animals

Article
Reproductive Biology of Three Important
Threatened/Near-Threatened Groupers
(Plectropomus leopardus, Epinephelus polyphekadion
and Plectropomus areolatus) in Eastern Indonesia and
Implications for Management
Miftakhul Khasanah, Nadiarti Nurdin Kadir and Jamaluddin Jompa *
Faculty of Marine Science and Fisheries, Hasanuddin University, Jl. Perintis Kemerdekaan km. 10,
Makassar 90245, Indonesia
* Correspondence: [email protected]




Received: 8 July 2019; Accepted: 26 August 2019; Published: 2 September 2019


Simple Summary: Indonesia needs basic data on the reproductive dynamics of economically
important fishery species, including groupers, in order to support sustainable fisheries management.
Data from the histological examination of grouper gonad samples were combined with data from:
participatory mapping and interviews with fishermen, monitoring data from known spawning
aggregation sites, and analyzed descriptively to provide basic data on the size at first maturity and
seasonal reproductive patterns of three economically important groupers. We suggest management
measures including size limits, temporal spatial closures, and a trading ban during the peak spawning
season (November–December).

Abstract: The three grouper species most heavily fished for the live reef fish trade (LRFT) in Indonesia
are Plectopomus leopardus (greatest catch volume), and two species classified as Vulnerable on the
International Union for Conservation of Nature (IUCN) Red List: Plectropomus areolatus and Epinephelus
polyphekadion. Understanding the reproductive biology of these fishes is essential for sustainable
management, but relevant data are limited. This study aimed to determine reproductive dynamics, so
as to inform management measures to maintain the reproductive capacity of these groupers. Grouper
gonad samples collected from fish caught for the LRFT were analyzed histologically. Data were also
collected from participatory mapping and interviews with fishermen, and underwater monitoring of
three known spawning aggregation sites in the Wakatobi National Park, Eastern Indonesia. Based on
observed gonad development, the respective lengths and weights at first maturity were: 37.7 cm and
759 g (P. leopardus); 36.65 cm and 771.2 g (P. areolatus); 36.95 cm and 889.9 g (E. polyphekadion). The
mean weight of the groupers market-based sampled was higher than the size at first sexual maturity.
Sex transition was observed in P. leopardus; sex reversal was not observed in E. polyphekadion, and the
sex pattern of P. areolatus was unresolved. Based on the fisher surveys and spawning aggregation
monitoring, spawning occurs around the new moon from September to April, with reproductive peaks
in November and December. Fisheries management measures that are suggested to sustain grouper
stocks include enforcing appropriate size limits, temporal spatial closures (spawning aggregation
sites), and a trading ban during the peak spawning season (November–December).

Keywords: sustainable fisheries management; threatened groupers; spawning aggregations; size at

