries ago in India, Philippines, Indonesia and Taiwan (Agbayani et al. 1989; Nelson 2007). The prolong season of fry appearance aids aquaculture development as fry can be collected and stocked in water ponds in many months of the year (Bagarinao et al. 1987). In recent decades, many Asian countries have used technological advances to move into milkfish mass aquaculture. Pacific Island Countries and Territories (PICTs), milkfish aquaculture is new, while in Nauru milkfish culture is part of their tradition for household food consumption (Spennemann 2002). Vanuatu is one PIC with no quantitative records of milkfish fry appearances or history and culture, however there is history of local people collecting juveniles on some islands for food (Pickering et al. 2012).

The knowledge of milkfish fry seasonality is lacking in some PICs although milkfish fry are available in their coastal areas (Dela Cruz 1997). This knowledge is important for PICs to adopt milkfish culture, as access to advanced hatchery technology is poor and culture may depend on availability of wild fry for stocking ponds. Hence, the purpose of this study is to determine milkfish fry seasonality in Vanuatu and evaluate the potential of milkfish aquaculture.

1.2 Reviews on milkfish (Chanos chanos)

Milkfish Chanos chanos (Forsskål, 1775) is the only fish species of the family Chanidae in the order Gonorynchiformes that comprises four families, seven genera and 27 species (Bagarinao 1994b; Lim et al. 2002; Nelson 2006). The specific 1

epithet chanos (Greek for a fish of remarkable voracity) was attributed to milkfish in 1775 by the Swedish naturalist Peter Forsskål, who made a clear description of the fish based on a specimen from the Red Sea that was preserved at the Zoological Museum of the Copenhagen, University in Denmark (Klausewitz 1965). In 1803, the French naturalist Bernard Germain de Lacépède elevated the specific epithet to the generic level, naming the species Chanos arabicus, and further works from other authors later described the milkfish under different names and synonyms (Bagarinao 1994b; Bagarinao 1991; Brian 2015). The review by Bagarinao (1994b) revealed that nine different names were used to describe milkfish by Cuvier and Valenciennes while other authors described it under eighteen other synonyms. The name Chanos chanos was validated and used first by Klunzinger (1870) and following authors did the same (Fricke 2008) .

A picture of a typical adult milkfish is shown in figure 1.1 whereby it has a single dorsal fin, large fork tail and silver or milk colour. The milkfish swim over the sandy bottom and around islands where coral reefs are present (Figure 1.1).

Study species: Chanos chanos

Kingdom: Chordata Phylum: Vertebrata Class: Osteichthyes Order: Gonorynchiformes Family: Chanidae Genus: Chanos (Lacépède, 1803) Species: Chanos chanos (Forsskål, 1775)

Figure 1.1 Picture of adult milkfish (Source: www.aqtinfo.com)

1.2.1 Systematics

Milkfish is considered a more ancestral ostariophysian which belongs to a Monotype gonorynchiform family and is related mostly to the freshwater Ostariophysi (Bagarinao 1994b). Although milkfish morphological characteristics have significantly contributed to a much better understanding of the species, molecular 2

studies determined the number of chromosomes in milkfish as diploids, 2n = 32 consisting of 7 pairs of metacentric, 2 pair of submetacentric and 7 pair of acrocentric pairs of chromosomes (Arai et al. 1976). The number of chromosomes is low when compared to other primitive teleosts (Brian 2015). The genetic identification has helped to distinguish milkfish populations, whereby 9 populations were described in the Indo-Pacific of which 4 populations were determined as the Indian, Thailand, Philippine-Taiwan-Indonesia and Tahiti (Bagarinao 1994b; Bagarinao 1991; Kumagai 1990; Senta et al. 1977). Samples of larvae and juvenile specimens obtained from Kiribati, Tonga, Hawaii and Panama have revealed four other populations and a suggestion was made that there is a possibility for one population in the coast of Africa (Bagarinao 1994b).

1.2.2 Morphology

Adult milkfish has been described as having a silvery colour, muscular, streamlined body and powerful forked tail (Bagarinao 1994b). Recently Brian (2015) stated that, “The mouth is small and lacks teeth. There is a notch on the upper jaw in the mid- line into which a lower jaw protuberance fits. The large eyes have an adipose eyelid. The intestine is very long with many folds. The lower part of the esophagus has a "gizzard”, an area with longitudinal folds. Lateral line scales 70-92, with 3-11 on the tail fin”.

There are also variant forms of milkfish reported, the “goldfish” form from Philippine and Indonesia and the “dwarf or hunchback –shade type” found in Hawaii, Indonesia and Australia, but the knowledge about these other forms of milkfish is limited since they rarely occur (Bagarinao 1994b).

1.3 Habitat and life history

Milkfish are usually found in offshore marine waters and shallow coastal embayment, but also frequently enter estuaries and occasionally penetrate freshwater streams (Bagarinao 1994b). Adults occur in small to large schools near the coasts or 3

around the islands wwhere reefs are well developed (Bagarinao 19991). Milkfish eggs and larvae are pelagicc for up to 2-3 weeks and older larvae migratte inshore and settle in coastal wetlands (mangroves, estuaries) occasionally enterinng freshwater lakes (Bagarinao et al. 19887). Juveniles and sub-adults return to the seaa where they mature sexually and spawn iin fully saline marine water (Nelson 2007). MilkfishM fry feed on phytoplankton and zzooplankton; juveniles and adults feed on cyyanobacteria, small benthic invertebratess, and even pelagic fish eggs and larvae (Bagarinao 1991). Milkfish at the differrent life stages frequent various habitats beforre they migrate into the open ocean in aduult stage where they spend the rest of their lives and continue to spawn (Figure 1.2).

Figure 1.2 Milkfish liife cycles, 1-7 indicates the seven developmennt stages in the milkfish life cycle (obbtained with permission from Martinez, Tsengg et al. (2006)

Milkfish fry enters a coastal lagoon and swamp before they migrate m further into juvenile habitat. Accoording to Martinez et al. (2006), milkfish fryy are more abundant in estuaries and near river mouth. The fry tend to migrate into rivvers in the direction of water flow (Lin ett al. 2003; Pillay et al. 2005) and other coasstal habitats such as swamps and lagoons to enter into juvenile stage.

1.3.1 Life history

The life history and habitat of milkfish in the wild was documeented in the past by many researchers, aalthough many errors have been pointed out o in more recent 4

research, like the review by Bagarinao (1994b). In the last few decades milkfish have been demonstrated to mature and spawn under various condition of captivity, and hatcheries have produced larvae to supply ponds (Bagarinao 1991). Nowadays the aquaculture industry has employed facilities and techniques that facilitate breeding in captivity and the transition of fry to juvenile in captivity, including mass propagation with minimum mortality (Bagarinao 1991; Nelson 2007).

Milkfish fertilized eggs are spherical and pelagic with a diameter ranging from 1.1 to 1.2 mm, and the development of the embryo takes 20 - 35 hours at a temperature range from 26 ºC to 32 ºC, and at a salinity between 29 to 34 parts per thousand (ppt) (Bagarinao 1991). Delsman (1926) was the first scientist who took note of milkfish eggs when he examined the oocytes and later collected 15 eggs from the Java sea; later his identification was tested and proven to be correct after induced spawning was done in captivity (Bagarinao 1994b; Liao et al. 1979) . The spawning grounds of milkfish are in clean and clear saline water with warm temperature (25-30 ºC) and shallow water (< 200 m) over coral reefs and sandy beaches at a distance of about 6 km offshore (Brian 2015). The spawning location is also based on the need to minimize predation (Brian 2015), and close to shore to enhance larvae migration to coastal habitat (Johannes 1978).

Milkfish larvae are categorized into 5 developing stages: i) yolk-sac larvae (3.3-5.4 mm total length (TL), lasts for 3 days); ii) pre-flexion larvae (3.4-6.2 mm TL, lasts for 5 days); iii) flexion larvae (4.4-9.9 mm TL, lasts for 6 days); iv) post flexion larvae (9.5-14.9 mm TL, lasts for 7 days); and v) transformation larvae (9.5 -16.5 mm TL also named as fry are free swimming larvae, last from 2 to 4 weeks), after a detailed description was made by various authors (Bagarinao 1991). Milkfish larvae are pelagic, while at hatching, the yolk sac larvae are about 3.5 mm TL; the lava begin to feed when their eyes are fully pigmented (3 days old) and the mouth will open although some yolk is still attached (Bagarinao 1994a). It takes 36 hours for the larvae to grow into 5 mm total length and to consume about 90 % of the yolk until day 5 when the yolk is completely exhausted. The size of the egg, larvae, amount of yolk and mouth is far greater in milkfish than any other tropical marine fish (Bagarinao et al. 1986). Younger larvae occur both onshore and offshore while older larvae are found near shore only (Bagarinao et al. 1987). A study on larvae

movement in the Great Barrier Reef by Leis et al. (1991) revealed that the larvae move to the juvenile habitat through passive advection and active migration. Active migration is employed by larvae that gain a certain degree of morphological characteristics, probably at 10 mm TL and age of 2 weeks old (Bagarinao 1994b).