first maturity; reproductive patterns

Animals 2019, 9, 643; doi:10.3390/ani9090643 www.mdpi.com/journal/animals

Animals 2019, 9, 643 2 of 18

1. Introduction
Indonesia is a major exporter of live reef fishes, especially groupers [1]. Various rules and
regulations at national and provincial levels have been put in place; however, these regulations are
poorly enforced and inadequate to support the sustainable management of these species [2]. High
profits in the live reef food fish trade and weak surveillance by law enforcement officers motivate
Indonesian fishers and traders to avoid compliance, encouraging the continued use of destructive
fishing practices [3]. Consequently, excessive extraction levels are prevalent and widely reported [4].
Negative impacts from overfishing and destructive practices affect grouper stocks directly and indirectly
through the degradation of coral reefs and their capacity to provide grouper habitat [5].
The leopard coral trout (Plectropomus leopardus), squaretail coral grouper (Plectropomus areolatus)
and camouflage grouper (Epinephelus polyphekadion) are three of the 10 fishes most commonly sought
by the live reef fish trade (LRFT) in Southeast Asia [6,7]. These fishes have been intensively exploited
throughout Indonesia over the past four decades [8]. The leopard coral trout contributes the highest
volume to LRFT exports, followed by the squaretail coral grouper and camouflage grouper [7]. The
squaretail coral grouper and camouflage grouper are relatively fast-growing species [9,10], maturing
early; however, the spawning aggregations of these species are easy to find and usually well known
to fishers, making these groupers especially vulnerable to overfishing [10–14]. The high demand for
leopard coral trout was initially prompted by its attractive body coloration (red skin and white meat),
making this grouper a menu of choice for Chinese banquets, especially on the eve of the Chinese New
Year, as the color red is a symbol of good fortune in Chinese culture [15–17]. Heavy exploitation of
spawning aggregations can pose a severe threat to marine fishes, affecting reproductive success and
the long-term sustainability of capture fisheries, and can eventually lead to the extirpation of fish
stocks and cessation of aggregation at overfished sites [18,19]. Based on the dramatic reduction in fish
populations due to excessive extraction, the IUCN Red List assessments designated P. areolatus and E.
polyphekadion as Vulnerable (VU A2bd) in 2018 [20,21]. These assessments state that steps to improve
the management of these species are urgently needed. Although the IUCN Red List assessment
placed P. leopardus in the Least Concern (LS) category [22], the management of this species needs to be
improved in order to prevent it qualifying for Vulnerable (or even Endangered) status. Although a
number of studies from several countries have been conducted on the reproductive dynamics of these
species [5,9,10,23,24], data on sexual patterns and spawning seasons in Indonesia are still lacking [9].
Size limitation is one of the least complicated fisheries management approaches to dealing
with overfishing and is relatively straightforward to implement [25]. Most countries with a LRFT
fishery (with the exception of Australia) lack regulations for the target species [9]. Australia has
enacted regulations on allowable catch size, restrictions on fishing gear, recreational bag limits, and
spatial–temporal closures for several groupers, including P. leopardus [15]. In practice, some live
grouper traders in Indonesia have set a 500 g minimum weight limit for the live groupers which
they purchase for the LRFT (Hj Said, personal communication, 18 April 2017). However, this weight
limit does not as yet have any legal basis; furthermore, there is some uncertainty as to the likely
effectiveness of this size limit (500 g), due to insufficient data on the average size of the fish caught and
reproductive dynamics, including size at first maturity and spatial and temporal data on spawning
aggregations [9,26]. Therefore, it is important to understand the reproductive dynamics of the three
most commonly traded groupers in the Indonesian LRFT (P. leopardus, E. polyphekadion, and P. areolatus)
as a basis for sustainable fisheries management approaches.
This study aimed to determine important reproductive parameters (minimum size at first maturity,
evidence of sex change and spawning seasons) to support sustainable fisheries management of three
important highly exploited (including two near-threatened) groupers (P. leopardus, E. polyphekadion,
and P. areolatus) in Eastern Indonesia. The data on reproductive biology were used to formulate
recommendations regarding potential fisheries management approaches for the studied grouper
species (leopard coral trout, squaretail coral grouper, and camouflage grouper) in Eastern Indonesia.
Animals 2019, 9, 643 3 of 18

2. Materials and Methods

Data on the weight (g) of traded groupers were collected from one private company (2015–2016)
for leopard coral trout and squaretail coral grouper. Data on the weight of camouflage grouper
were collected during 2016 from 5000 fishers in 61 locations within Eastern Indonesia (from East
Kalimantan to Papua). Gonads of the three species studied (leopard coral trout, squaretail coral
grouper, and camouflage grouper) were collected from groupers caught by hook and line during the
reproductive season (between 24 October 2017, and 4 April 2018) in five areas within Eastern Indonesia.
These areas were Kapoposang Marine Tourism Park, Takabonerate National Park, Wakatobi National
Park, Kei Islands and Karas Islands (Figure 1). The reproductive season for the three groupers was
estimated based on previous studies. The leopard coral trout is reported to spawn around the new
moon during September–November across much of its wide Indo-Pacific distribution, including the
Takabonerate Archipelago [9,26]. Squaretail coral grouper are reported as forming large spawning
aggregations during September–February in Selayar [9,26], as well as February–May [10] around the
third quarter or new moon phase in Pohnpei [20]. Camouflage grouper spawning has been reported
during October–November and January–March in Sinjai [26].
The gonads of the sampled groupers were extracted, weighed (±0.1 g) and preserved in 10%
formalin. Gonad samples were transported by sea and airfreight from the sampling locations to the
laboratory of the Balai Besar Veteriner, Maros, in South Sulawesi. Histological slides were prepared
as follows. Tissue was excised from a central portion of one gonad lobe of each gonad. The samples
were subsequently dehydrated through a series of increasing concentrations of ethanol, cleared in
xylene, infiltrated, and embedded in paraffin wax. Transverse sections of 7 µm, made using a hand
rotary microtome, were mounted on glass slides using Mayer’s egg albumin. The samples were then
rehydrated and stained with Harris hematoxylin, and counterstained with eosin. Both ovaries and
testes were examined under a light microscope in the School of Biological Science, at The University of
Hong Kong.
The histological structure of gonads extracted from the three studied groupers species was similar
to that described for the Nassau grouper (Epinephelus striatus) (Table 1) [27]. Therefore, assignment of
sexual categories and maturity development was based on a modification of the criteria developed
for the Nassau grouper (Epinephelus striatus) [27]. The gonads were then classified as immature, or
as one of four stages of sexual maturation, from ‘resting’ to ‘post-spawning’ (Table 1). When gonad
tissue contained male (testes or seminiferous tubules) and female (ovarian gonadal structures include
lamellae, lumen and numerous stages of oocytes of gonad maturation) characteristics, the gonad was
classified as ‘bisexual’; however, such characteristics could be unrelated to hermaphroditism [27–29].
Data on gonad development was limited to gonad morphology and features providing evidence of
‘sexual change’ in the samples, and were not related to sexual function [27,29].
Whenever possible, the total length (TL in cm) and weight (whole body mass, W in g) of the
groupers were measured. These data were used to calculate the length–weight relationship (W = aLb )
parameters a and b. In the live reef food fish trade (LRFFT) in Indonesia, fish size (for buying and
selling and for reporting) is commonly based on weight (whole body size), not length. The mean size
at first maturity (Lm50 ) for female groupers of each species was estimated by fitting a log transformed
regression function to the proportion of mature fish in each size cohort [30]. Because live groupers
are typically measured by weight, while the regression used requires length, the weight data were
converted to length using the length-weight relationship equation W = aLb , where W is fish bodyweight
(g), L is total length (cm), a means a constant obtained from the intercept of the regression model, and b
is the regression coefficient.
Participatory mapping interviews with the target of 50 fishers per site, for 5 sites, were used to
obtain an initial indication of grouper spawning seasons at each site, as suggested by the spawning
aggregation sites (SPAGs) monitoring manual from the Society for the Conservation of Reef Fish
Aggregation (SCRFA) guidebook [31]. The respondents were asked questions regarding any evidence
of spawning aggregation sites, types of grouper, spawning periods, water depth at spawning sites,
Animals 2019, 9, 643 4 of 18
Animals 2019, 9, 643 4 of 20

andspawning
any changes
sites,inand
spawning aggregations
any changes overaggregations
in spawning time. Interviews were Interviews
over time. conductedwere
withconducted
fishers from
with fishers
different from different
age groups age groups
and generations and generations
(Appendix A). (Appendix A).
Additional
Additional data data on spawning
on spawning seasons
seasons werewere obtained
obtained from
from the the Wakatobi
Wakatobi NationalNational Park
Park Authority
Authority surveys conducted during spawning seasons from 2005–2011 under
surveys conducted during spawning seasons from 2005–2011 under a joint monitoring program a joint monitoring
program (2005–2011)
(2005–2011) by The Nature by The Nature Conservancy
Conservancy (TNC), the(TNC),
Worldthe World
Wild FundWild Fund and
(WWF) (WWF)the and the
Wakatobi
Wakatobi National Park Authority. Three spawning aggregation sites of the squaretail
National Park Authority. Three spawning aggregation sites of the squaretail coral grouper were coral grouper
were monitored using the methods in the SCRFA guidebook [31]. Underwater visual census surveys
monitored using the methods in the SCRFA guidebook [31]. Underwater visual census surveys were
were conducted at least once every two months with each survey lasting one week starting from the
conducted at least once every two months with each survey lasting one week starting from the waning
waning gibbous moon phase. The surveyors collected data on species identity and fish behavior over
gibbous moon phase. The surveyors collected data on species identity and fish behavior over the
the spawning period, such as aggression, courtship, visibly gravid fish, and spawning activity. They
spawning period, such as aggression, courtship, visibly gravid fish, and spawning activity. They also
also estimated the sizes (total length) of individual fish and number of fish per species in each
estimated the sizes (total length) of individual fish and number of fish per species in each aggregation.
aggregation.

Figure
Figure 1. Site
1. Site maps
maps of of
thethe study
study areas
areas forfor gonad
gonad sampling
sampling andspawning
and spawningaggregation
aggregationsite
sitemapping;
mapping;(A)
(A) Kapoposang Marine Tourism Park, (B) Takabonerate National Park, (C) Wakatobi
Kapoposang Marine Tourism Park, (B) Takabonerate National Park, (C) Wakatobi National Park, National Park,(D)
(D) Kei Islands, and (E) Karas Islands, Fak-Fak
Kei Islands, and (E) Karas Islands, Fak-Fak Regency. Regency.