The juvenile milkfish has a minimum length of 20 mm and the shape and structures of adult (Bagarinao 1994b; Bagarinao 1991). The juveniles, length between 2 to 10 cm TL, are called “fingerlings”, particularly in the aquaculture industry (Bagarinao 1994b). Milkfish larger than 30 cm are found in a variety of habitats such as estuaries, coral lagoons, tidal creeks, mud flats and tide pools that are protected areas and characterized by rich food supply (Buri 1980; Kumagai et al. 1985; Kumagai et al. 1981). The juveniles feed mainly on food from the bottom such as cyanobacteria, diatoms, detritus, filamentous green algae and invertebrate such as worms and crustacean (Bagarinao 1994b; Bagarinao 1991). The wild juveniles feed during the day as determined by gut content analysis, whereby morning guts were empty while those during the day were loaded with food (Kumagai et al. 1985).

An adult milkfish has a length of 50-150 cm TL and weight range of 4-14 kg with age from 3-15 years (Bagarinao 1991). More recently, Brian (2015) has noted in a review of the milkfish of Iran that this species has the size of up to 1.85 m and 18.6 kg. Adult milkfish are powerful swimmers and are seen in large schools near islands and the coast where reefs are well developed, and also in coastal lagoons where they often swim with their dorsal fins above water like sharks (Bagarinao 1994a; Brian 2015). Adults are seen and caught near shore usually during breeding season (Kumagai 1990; Tampi 1957). Milkfish reproduction in the wild is not well understood, although milkfish has been successfully bred in captivity in the Philippines, Taiwan, Hawaii and in Indonesia, and in Kiribati (Bagarinao 1994b). In nature milkfish may reach sexual maturity at age 3-5 years and in captivity at age 8- 10 years (Bagarinao 1994b; Brian 2015). The females may spawn more than twice per year, both in captivity and in the wild (Marte et al. 1986b; Schuster 1960; Silas et al. 1982; Tampi 1957), and 3-13 kg female may produce 0.5 to 6 million eggs. Spawning usually happens at night and is triggered by lunar and seasonal periodicity (Kumagai 1990; Marte et al. 1986b).

1.4 Food, Growth and Feeding Habit

Milkfish larvae normally feed on phytoplankton and zooplankton (Bagarinao 1991; Brian 2015), and juveniles in a variety of food, including cyanobacteria, detritus, diatoms, filamentous green algae, fish eggs and larvae, and some invertebrates such as crustaceans and worms (Bagarinao et al. 1986). Milkfish larvae are particulate visual feeders. They feed by snapping prey such as rotifer, water flea, copepod and brine shrimp (Bagarinao 1991). However, Carreon et al. (1984), evaluated the feeding response of larvae on diets of plankton and detritus (containing bacteria, various species of cyanobacteria, diatoms, rotifers and protozoans) in race way tanks whereby growth, health and survival was better in larvae supplied with natural planktons in comparison to those reared on detritus.

An adult milkfish feeds on plankton, benthic plants and animals (Bagarinao 1994b), they feed on plankton by swimming through plankton masses and also larval fish schools. They also graze on the rock's surface and floating algae therefore, adult milkfish is an opportunistic generalist (Bagarinao 1994b). Although juveniles and adult milkfish feeds on both day and night they are more active during the day (Kawamura et al. 1981).

1.5 Milkfish distribution

Milkfish are widely distributed in tropical, subtropical seas and around Oceanic islands, where seawater temperatures are greater than 20 °C, ranging from Red Sea and African coast to East Pacific, South Pacific coast of the U.S.A, Central America, Central and Western Pacific, Northern Pacific, South Pacific to New Zealand, the Indian Ocean and around Australia (Figure 1.3). Milkfish is said to be an Indo-West Pacific fish species, although it ranges across, to the eastern Pacific (Brian 2015). The adults can swim at the speed of 2 km/h although they were never caught in the high seas (Brian 2015). It is thought that milkfish recolonized the eastern Pacific by dispersal from the Indo-West-Pacific via the equatorial counter current (Briggs 1961), even though there is still debate regarding these theory (Bagarinao 1994a;

Bagarinao 1991; Brian 2015; Leis 1984). Milkfish are not found in tropical waters that are affected by cold ocean currents (Bagarinao 1991).

1.6. Milkfish aging, spawning and fry seasonality

Otoliths are fish ear bones, small calcified structures found inside the inner ear region to help with hearing and orientation (Begg et al. 2005; Popper et al. 2005).

The shapes are different for each three type of otolith such as the lapilli, sagittae and asterisci (Panfili et al. 2002). The sound wave is detected from the movement of otolith and the fish movement that send mechanical signals which assist in hearing and orientation (Popper et al. 2005).

Otoliths have been used widely to determine age, growth rate, development and life history of fish and are helpful in understanding fish systematics and evolution (Fey et al. 2005; Popper et al. 2005). Otoliths are formed at the hatching of the eggs and grow daily rings made of calcium ion from the endolymph precipitate (Warner et al. 2005). The rings continue to grow around the core and form a layer called microstructures, increment or growth rings. The daily growth rings or increments become narrower as the fish age increase, which also pose a difficulty in determining the life history of older fish (Baumann et al. 2005).

The growth of milkfish larvae follows a sigmoid growth curve (Liao et al. 1979). Kumagai et al. (1981) stated that fry in shore waters grow at a rate of 0.5 mm per day. Tzeng et al. (1988) have determined that the first otolith increment in reared milkfish larvae is formed 2 days from hatching and continues on a daily basis. A subsequent study by Tzeng et al. (1989) on wild caught larvae using the oxytetracycline method has validated that increments form at the rate of 1 per day regardless of growth rate. Kumagai (1990) has determine the size distribution frequency of 10,000 milkfish fry collected in shore waters where he concluded that shore caught fry grow at the rate of 0.5 mm/day and larvae remains at 5 mm for about 4 days from hatching. Therefore the relationship between size and age was derived from the following equation (Equation 1) from Bagarinao (1991).

Equation 1: TL = 5.0 + 0.5 (D-4) 8

Where, TL=total length and D=day from hatching

Furthermore, the growth of shore caught milkfish fry is temperature dependent and the transformation stage begins in 5 days of rearing (Villaluz et al. 1983a). The growth of fry also varies with factors such as diet and feeding (Bagarinao 1991).

1.6.1 Milkfish spawning

The spawning age of milkfish was determined to be 3 to 10 years in a temperature range of 25 to 30 °C and salinity of 29 to 34 ppt (Bagarinao 1991). Milkfish females can produce large amount of eggs with ovaries weighing 10 % to 25 % of the body weight when mature (Bagarinao 1991). The frequency of spawning as mentioned previously occurs probably more than once per year, based on indications from a female caught in the Philippines that contained 3-4 batches of oocytes with different sizes (Bagarinao 1991).

In captivity, female milkfish at the Southeast Asian Fisheries Development Centre/Aquaculture Department (SEAFDEC/AQD) can spawn to a maximum of three times per year naturally (Marte et al. 1986a). Spawning occurs around midnight and this was confirmed based on the egg development stages that were collected in the Panay island in the Philippines (Bagarinao 1991), although spawning at day time can occur less frequently. Spawning behavior of milkfish was observed in floating cages at the SEAFDEC/AQD in Philippines whereby an increase in swimming activity, chasing, leaping and slapping of water is seen from afternoon to early evening.

Milkfish spawning was assessed in the Philippines in Moon phases whereby the spawning occurred around midnight of every 1st and 3rd quarter Moon phase (Bagarinao 1991). It was also suggested by Delsman in 1929 that, spawning during neap tide minimize flushing of the eggs and facilitate larvae to remain in near shore waters (Bagarinao 1991), and the same was also suggested by Johannes (1978) for marine species in the tropical region with pelagic eggs. The young milkfish larvae are more abundant in quarter Moon phases and older ones on the New and Full Moon phases while fry were more abundant during New Moon and Full Moon of 9

which the same occurrence happened in the Philippines and other localities (Bagarinao 1991; Kumagai 1984).