Table 1. Descriptive classification of maturity stages in female and male gonads of three grouper
Table 1. Descriptive
species based on classification of maturity stages
histological characteristics, in female
adapted fromand
the male gonads offor
classification three grouper
Nassau species
grouper
based on histological
(Epinephelus striatus)characteristics,
[27]. adapted from the classification for Nassau grouper (Epinephelus
striatus) [27].
Gonad
Gonad Development
Histological Description
Histological Description
Development
The whole gonad was small and compact; the gonads dominated with primary growth stage 1/2
Immature
Immature female The whole gonad was small and compact; the gonads dominated with
oocytes (Figure 2A).
female primary growth stage 1/2 oocytes (Figure 2A).
The oocytes stage 1/2 and three presented with occasional early stage 4 oocytes (Figure 2B). This
Mature resting female
The oocytes
stage was stage 1/2 and
mostly found three
between presented
spawning with
seasons, but itoccasional early
was not possible stage 4
to determine
whether this stage of ovary had previously ovulated or was maturing for the first time. In this
Mature oocytes (Figure 2B). This stage was mostly found between spawning
research, the mature resting female was dominated by large species.
resting seasons, but it was not possible to determine whether this stage of ovary
All gonads dominated with oocytes stage 1/2, 3, 4, and vitellogenic oocytes present in various
female had previously ovulated
proportions, the ovarian wallormaywas maturing
become for 2C).
thin (Figure the There
first are
time.
onlyIn this
a few (if any)
Mature ripe female post-ovulatory follicles visible. At ovulation stage, hydrated eggs released from their follicular
research, the mature resting female was dominated by large species.
developed into the ovary lumen forming post-ovulatory follicles (that were only found around
24 h after ovulation) [32].
Animals 2019, 9, 643 5 of 18

Table 1. Cont.

Gonad Development Histological Description

Gonad contains degeneration of vitellogenic oocytes and 1/2 stages oocytes and lamella wall
Post spawning female
becomes thick (Figure 2D).
Gonad contained stages 1/2 oocytes and1/2 spermatocytes (immature bisexual) (Figure 3E); 3/4
Bisexual
oocytes and 3/4 spermatocytes (mature bisexual) appear together in similar amount (Figure 4F).
All gonads were small and compact, contained a central lumen, and were dominated by
Immature male seminiferous tubules. Scattered cysts of spermatocyte stage 1/2 were evident, but not common
(Figure 3A). An occasional stage 1/2 oocyte was present.
Gonad was dominated by early stages of spermatogenesis (i.e., spermatocyte stages 1 and 2)
with scattered cysts of sperm increasingly evident as the spawning season approached.
Mature resting male
Occasional dispersed phase 1/2 oocytes were present. The testis lumen became smaller than in
immature phase (Figure 3B).
Late stages of spermatogenesis were dominant and spread to fill almost all parts of the testis.
Mature ripe male Sperm occurred in expanded lobules, and spermatocyte stages 1 and 2 were relatively less
abundant (Figure 3C).
Most of the late stages of spermatogenesis have released. Spermatocyte stages 1 and 2 were
Post spawning male relatively less abundant than the mature male phase. The gonad often had a thickened wall;
there was space between the muscular wall and sperm (Figure 3D).
Animals 2019, 9, 643 6 of 20

Figure
Figure 2. 2. Histologicalslides
Histological slidesofofgrouper
grouper ovaries.
ovaries. (A)
(A)Immature
Immaturefemale
femaleleopard
leopardcoral trout,
coral collected
trout, 12 12
collected
December 2017, total length (TL) = 35 cm; (B) Mature resting female leopard coral trout
December 2017, total length (TL) = 35 cm; (B) Mature resting female leopard coral trout collected collected 12 12
December 2017, TL = 45 cm; (C) Mature ripe female squaretail coral trout collected 21 December
December 2017, TL = 45 cm; (C) Mature ripe female squaretail coral trout collected 21 December 2017, 2017,
TL TL = 43
= 43 cm;cm; (D)
(D) Postspawning
Post spawningfemale
female camouflage
camouflage grouper
groupercollected
collected1111November
November 2017, TLTL
2017, = 47
= cm;
47 cm;
O2, stage1/2 oocytes; O3, stage 3 oocytes; O4, stage 4 oocytes; VO, vitellogenic oocyte; DO,
O2, stage1/2 oocytes; O3, stage 3 oocytes; O4, stage 4 oocytes; VO, vitellogenic oocyte; DO, degenerated
degenerated oocyte; POF, postovulatory follicles; SC, spermatocytes.
oocyte; POF, postovulatory follicles; SC, spermatocytes.
Animals 2019, 9, 643 6 of 18