1.6.2 Seasonality of fry

Many authors have determined seasonality and the occurrence of milkfish fry in different countries. Villaluz (1986) stated that milkfish fry seasonal occurrence may be predictable but the abundance will vary from year to year. The season is long near the equator and becomes shorter at the higher latitudes and in regions affected by trade winds or monsoon. The peak season may coincide with one or both of the yearly wind shifts (Villaluz 1986).The seasonality of milkfish fry has been determined for Fiji with a seasonal peak from December to February (Villaluz 1990) and for Kiribati with two (2) seasonal peaks, one from late December to early February and the other from May to September (Wainwright 1982). The identification of peak seasons is a preliminary step necessary to develop milkfish fry collections for aquaculture and has been done in Fiji and Kiribati (Bagarinao 1994a; Billings et al. 2010). In Hawaii, a study conducted on the gonadosomatic index (GSI) of milkfish over 36 months has indicated that breeding season is between June and August with a synchronized spawning behaviour observed (Kuo et al. 1979). Among the thirteen countries for which the seasonality of milkfish fry was established (Figure 1.4), Fiji is the only one that lies along the same latitude of Vanuatu and thus may help to predict the fry occurrence.

Figure 1.3 A graphiccal representation of peak and lean season off milkfish fry occurrence in severall countries with data retrieved from Kuo et al.a (1979),Villaluz (1986) and Vuto et all. (2014).

1.7 Migration and mmovement of fry

The fry swimming veelocity is within the range of 9-11 cm/s (Kawwamura et al. 1984). Bagarinao (1991) hhas stated that milkfish fry near shore watters do not school; however, they becomme loosely aggregated. The fry arrive in batchees at different times and distribute evenlyy along extensive sections of beach. There iss a need for further

studies to determine mechanism of larval transport to understand the natural recruitment of fry along inshore habitat (Bagarinao 1991). Some parameters that influence the abundance of fry along the coast were discussed by Kumagai (1984) such as currents, tides, bottom profile and proximity to inland waters whereby currents and tides were the factors that influence most the abundance along the shoreline. Fry are more abundant at flood tide due to the effects of the strong current and collected in large numbers at the surf zone (Bagarinao 1991). The time of day and wind speed does not have any influence on the fry abundance (Bagarinao 1991). The arrival of fry at the shoreline is in the form of small batches and this movement was suggested to be passive, driven by physical factors as such as longshore current (Kumagai 1984; Villaluz 1986). Although Buri & Kawamura (1983) claimed that fry in inshore waters were actively migrating into coastal habitat, Kawamura (1984) suggested that migration was also passive towards nursery habitat. More recently, Bagarinao (1994a) stated that, milkfish fry move inshore by both passive advection and active migration.

1.8 Predation on milkfish fry

The most important predators of milkfish larvae and fry are the Hawaiian tenpounder Elops hawaiensis and the Indo-Pacific tarpon, Megalops cyprinoides (Buri 1980; Pickering et al. 2012). Other common predators of milkfish are Ambassis sp., Terapon sp., Epinephelus sp., Lutjanus sp., Sphyraena sp., Chaetodon sp., Meiacanthus grammistes, Oxyurichthys microlepis, and Scatophagus argus (Buri 1980). Bagarinao (1991) suggested that milkfish fry may be eaten also by mugilids and siganids. The fry of tarpon and tenpounder are very similar to milkfish but they are predators of milkfish fry and fingerlings. These two species can be distinguished from milkfish as they have longer and wider bodies and much larger mouth, different swimming movement and light amber colour (Villaluz 1986).

1.9 Overview of main methods of fry collection

Milkfish fry are well-captured using gears operated by filtering water, such as plankton nets. Milkfish fry have well developed vision so they may respond negatively (i.e. avoidance) if they recognize the gear or positively (driven) with the gear if they see the net as some form of shelter (Bagarinao 1991). Black nets were observed to be more effective in catching fry although white nets are preferred due to easy identification of fry during capture (Kawamura et al. 1980). Experiments in tanks on the behavior of the fry response to gear revealed that, milkfish fry gear with herding design will function well rather than those with filtering (Kawamura et al. 1980). The good condition of the fry is identified through their ability to swim in one direction facing the current (Villaluz 1984).

The selection of a more appropriate and effective method to collect milkfish larvae or fry from the wild depends also on the topography of the site, currents, wind direction and tidal fluctuation (Villaluz 1986). However, the effectiveness of the methods can also be affected by small population size as shown in small lagoons with small population (Buri 1980).The fry are caught better with driving gears than filtering, and the gears may perform well in shallow part of the fry ground. It has been reported that milkfish fry used to congregate close to the fish shelter located offshore where they can be caught in sizable quantities (Villaluz 1986). The fry always stay at the surface, even when they are scared so the depth of the catching gear will not have to be increased to catch more fry (Villaluz 1986; Villaluz et al. 1983b). The gears used in fry collection are fine nylon nets with a mesh size ranging from 0.3 to 1.6 mm. The bulldozer net method is a very useful fry collection gear, which is normally operated in shallow lagoon. The bulldozer net gear was use first in the Philippines and it was reported to be effective in the night (Villaluz 1986). However, more recently, Vuto et al. (2014) showed that there is no significant difference in density of fry between the day and night.

Milkfish fry swim at the low speed of 9-11 cm/s (Kawamura 1984). They are caught in large numbers with juveniles and also the larvae of other predatory species (Bagarinao 1991). Sampling methods used in different countries are selected based on the topography of the sites, such as skimming net, hand scoop net, seine net collector, beach drag seine net, push net or bulldozer, barrier net and plankton net (Garcia 1990; Pickering et al. 2012). A skimming net is used in mud flats and

mangrove areas, and the hand scoop net is used mainly in mudflats, pools and canals. The seine net collector is used mainly in mud flats. A beach drag seine net is used mainly in deep waters of the shoreline. The barrier net is used mainly in estuaries with bridge as deploy platform for high and low water influx (Villaluz 1984). Plankton nets are used in deep waters and also on the bridge as a barrier for trapping fry during high tide (Kawamura 1984). The best design for fry collection in sandy beaches is the bulldozer net whereby one person will push the dozer net while the other person will check the back of the net for fry using a plastic basin. The fine mesh size net of 30 μm is used for collecting the fry constructed onto a bamboo frame. The separation of the fry will be made in a white basin whereby milkfish fry will be spotted as having two spot on the eyes, a fork tail, and a black spot on the middle and the characteristic is of a darting swimming behavior into the current when the water swirled (Pickering et al. 2012).

1.10 Food demand in the Pacific island countries

In the PICs and Territories, fish consumption is common and a major source of protein food as many of the villages are located along the coasts. The average fish consumption per capita in PICs and Territories is 16 -18 kg per person per year (Bell et al. 2009). The decrease of protein food supply from the oceans is an ongoing problem all around the world due to increased demand from the growing human population, and aquaculture is deemed to be an efficient approach to address food demand in the coming future (World Bank 2013). Capture fisheries have so far contributed to food production, but unfortunately they are unable to supply the growing demand as more people request fish and fish products (Tidwell et al. 2001). Aquaculture alone has accounted for 47 % of fish supply as capture fisheries leveled off in the last twenty years (FAO 2016) . Overfishing plays a major role in the decline of coastal ecosystems (Myers et al. 2003) and climate change may play a significant part as well (Williams et al. 2010). Research into aquaculture is increasing globally to identify new approaches that may support food security and sustain community livelihood through sustainable production. Different marine species are studied which increases chances for potential later application in

aquaculture. Milkfish is one of the marine species farmed previously as traditional industry in Asian countries where annual restocking of fingerlings was reared from wild caught fry (Nelson 2007). The culture of milkfish was reported to spread to some PICs and Territories, and the potential is recognized to address food demand (Pickering et al. 2011). In Vanuatu, milkfish forms an extremely small component of substance diet when compared to its multispecies fisheries. There has been a history of collecting seasonal shoals of milkfish fry and juveniles for food (Pickering et al. 2012). In the Solomon Islands, milkfish aquaculture is based on fry collection from the wild, and the economic analysis done by Sulu 2016 has confirmed that subsistence farming is possible if labor cost is zero (Sulu et al. 2016). As such, milkfish subsistence farming may be predictable also to contribute to subsistence diet in Vanuatu. Moreover, reef fish is becoming very expensive in Efate, which is the most populous island in Vanuatu and where the capital of Vanuatu is located, therefore, if lower income households were able to culture milkfish this could improve both their food security and culture options.

The Vanuatu government has recognized milkfish as a priority species for culture in the Aquaculture Action Plan (SPC 2008), but no attempt has been made yet on culturing the milkfish Chanos chanos species. In some PICTs such as Fiji, Kiribati and Solomon Island attempted were made by collecting the fry from the wild for culture based on the knowledge of the seasonality (Billings et al. 2010; Vuto et al. 2014; Wainwright 1982). The Australian Centre for International Agricultural Research (ACIAR) funded project FIS/2012/076 – Improving community based- aquaculture in Fiji, Kiribati, Samoa and Vanuatu, is now supporting the government action plan. In this master’s thesis the seasonality of milkfish fry will be determined as part of the plan for Vanuatu. Upon completion of this, the Vanuatu government will have at disposal useful information for further work, such as trial culturing and assessing the economic viability for milkfish culture. The knowledge of milkfish fry seasonality is the primary basis of the aquaculture process where there is a lack of advanced breeding technology, such as in the PICs and Territories. There has been a history of collecting seasonal shoals of milkfish fry and juveniles for food (Pickering et al. 2012) but to date there has been no attempt to quantify milkfish fry seasonality in Vanuatu. Hence, the knowledge of milkfish fry seasonality and abundance is important for further engagement in milkfish farming in Vanuatu. 15

1.11 Conclusion

The literature review has re-affirmed importance of quantifying the seasonality of milkfish fry in Vanuatu in order to establish baseline information for fry collection, which is a prerequisite for milkfish aquaculture development. Environmental variables can also affect the fry seasonality and spawning time and density. Temperature is known to be a variable that influences milkfish spawning. Milkfish fry seasonality is latitude dependant with two peaks close to the equator and one peak at higher latitudes. This chapter provided some knowledge of milkfish studies in the past that will help to establish the seasonality study in Vanuatu. The methods of fry collection, time of spawning, and other variables that influence fry appearance and abundance were highlighted in the review. The methods for fry collection were reviewed and the best method based on the topography of the sites will be adopted in this study.

1.12 Research objectives

The main aim of this research is to determine the availability of milkfish fry and their seasonality and abundance in a coastal area of Efate Island, Vanuatu.

The following is a list of questions that this study is addressing:

1. Is milkfish present in the selected study areas on Efate Island, Vanuatu? 2. What is the seasonality and abundance of milkfish fry in the selected study area on the coast of Efate Island, Vanuatu? 3. What is the age of milkfish fry at the coast of Efate, Vanuatu?

There are three hypotheses to be tested:

1. Fry abundance in Vanuatu shows a lunar and annual seasonality 2. Environmental factors such as temperature, cloud cover, rainfall, wind speed, current speed, and turbidity are correlated with fry abundance, length and weight.

3. Fry abundance in the New Moon, 1st quarter Moon, Full Moon and 3rd quarter Moon are significantly different. The following is a list of objectives to answer the questions raised above.

1. To determine the presence, seasonality and abundance of milkfish fry at different sites related to the Moon phase in the coastal zones of Efate Island. 2. To estimate the age at which fry appear along the coast of Efate Island. 3. To determine the impact of environmental variables on fry abundance, length and weight.

Chapter 2: Methodology

2.0 Site selection and description

Efate Island is Vanuatu's third largest island and it is where the capital, Port Vila is situated (Figure 2.1). Efate Island has the highest population due to migration of people from the outer island in Vanuatu to Port Vila town for employment and education (Amos 2007). A site inspection was conducted around Efate Island to check for the eligible environmental characteristic such as presence of rivers, lagoons, sandy shoreline, coral reefs, mangrove swamps and brackish shoreline for fry occurrence. The Coast of Efate Island has bays and lagoons with mangrove swamps and rivers. The fringing reefs are narrow and sandy beach is present in the shoreline, as observed during site inspection. The river inputs into the lagoons create turbid in shore water, which is preferred for milkfish fry recruitments. The four closest sites to Port Vila were selected.

The four sites selected for the study were Erakor, Kawenu, Mele and Teouma (Figure 2.2). Their coordinates according to Google Earth are as follows: Erakor: 17°46'54.04"S, 168°19'30.52"E, Kawenu: 17°43'49.30"S, 168°18'18.50"E, Mele: 17°41'31.02"S, 168°15'58.24"E, Teouma: 17°47'27.14"S 168°22'49.73"E. All the four sites have input of freshwater from rivers and lakes into the coastal shorelines, with Teouma having two rivers flushing into the lagoon, one from the East and the other from the West. The similarities between sites were the coastal sandy beaches, brackish water and coral reef within the lagoon. The differences were the size of the lagoon, the source of fresh water (river and swamp), the presence and size of mangrove canopy and the distance of the sites relative to Port Vila town (Figure 2.2).

Figure 2.1 Map of Vanuatu where the study is based. Retrieved from http://www.nationsonline.org/oneworld/map/vanuatu-map

Figure 2.2 Map of the four sites Erakor, Kawenu, Mele and Teouma where milkfish fry was assessed during the preliminary study, and the Vanuatu capital (marked with a star).

2.1 Sampling materials and methods

2.1.1 Sampling materials

A Secchi disk to test water turbidity was made from timber, and the rope on which the disk was mounted was marked up to 100 cm with ±0.5 cm precision. A Kedida concentration tester (CT) -3080 pen type salt and temperature meter were used to measure salt concentration in mg/l with precision of ± 1 mg/l and temperature in degree Celsius (°C) with ± 0.1 °C precision. A thermco water bath thermometer of ± 0.5 °C precision was used later to measure temperature during continuous sampling.

A plastic bottle, stop watch and a nylon rope of 5 m were used to measure current speed along the surf zone of the coastline by deploying a plastic bottle and letting it drift along a 5 m distance while taking the time with a stop watch. Additional environmental parameters such as rainfall, cloud cover and wind speed were obtained from the Vanuatu meteorology and Geo-hazard department (VMDG).

2.1.2 Bulldozer net construction

The bulldozer net was built from bamboo, and tire rubber was used to tie the bamboo sticks together (Figure 2.3). The net was seamed with a 100 μm fishing line onto the nylon rope to keep the edges from wearing and also to retain the shape. A wire was used to attach the net at each corner of the frame and a fishing line was used to tire the net into position. The pouch was a fine mesh size of 150 μm while the wings of the net were made of a larger nylon mesh size of 250 μm.

The diagram of the frame with the measurements is shown in figure 2.4. The length of the bamboo frame was 2.6 m and the back of the frame was narrow to allow for easy pushing and collection of the fry. The front was wide which was used for the wing controlling the fry.

The diagram of the net and measurement are shown in figure 2.5. The pouch was short and covered about 1/3 of the total length. The back of the net was deep to enable fetching when hauled to check for milkfish fry (Figure 2.5).

Figure 2.3 The bulldozer net built and used for this study. Picture shot at Teouma on March 2016.

Figure 2.4 The bamboo frame design for the bulldozer net (Pickering et al. 2012)

Figure 2.5 Net size and design modified from Pickering et al. (2012)

2.1.3 Sampling method

The net was first tested for its effectiveness and efficiency at the Kawenu lagoon on the side towards Port Vila. The first deployment showed the catching of a juvenile grouper after three trials pushing over 25 m along the surf zone. Sampling trips were made once in every Moon phase for one-year period. Overall, 69 sampling trips were made during the period of this study from February 17th 2016 to February 15th , 2017 of which 18 trips were made for the preliminary site assessment. A total of 7 to 8 net deployments were made per sampling trip whereby each deployment covers a push over 25 m distance at a time of 5-7 minutes. Each deployment is referred to as effort whereby the net was checked for the fry presence for each deployment.

Fry abundance was checked in the surf zone. Net deployment was done only during daylight hours between 6 am to 6 pm during the 1st and 2nd hour of high tide. The abundance of larvae is expressed as number collected per effort (Morioka et al. 1993) where the effort is the number of bulldozer net deployments.

2.2 Fry identification

Milkfish fry identification was made visually with the help of a white plastic basin on the field. Milkfish fry (Figure 2.6) is characterized by lack of pigmentation, two dark spots (the eyes) on each side of the head, a dark spot (the air bladder) on the middle of the body and also a light grey spot on the caudal fin(Pickering et al. 2012). The fry uses a darting swimming motion with the tendency to swim against the current.

Figure 2.6 Milkfish fry collected in a white plastic basin for visual identification. The black dot from left to right showed position of the grey spot on the caudal fin, dark spot in middle of body (air bladder) and two dark spot (eyes) on each side of the head.

2.3 Fry length and weight measurement

After identification, the larvae were dipped into 95% ethanol for 10 min to enhance hardness for measuring and weighing. After 10 min, the TL of the larvae was measured with a ruler and expressed in millimeters and weighed in grams with an electronic balance (FX-1200 SN1010061) with a scale of 3 decimal places and precision of 0.1 g. After mass and length measurements were done,

each larva was transferred into small containers of 20 ml in 95 % ethanol until further use (Miller et al. 1994).

2.4 Otolith analysis

Before otolith analysis, a maximum of 5 fry from each sample collected in each month was measured and TL measures were converted to equivalent fresh-state using the equation from Morioka et al. (1993)on milkfish length conversion applying the isopropanol equation. The equation for isopropanol was used as there was none for ethanol available. The isopropanol equation employed was, (Equation 2):

Equation 2: ܶܮ ൌ ͵ǤͶͳ͹ ൅ ͲǤͺͳͶܮ௜

Where TL is the total length of fresh specimen and Li is the length of the specimen in isopropanol (Morioka, Ohno et al. 1993).

Examination of otolith was made on the sagittae on increment of Teouma wild caught fry excluding the ones from Erakor, Kawenu and Mele, as the seasonality study only focused on one site. A total of 91 fry was randomly selected from the samples collected in Teouma for the examination.

The sagittae were removed from both sides of the milkfish fry cranium by teasing them with an insulin syringe. Each sagitta was then mounted onto microscope slides and immersion oil was used for otolith observation. Count of the total number of increments was done under an Olympus dissecting light microscope SZ-ST type of magnification ranging from 100 to 500x. The photos were taken with an Infinity 1 canon camera (A-1) connected to the microscope and counts were done twice, three days apart, by using the same photographs. A third count was done under the microscope. The three counts were validated using analysis of variances (ANOVA) single factor in excel and only the counts from the microscope were used in the age estimate.

The age of milkfish was determined from the reading of otoliths. In milkfish the first increment is formed two days after hatching during yolk sac re-absorption period and the increments continue to grow daily regardless of the growth rate 25

(Bagarinao 1991). The relationship between the number of increments and the age in days is the following (Equation 3):

Equation 3: D = N + 1

Where N = number of increments and D = Days from hatching

The equation, developed by Bagarinao (1991) was used to backdate the time of hatching and thus the date of spawning was calculated from the counting done under the microscope.

2.5 Data analysis

The comparison of milkfish fry abundance in the different sites, months and Moon phases were determined using ANOVA in R version 3.4.0 (R Core Team 2007) with a significance level of 0.05. The difference in weight and length between Moon phases was also determined by the same test. The fry seasonality data was presented from August 2016 to January 2017 and from February 2016 to July 2016 to better depict the seasonal occurrence of milkfish fry.

The environmental data collected during sampling and those collected on sampling day by VMDG were analyzed to evaluate their effect on the milkfish fry abundance, length and weight. Regression analysis with multiple predictors, also known as multiple regressions, was run and model selection was made by comparing the most complex model with simpler ones in R for all variable data such as sea surface temperature measured in situ, cloud cover, rainfall, turbidity and wind speed. The Pearson correlation test for statistical software version 10 (Statistica Inc 2011) was conducted to verify the correlation coefficient and significance level at 95% significance level and then used as final. Lastly the correlation between age, length and weight of fry was done in Microsoft excel version 15 (Microsoft Officer 2013).

Chapter 3: Results

A total of 47 fry were captured in the four sites altogether for the site assessment, with 369 fry collected in the Teouma bay during a one year period and were used for the following analysis except for aging, which was done on a subsample of 91 fry.

3.1 Site assessment and selection

Results for the four environmental parameters (i.e. turbidity, current speed, salinity, and temperature) assessed over a month in Erakor, Kawenu, Mele and Teouma are summarized in table 3.1. Erakor, Kawenu, Mele and Teouma, had a mean turbidity of 80 ± 12.2 cm, 75.8 ± 10.3 cm, 89.3 ± 4.0 cm and 69.5 ± 11.2 cm respectively. During the first 1-2 hour of high tide, Teouma had a mean current speed of 0.01 ± 0.0 m/s and Mele, Kawenu and Erakor of 0.01 ± 0.1 m/s. In ascending order Teouma, Kawenu, Erakor, and Mele had mean salinity of 8.4 ± 2.5 parts per thousand (ppt), 14.1 ± 3.0 ppt, 14.5 ± 3.5 ppt and 15.3 ± 3.5 ppt respectively. The mean monthly sea surface temperature (SST) measured showed Mele had the lowest (30.4 ± 1.7 qC), while Teouma, Kawenu and Erakor had a higher temperature (30.7 ± 0.5 qC, 31.1 ± 3.5 qC and 31.3 ± 2.0 qC, respectively).

Sites Landscape Turbidity Current Salinity SST features (transparency) speed (ppt) (ºC) (cm) (m/sec)

Mele Sandy beach, 89.3 ± 4.0 0.1 ± 0.1 15.3 ± 3.5 30.4 ± 1.7 coral reef, two rivers Kawenu Sandy beach, 75.8 ± 10.3 0.1 ± 0.1 14.1 ± 3.0 31.1 ± 3.5 coral reef, spring

water, mangrove habitat Erakor Sandy beach, 80.0 ± 12.2 0.1 ± 0.1 14.5 ± 3.5 31.3 ± 2.0

coral reef, lagoon, swamps Teouma Sandy beach, 69.5 ± 11.2 0.1 ± 0.0 8.4 ± 2.5 30.7 ± 0.5 coral reefs, two rivers

Milkfish fry appeared in the four sites with the least abundance recorded in Erakor, Kawenu and Mele (1, 2, and 1 fry, respectively) and was significantly different (p = 0.01) from those in Teouma with the highest (43 fry) (Figure 3.1). The lowest number of fry at Erakor, Mele and Kawenu were not significantly (p > 0.99) different from each other.

The study proceeded only in Teouma, based on low numbers of abundance in the other sites. Thus, the following results refer only to Teouma.

Figure 3.1 Milkfish fry total abundance (number of fry captured during the exploratory trial done on Erakor, Kawenu, Mele and Teouma). The different letters indicate significant difference in abundance (p < 0.05) while the same letter indicates no significant difference (p > 0.05).

3.2 Milkfish fry occurrence in Teouma

3.2.1 Monthly occurrence and abundance

Milkfish fry occurrence in Teouma started in October and ended in early May (Figure 3.2). The month of November and April showed the two highest peaks in abundance (120 and 92 fry respectively) with no significant difference where p = 0.67 (Appendix 1). In the months of October, December, January, February, March and May the abundance was low (19, 24, 3, 55, 41 and 15 fry, respectively) and significantly different from those November (92) and April (120) where p < 0.05 (Appendix 1). The months of June, July, August and September showed no occurrence of fry, but were not statistically different (p > 0.05) from the months with low abundance October, December, January, February, March and May except for July and February which were significantly different to each other where p = 0.03 (Appendix 1). The sea surface temperature (SST) collected from both Meteorology department and in situ shows milkfish fry occurrence at monthly mean SST of 26℃ and above while no occurrence is observed at mean SST below 26℃.

Figure 3.2 Milkfish fry abundance and occurrence with SST by month for Teouma. The different letters indicate significant difference in fry abundance (p < 0.05).

3.2.2 Milkfish fry length and weight

The mean total length (TL) of the collected fry was 12.8 ± 1.2 mm (Appendix 3). The Moreover, 59 % of the fry had length from 13 to 14 mm and 33 % had the length between 11 and 12 mm (Figure 3.3). The minimum length recorded was 8 mm and the maximum was 15 mm.

The weight of the milkfish fry ranged from 3 mg to 9 mg, with an average weight of 5.8 ± 1.6 mg (Figure 3.4, Appendix 3). A total of 65 % of fry in Teouma had a weight range between 5 mg and 7 mg while 17 % of the fry caught had a weight of 8 mg and 9 mg.

The fry TL by months shows that May had the highest mean TL (Figure 3.5, A) and is significantly different in April and October (p = 0.04 and p = 0.04 respectively)

with the lowest meann TL. The comparison of weight by monthh (Figure 3.5, B) of sampling shows no sttatistical significance (p > 0.05) with the weigght of fry collected.

Figure 3.3The total leength frequency of milkfish fry (N = 369) in theth Teouma

Figure 3.4 The frequency distribution of milkfish fry (N = 369) weeight in Teouma

Figure 3.5 Milkfish frfry mean total length (A) and weight (B) per samplings month. The different letters inndicate significant difference (p<0.05). The errore bars represent the standarrd deviation of the mean.

3.3 Effect of Moon pphases

3.3.1 Abundance by Moon phase

The distribution of cuumulative fry abundance at the four consecutiive Moon phases showed that the higheest fry abundance (N = 160) was observed duuring New Moon, followed by 3rd quarteer (N = 120), with no significant difference (pp = 0.44) between the two (Figure 3.6). Milkfish fry abundance was lower in the 1st quarter and Full Moon (N = 57 and 344 fry, respectively), and were significantly diffferent from the abundance in New MMoon (p = 0.001 and p = 0.001 respectively). TheT fry abundance in the 1st quarter and 3rd quarter Moon phase were not significantlly different (p = 0.12).

Figure 3.6 Milkfish fry cumulative abundance in all Moon phases for Teouma throughout the year. The different letters indicate significant difference is the abundance between Moon phases (p < 0.05).

3.3.2 Total length and weight in Moon phase over one year

The mean total length of the fry captured at 1st quarter, Full Moon and 3rd quarter (13.1 ± 0.9 mm, 13.5 ± 1.0 mm and 13.1 ± 1.2 mm, respectively) were not significantly different (p > 0.05) from each other (Figure 3.7.A, Appendix 1). The fry in 1st, 3rd and Full Moon had a mean total length higher than the fry captured at New Moon (12.2 ± 1.2 mm) and were significantly different (p < 0.05) from each other (Figure 3.7).

The fry captured in the 1st quarter Moon phase had the lowest mean weight (4.7 ± 1.5 mg) followed by the Full Moon (4.9 ± 1.8 mg) and not significantly different (p = 0.61) from each other (Figure 3.7, B). In the 3rd quarter and New Moon the fry had the highest mean total weight (5.3 ± 1.7 mg and 5.4 ± 1.6 mg, respectively) and these were not significantly different (p = 0.10) from each other. The fry sampled at the Full Moon had a mean weight not significantly different (p > 0.05) from the other Moon phases (Appendix 1).

Figure 3.7 Milkfish frfry mean total length (A) and weight (B) in fouur Moon phases. The different letters sshow significant difference in mean total lenggth or weight (p < 0.05) and the error baars represent the standard deviation of the mean.m

3.4 Comparison of ffry abundance with other indices

3.4.1 Environmentall variables correlation with fry abundancee

The environmental vaariables assessed (temperature, rainfall, turbiddity, wind speed and current speed (haad no significant influence on the milkfish fryy abundance along the coastal shoreline aas shown by the low correlation values (p > 0.05)0 (Figure 3.8). However, cloud coveer had a significant influence on the milkfish fryf abundance (p < 0.05) whereby fry abuundance increased with cloud cover.

Figure 3.8 The enviroonmental variables, rainfall, cloud cover, winnd speed, temperature, turbidityy (transparency) and current speed, correlateed with milkfish fry abundance.

3.4.2 Environmentall variables correlation with length and weiight of milkfish fry

Milkfish fry length shhowed non-significant correlations with tempperature, cloud cover, rainfall and turrbidity (p > 0.05) (Figure 3.9). The wind speeed and current speed showed a significant (p < 0.05) positive correlation (r = 0.244 and r = 0.21, respectively) with fryy length.

Figure 3.9 The enviroonmental variables, temperature, cloud coverr, rainfall, turbidity (transparency), wind speed and current speed correlation with milkfish fry total length

Milkfish fry weight showed non-significant (p > 0.05) correlationss with temperature, cloud cover, wind speeed and current speed (Figure 3.10). There was a significant (p = 0.02) positive correelation in milkfish fry weight with rainfall andd turbidity as transparency increasee.

Figure 3.10 The environmental variables, temperature, cloud cover, rainfall, turbidity (transparency), wind speed and current speed, correlation to milkfish fry weight

3.5 Otolith analysis and age of the fry per month and spawning estimates

The age of the fry was assessed by measuring the number of increments in the otoliths. In figure, 3.11 are shown examples of this assessment. In more detail, the increment of a young larvae of 14 days old (Figure 3.11, a) has wider rings indicated with a series of black dots while older larvae of 22 days (Figure 3.11, c) have narrow increments. The star at the center of otolith indicates the core which was formed before the first increment and the discontinuity zone shown by the light zones indicated with the bar (Figure 3.11, a). There are four dimensions on the otolith sagittae (Figure 3.11, a) of a milkfish fry, the posterior, ventral, dorsal and the anterior. The 3 photos (Figure 3.11, a, b, c) indicate a typical 14 days, 15 days and a 22 day old milkfish fry otolith increments.

Figure 3.11 Photographs a, sagittae otolith from 14 days old larvae, 11 mm total length (TL). (x400), b is an otolith of a 15 day larva of 11 mm TL and, c is an otolith of a 22 day larva of 14 mm TL. In photograph a, letters. A, anterior; P, posterior; D, dorsal; V, ventral. The dots (●) are the increment dark zone, the star (ღ) indicates the core and the arrow (↓) indicate the discontinuity zone.

3.6 The age distribution

The milkfish fry at Teouma coast were of age that ranged from 13 days to 25 days old (Figure 3.12). The fry age that ranged from16 days to 18 days accounted for 57 % of the total fry caught while 29 % of the fry were aged between 19 to 22 days.

Figure 3. 12 The age frequency of milkfish fry in Teouma coastal shoreline

The age distribution between months revealed by otolith analysis two distinct age groups, the first from October to December and the second was from February to May (no otolith analysis was performed with samples from January) (Figure 3.13). The oldest fry appears in March, April and May.

Figure 3.13 The mean age of fry per month of occurrence in Teouma. The error bars represent the standard deviation of the mean.

The relationship between total length and age shown in figure 3.14 indicates that the length was directly proportional to the age where r = 0.46. Thus length also increases with age at occurrence of fry and were significantly correlated (p < 0.05).

Figure 3.14 Correlation between the age and length of milkfish fry

3.7 Spawning time estimates for Teouma over one year period

The backdating of the otolith data for each sampling trip revealed that milkfish- spawning season starts in September and ends in April (Figure 3.15). Moreover, there were two spawning groups or seasons, one from September to November and the other from January to April. Milkfish spawning does not occur in December, May, June, July and August. It is observed that milkfish spawning activity in Teouma is link to the mean Monthly SST of above 25 ℃, and is the predicted spawning threshold (Figure 3.15).

Figure 3.15 The spawning season of milkfish fry (N =63) based on different days over a one year period with mean Monthly SST

3.8 Comparison of fry abundance in the Melanesian PICs The fry abundance in Teouma for one day over 1 hour of sampling using bulldozer net is very low (0-79 fry) compared to the fry abundance reported in Fiji and the Solomon island with 200-100 and 0-1000 respectively over one day of sampling (Table 3.2).

Table 3.2 The fry abundance between Fiji, Solomon Islands and Vanuatu

Chapter 4: Discussion

The main goals of this study were to identify milkfish fry seasonality, abundance, and spawning season, and to identify the environmental parameters such as Moon phases, cloud cover, current speed, rainfall, turbidity, temperature and wind speed for their effects on milkfish appearance and abundance in Vanuatu. The knowledge of fry seasonality is crucial for Vanuatu, as hatcheries are too expensive to operate due to the high cost of induced spawning technology, bloodstock hatchery, and management. Similarly, according to FitzGerald (2004), the high cost of capital investment along with investment on technically trained staff will make the development of hatchery not advisable in the initial stage of milkfish aquaculture. Therefore, the study can aid policy implementation based on this information for milkfish culture purposes in Vanuatu.

Initially, four sites close to Port Vila on Efate Island, namely Erakor, Kawenu, Mele, and Teouma were selected for a preliminary assessment. The sites were selected due to the presence of rivers, freshwater sources, and landscape features such as a lagoon, sandy beach, coral reef and coastal mangrove forest. The presence of rivers and lagoon creates favorable environmental conditions known to be important for milkfish fry appearance (Bagarinao et al. 1987; Bagarinao 1991; Brian 2015). All the selected sites have similar topographic features of a gentle slope on the surf zone whereby a bulldozer net method of fry collection was selected as it was used for the first time in the Philippines in 1983 and was deemed effective along surf zones of sandy coastal shorelines (Villaluz 1986). The pushes are made parallel to the coastline facing the direction of the wind. The bulldozer net design used in this study was a modification from the one used by Pickering et al. (2012). In Vanuatu, the bulldozer net was never previously used for any purpose and that includes milkfish fry collection, nevertheless, sampling done with the bulldozer net was proven efficient in this study and thus its further use is highly recommended in locations with similar characteristics.

After the completion of one-month assessment in the four sites, it was observed that milkfish fry was present in all four sites. However, Erakor, Kawenu, and Mele, the 42

sites closest to Port Vila, had the lowest abundance in milkfish fry. Teouma was the site furthest from Port Vila where the council of chiefs has control of the coastal resources. Recently they imposed regular seasonal banning of fishing from November to April as they believe it is the season when much of the spawning occurs. In Pakistan, a study on the breeding season of Schizothorax plagiostomous by Jan et al. (2017) using GSI techniques resulted that spawning occurs twice a year, once in March-April and October-November. Similarly, an evaluation of closed area for fish stock conservation in Europe by Horwood et al. (1998) revealed that closure areas were likely to have benefited many commercial and unregulated fisheries species. In addition, a study of the sustainability of two coral reef fish (Amphiprion percular and Chaetodon vagabundus) larvae recruitment in a Marine Protected Area (MPA) in Papua New Guinea revealed that even small MPAs may be self-sustaining in larvae recruitment from the adult spawning in the same location (Berumen et al. 2012). This may be the same case for the seasonal closure in Teouma where adult milkfish around the coast will only sustain fry recruitment along the Teouma shoreline. However, this needs to be verified with a more comprehensive research. In addition, the presence of two rivers within the lagoon in Teouma also makes a good fry collection ground. This is similar to what was found by Sulu et al. (2016) in the Solomon Island that better fry collection grounds are usually located close to river mouths and swamp outlets. Furthermore, Bagarinao (1991) has stated that the most important component of the water chemistry is the muddy smell, composed of organic compounds called geosmin which is formed from the bacterial breakdown of detritus and benthic zooplanktons that triggers most of the fry appearance to the coastline to feed where conditions are favorable.

The physical parameters of turbidity, current speed, salinity, and temperature, were measured at sampling during the site assessment and some differences between the sites were recorded, that may aid to explain the differences in milkfish fry abundance. It was evident in Teouma that physical parameters such as temperature, current speed, turbidity, and salinity do not fluctuate when compared to the other three sites Erakor, Kawenu, and Mele. The stability of the environmental conditions is crucial for milkfish larvae survival, similarly, the high survival rate of milkfish

juvenile was observed by Jaspe et al. (2012) in the pond when there is enough food supply and water quality is stable.

Milkfish fry appearance in Teouma started in October and continued to occur until early May which is similar to their occurrence in Fiji from September to May (Pickering et al. 2012) and has two peaks which are similar as in the Solomon island (Sulu et al. 2016). A study on the gray mullet Mugil cephalus fry seasonality in South Africa by Bok (1979) revealed that recruitment along the coastline occurs from May to November and the peak period of occurrence took place from July to October. The seasonality of Mugil cephalus in South Africa is different to milkfish fry occurrence in Vanuatu. In India, Sarojini (1951) has determined that the fry of Mugil tade and Mugil parsia usually occur from November to February (Oren 1981). On the Teouma coast of Efate, milkfish fry recruitment had two peaks of occurrence, one in November and the other in April (120 and 94 fry respectively) which is similar to the number of peaks recorded in Kiribati although the peaks were shorter compared to Kiribati with peaks from May to September and December to April (Wainwright 1982). However, fry seasonality in Kiribati is similar to those of Solomon Islands (Sulu et al. 2016), as it occurs throughout the year, which is different to Fiji and Vanuatu with 8 and 9 months respectively. In October, December, January, February, March, and May a low number of milkfish fry was observed in Teouma. The months of October and January were the beginning of the first and second period of fry seasonal appearance, which later leads to the peak occurrence in November and April. The reduction in fry abundance in March may be related to the effects of storm surge due to cyclone Winston which started early in February in North West of Port Vila in Vanuatu. Similarly, a visual census carried out in 8 coral patch reef in Lizard Island of great barrier reef during larval seasonal peak occurrence 12 days before and 2 days after a cyclone resulted in fewer juveniles recruited into the coral reef due to storm surge (Lassig 1983). The study has concluded that cyclone has marked effects on the fish assemblage as a whole. In Teouma, the sampling done 2 days after the week of storm resulted in no fry and they become available only on the sampling day, which is 10 days after the storm surge of the cyclone. Therefore, the larvae could have suffered additional mortality,

which resulted in a decline of fry abundance in March. No fry was captured in the months of June, July, August, and September. The close range in length of fry occurrence in Fiji and Vanuatu may be related to the closeness in latitudinal position between the two countries as it was stated that countries with similar latitude would have similar periods of occurrence (Bagarinao 1994b). Milkfish fry in the coastal shoreline of Teouma occurs mainly in the summer months, which may be the same for their occurrence reported in Fiji. However, the low abundance observed in this study could also be related to sampling chance, as there is no significant difference in abundance in the month of low fry occurrences and those of no fry occurrence. A different method of sampling is required to avoid errors in sampling chance as in the Solomon Islands and Fiji more fry was caught using other methods (hand dip net, scoop net) in narrow, shallow lagoon compared to bulldozer net (Sulu et al. 2016). By comparing the fry abundance in Teouma to Fiji and Solomon Island’s data, fry abundance in Teouma (0-79 fry per hour) may not be so different using the bulldozer if sampling is done for more than one hour per day. In Fiji and Solomon islands, fry were collected at 200-1000 and 0-1000 fry in a sampling day respectively (Napulan 2012; Sulu et al. 2016).

Milkfish fry TL for Teouma coast ranged between 8 mm and 15 mm. This is similar to those appeared in Japan and Philippine which ranged from 10 mm to 17 mm (Kumagai 1984; Senta et al. 1981), however, they are more similar to those occurred in Solomon island with a length range of 10 mm to 15 mm (Sulu et al. 2016). In the Philippines, milkfish larvae of less than 9-10 mm TL are present in offshore surface waters but are driven onshore by wind-driven current and tidal current (Kumagai 1984). The weights of the fry in Teouma ranged from 3 mg to 9 mg and were similar to those reported in Taiwan by Liao et al. (1977) with the weight range from 3.2 mg to 11.2 mg. In regards to seasonal abundance, milkfish fry was abundant in summer compared to winter. The two peaks in fry seasonality were in line with previous findings (Kumagai 1984), where it was determined that fry seasonality is long near the equator with two peaks of which April-May is higher and progressively shorter at high latitudes. The fry seasonality in Teouma occurs at monthly mean sea surface temperature (SST) above 25℃ (figure 3.2) and most spawning activity also occurred

at temperatures above 25℃, similarly, milkfish fry occurrence in Fiji, Solomon Islands, and Kiribati occurs at the same temperature (Bagarinao 1991; Sulu et al. 2016). Therefore, the SST threshold value for prediction of milkfish fry occurrence is 25℃ and above and is noted in the Philippines (Bagarinao 1991).

The milkfish fry occurrence in the different Moon phases showed a high frequency in New Moon and 3rd quarter Moon phase (120 and 94 fry respectively). This pattern observed in Teouma was different to the pattern of fry occurrence determined in the Philippines whereby spawning occurs in quarter Moon period and fry are most abundant in Full and New Moon period (Bagarinao 1991; Kumagai 1984). In addition, in the Solomon Islands, the catch during the New Moon period is higher than the Full Moon, which is similar to those observed in Teouma but a non- significant difference was identified (Sulu et al. 2016). It was anticipated by Garcia (1990) that milkfish fry is more abundant between full Moon and New Moon periods after observing the seasonality of fry in the Philippines. However, this study carried out in Vanuatu supports that fry is more abundant in New Moon period than in the Full moon period. This is different to what is observed in Solomon Island and in the Philippines. A study conducted by Horký et al. (2006), on the behavior of fish in Moon phase using pikeperch, in the behavior revealed that New Moon has increased diurnal activity compared to other Moon phases, similarly as the sampling was done during the day, more fry was caught in New Moon. The results of this study also show that smallest fry are more abundant in quarter moon period and otolith analysis also proved that younger fry occurs in the similar period. Concerning the weight of the fry in Moon phases, those fry appeared in New Moon have more weight. According to Horký et al. (2006), fry activities increase in the New moon period. This study supports that as fry activity increased in New Moon period, feeding could also increase which result in more weight of fry in New Moon.

The environmental variables assessed (temperature in situ, rainfall, turbidity, wind speed and current speed) had no significant influence on milkfish fry abundance along the coastal shoreline of Teouma. The monthly mean SST (figure 3.2, 3.15) indicates a possible link with fry seasonality although there was no correlation, and this may need further assessment to validate. Fry were caught only on the months when monthly mean SST was above 25 ℃ and this could be the threshold for

prediction fry occurrence. Similarly, countries like Fiji, Solomon Island and Kiribati have records of fry occurrence at monthly mean SST above 25℃ (Bagarinao 1991). However, cloud cover had a significant influence on milkfish fry abundance whereby fry abundance increased with cloud cover that may reduce fry capability of seeing and escaping the net. It was also noted by Kawamura (1984) after testing fry visibility with black and white net mesh that fry escape white net mesh while they retain more in the black mesh net. Nevertheless, a white color net mesh was used in this study to ease milkfish fry identification, and as a result, fry tends to be caught in higher numbers during high cloud cover possibly because net visibility is reduced. In future studies, a black net should also be tested in Teouma to address the low capture numbers and evaluate if these are related to the color of the mesh used.

The correlation of environmental variables studied with the length of fry over one year period revealed that milkfish fry length was not significantly correlated with temperature, rainfall, turbidity and cloud cover. However, wind speed and current speed showed a significant correlation whereby larger fry were caught with increasing wind speed and current speed. The wind speed drives fry into the net by blowing onto the surface current causing larger fry difficulty to escape the net as the net was deployed facing the wind direction. It was also reported in the Philippines that milkfish fry were more abundant in favorable wind direction, particularly when the wind blows towards the shoreline (Senta et al. 1981; Villaluz et al. 1983b).

The environmental variables, temperature, cloud cover, wind speed and current speed, showed no significant correlations to milkfish fry weight. However, there was a significant positive correlation of the fry weight to rainfall and increased turbidity. Rainfall is important to fry as it increases river flow, causing suspension of detritus in the coastline where more zooplanktons feed on them. Similarly, Edwards (2001) has determined that zooplankton feeds on detritus and as more zooplanktons are available, the fry feeding activity increases and thus the weight increases. In addition, rainfall increases saturated oxygen concentration (Li et al. 2015), whereby it was determined by Mallya (2007) using a case study on Atlantic halibut cultured under various saturated oxygen concentration whereby growth and feed conversion increase with saturated oxygen.

The age of the milkfish fry were assessed by measuring the number of increments from 92 milkfish larvae. The fry (8-15 mm TL) captured on Teouma coast were of age ranging from 13 days to 25 days old which is similar to the one determined in the coast of Japan documented by Kawamura et al. (1984) where milkfish fry within 10 - 17 mm TL were aged between 14 to 29 days. Similarly, the fry result from Teouma showed that more fry were aged between 16 and 18 days old which is not different to those determined by Kawamura et al. (1984) whereby the fry length 12.5 -14.0 mm TL were aged 18 to 21 days old. The result of the study revealed that milkfish fry spend 2 to 4 weeks before disappearing for the nursery which correspond to the size at transition stage reported by Bagarinao (1991). The fry length was positively correlated to age, similarly, (Sackett et al. 2013) have stated that fish length increases with age.

The spawning season of milkfish fry was determined by backdating of the otolith data and revealed that milkfish spawning starts in September and ends in April, this links to the period of fry occurrence mentioned previously in the first section, whereby fry occurs in October following the spawning activity in late September. Furthermore, there were two spawning groups or seasons, one from September to November, and the other from January to April, which relates to the two peaks of fry occurrence. The peak period of fry occurrence is short in the two periods of spawning season. Helfman et al. (2009) found that typically pelagic fishes spawn over a 4 month period with a short period of maximal activity, after determining the spawning period of Cod in the North Sea of the Southeast coast of England where spawning occurs between January and May where 70% of eggs produced during 6 weeks period. Milkfish spawning in Vanuatu does not occur in December, May, June, July, and August. According to the results of the backdating, there is a period of 44 days lapse between the first phase of spawning and the second phase of spawning activity. The spawning season of mullet in Australia was studied by Horký et al. (2006) who determined that Mugil chepalus spawning occurs between March and September which is different to that of milkfish Chanos chanos in Teouma coast of Vanuatu. The Mugil cephalus of the Mediterranean shore of Israel spawn from July to December (El Meseda et al. 2006). It is seen that different species of mullet spawn at different times of the year based on salinity and geographical location as determined for Mugil cephalus (El Meseda et al. 2006). Similarly, Martinez et al. 48

(2006), has reported that different localities have different milkfish spawning seasons whereby spawning can occur more than once during the annual spawning season. In addition, an oocytes analysis of a hormone-induced trial on milkfish by Lee et al, (1986) indicated that milkfish can spawn several times during the annual breeding season (Garcia 1990). The otolith analysis resulted that some months fry appear onshore were then to be older. The cause of the difference in age maybe related to many factors.

Otolith analysis revealed that fry caught in November and February had the lowest mean age. Older fry were caught in two periods, the first was in December and the second period was in March and April. Further research is needed to determine factors correlated to differences in fry age between the months of occurrence.

However, by synthesizing data from both seasonality of fry occurrence, age and spawning period, it may be possible that this data can be used for management purposes to predict fry seasonal occurrence. The months when occurrence peaks can be used to determine abundance in other islands and possible fry collection ground may need to be banned from catching of milkfish during the breeding season. Therefore, milkfish aquaculture development in Vanuatu can be enhanced with this knowledge of fry seasonal occurrence.

Chapter 5: Conclusion

This study has concluded that milkfish fry is available along the coast of Efate Island in Vanuatu and it was present in all four sites assessed, though the abundance recorded was low and thus unlikely to be sufficient to support milkfish aquaculture. The sites close to Port Vila, Erakor, Kawenu and Mele showed low abundance in milkfish fry compared to Teouma, which is the furthest site from Port Vila and was selected for the seasonality study. The season of milkfish fry occurrence on the Teouma coast of Efate started in October and ended in May, with two peaks of occurrence identified, one in November and the other in April. The fry occurrence was correlated with the spawning, which probably occurred from September to November and from January to April, as inferred from aging analysis. Since the study was done in the central province of Vanuatu, it is recommended that further assessment of milkfish fry should be carried out also in other islands where conditions are favorable for their occurrence.

The abundance of wild fry collected over the period of this study may not be sufficient to support a subsistence culture of milkfish, as the numbers were quite low. A comprehensive assessment of the abundance and seasonality is needed across the Vanuatu groups of islands for the presence of milkfish fry and their occurrence in order to close the seasonality of occurrence in Vanuatu and other methods of fry collection may also needed to be tested on the abundance. A thorough study on fry abundance will help determine whether milkfish culture in Vanuatu can rely on the collection of fry from the wild. Additionally, the absence of fry in some months could also be related to sampling chance, as there is no significant difference in fry abundance between the month of low occurrence and absent. Most of the environmental variables did not correlate with abundance, length, and weight of milkfish fry, which may require further research to prove. Concerning Moon phases, fry were more abundant in the 3rd quarter and New Moon compared to 1st quarter Moon phase and Full Moon.

The period of fry occurrence is 8 months long, which is similar to that of Fiji. However, the fry abundance was too low so the aquaculture of milkfish is still a question that needs further research although the seasonality can be predictable. A

nationwide assessment can be done using this study as a pilot for spawning and fry occurrence. The island of Santo, Malekula and Tanna would be the other sites that look promising for the assessment, as there is plenty of bay and rivers connected to the sea. However, it is recommended that after a complete assessment the seasonality study should be done repeatedly over years to be able to build a complete cycle of milkfish fry occurrence in Vanuatu.

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Appendix

Appendix A. Table of p-values for the ANOVA test

The following data were tested with ANOVA for the level of significance in R- software Sites Compared Milkfish fry abundance Significance level = p-value (95%) to (number) Kawenu Erakor 1.00 Mele Erakor 1.00 Teouma Erakor 0.00 Mele Kawenu 1.00 Teouma Kawenu 0.01 Teouma Mele 0.01

Month Compare Milkfish fry abundance Significance level = p-value (95%) to (number) January November 0.00 February November 0.02 March November 0.03 May November 0.00 October November 0.00 December November 0.00 January April 0.00 February April 0.00 March April 0.00 May April 0.00 October April 0.00 December April 0.00 November April 0.67 October June 0.99 December June 0.94 January June 1.00 February June 0.16 March June 0.27 May June 1.00 October July 0.96 December July 0.82 January July 1.00 February July 0.03 March July 0.08 May July 0.99 October August 0.98 December August 0.89 January August 1.00 February August 0.08 March August 0.17

May August 1.00 October September 0.98 December September 0.90 January September 1.00 February September 0.08 March September 0.17 May September 0.99

Month Compare Milkfish fry length (mm) Significance level = p-value (95%) to January April 0.89 February April 0.02 March April 0.59 May April 0.03 October April 0.92 November April 0.04 December April 0.38 January December 1.00 February December 1.00 March December 1.00 May December 0.95 October December 0.17 November December 1.00 January February 1.00 March February 0.96 May February 0.97 October February 0.03 November February 0.99 March January 1.00 May January 1.00 October January 0.68 November January 1.00 May March 0.67 October March 0.29 November March 1.00

Month Compare Milkfish fry weight (mg) Significance level = p-value (95%) to January April 0.67 February April 1.00 March April 1.00 May April 1.00 October April 0.28 November April 0.61 December April 0.98 January December 0.91 February December 1.00

March December 0.96 May December 1.00 October December 0.96 November December 1.00 January February 0.97 March February 1.00 May February 1.00 October February 1.00 November February 1.00 March January 0.62 May January 0.79 October January 1.00 November January 0.91 May March 1.00 October March 0.28 November March 0.69

Moon Compared Milkfish fry abundance Significance level = p-value (95%) to (number) Full 1st moon 0.86 New 1st moon 0.01 3rd Moon 1st moon 0.12 New moon Full moon 0.00 3rd Moon Full moon 0.02 3rd Moon New moon 0.44

Moon Compared Total length for fry Significance level = p-value (95%) to (mm) Full 1st moon 0.60 3rd Moon 1st moon 0.10 New moon 1st moon 0.00 3rd Moon Full moon 0.30 New moon Full moon 0.00 New moon 3rd moon 0.00

Moon Compared Total weight of fry Significance level = p-value (95%) to (mm) Full 1st moon 0.29 New moon 1st moon 0.00 3rd moon 1st moon 0.00 New moon Full moon 0.53 3rd moon Full moon 0.61 3rd moon New moon 1.00

Appendix B. The dailyy SST from VMDG and monthly mean, maximmum, minimum, median and standardd deviation beginning from February 17th 20116 to February 17, 2017.

Appendix C. Milkfish fry mean length and weight plus the descriptive statistic for Teouma over one year period

Descriptive Length (mm) Weight (mg) statistics Milkfish fry Mean 12.8 5.78 Standard Error 0.1 0.08 Median 13.0 6 Mode 13.0 5 Standard 1.2 1.6 Deviation Sample Variance 1.5 2.5 Kurtosis 0.4 -0.5 Skewness -0.6 0.2 Range 7 6 Minimum 8 3 Maximum 15 9 Sum 4709 2135 Count 369 369