Animals 2019, 9, 643 7 of 20

Figure 3. Histological slides of grouper testes and intersex gonads. (A) Immature male camouflage
Figure 3. Histological slides of grouper testes and intersex gonads. (A) Immature male camouflage
grouper collected 26 November 2017, TL = 39 cm; (B) Mature resting male leopard coral trout collected
grouper collected 26 November 2017, TL = 39 cm; (B) Mature resting male leopard coral trout collected
7 January 2018, TL = 45 cm; (C) Mature ripe male leopard coral trout collected 22 February 2018, TL =
7 January 2018, TL = 45 cm; (C) Mature ripe male leopard coral trout collected 22 February 2018, TL =
60 cm; (D) Post spawning male leopard coral trout collected 20 February 2018, TL = 56 cm; (E)
60 cm; (D) Post spawning male leopard coral trout collected 20 February 2018, TL = 56 cm; (E) Immature
Immature female dominant bisexual leopard coral trout collected 10 November 2017, TL = 27 cm; (F)
female dominant bisexual leopard coral trout collected 10 November 2017, TL = 27 cm; (F) Mature
Mature bisexual leopard coral trout collected 27 December 2017, TL = 42 cm; S2, stage 2 spermatocytes;
bisexual leopard
SC, stage coralSP,
1 oocytes; trout collected 27 December
spermatid/spermatozoa; ST, 2017, TL = 42tubules.
seminiferous cm; S2, stage 2 spermatocytes; SC,
stage 1 oocytes; SP, spermatid/spermatozoa; ST, seminiferous tubules.
Animals 2019, 9, 643 7 of 18
Animals 2019, 9, 643 8 of 20

26 post mature male

24 A
No. of Plectropomus leopardus 22 mature ripe male
20
18 mature resting male
16
14
immature male
12
10
mature bisexual
8
6
4
2
0
21–25 26–30 31–35 36–40 41–45 46–50 51–55 56–60 61–65
Total length (cm)

10 mature ripe male

mature resting male
9 B
No. of Plectropomus areolatus

mature ripe female

8 mature resting female
7 immature female
6
5
4
3
2
1
0
31–35 36–40 41–45 46–50 51–55
Total length (cm)

8
mature ripe male
No. of Epinephelus polyphekadion

7 C immature male
6 post spawning female
mature ripe female
5
mature resting female
4 immature female
3
2
1
0
26–30 31–35 36–40 41–45 46–50 56–60 76–80
Total length (cm)

Figure 4. Stages of gonad maturity of leopard coral trout by size class (TL) (A), squaretail coral trout
Figure 4. Stages of gonad maturity of leopard coral trout by size class (TL) (A), squaretail coral trout
(B), and
(B), andcamouflage
camouflagegrouper
grouper (C).
(C).
3. Results

3.1. Histological Characteristics of Gonad Developmental Stages

The sampled leopard coral trout gonads examined microscopically came from wild grouper
caught in the study areas by fishermen, as follows: Kapoposang Marine Tourism Park (MTP) (n = 34),
Animals 2019, 9, 643 8 of 18

Takabonerate National Park (NP) (n = 18), Karas Islands (n = 31), and Kei Islands (n = 5). Squaretail
coral trout gonad samples were collected from the Takabonerate National Park (n = 9), Wakatobi
National Park (n = 15), and Karas Islands (n = 1). Camouflage grouper samples were also collected
from the Takabonerate National Park (n = 15) and Wakatobi National Park (n = 5) (Table 2).

Table 2. Total sample of three groupers species per month and species during this study (n = 133). F
refers to female, M refers to male, and T refers to transitional.

Sexual 2017 2018

Species Total
Differentiation May Jun Oct Nov Dec Jan Feb Mar Apr
F 0 4 4 21 25 7 9 0 0 70
Plectropomus
M 1 0 3 2 2 1 1 0 0 10
leopardus
T 1 0 0 2 1 1 0 0 0 5
F 0 0 0 7 11 5 0 0 1 24
Plectropomus
M 0 0 0 1 1 0 0 0 0 2
areolatus
T 0 0 0 0 0 0 0 0 0 0
F 0 0 3 6 1 7 0 0 1 18
Epinephelus
M 0 0 1 1 0 0 0 0 2 4
polyphekadion
T 0 0 0 0 0 0 0 0 0 0
Total 133

3.1.1. Leopard Coral Trout (Plectropomus leopardus)

All stages of leopard coral trout gonad maturation development were observed during this
research with the exception of immature males. Reproductively inactive females had previtellogenic
oocytes and included immature and resting mature females (Figure 2A,B). The cell wall was still
somewhat irregular in shape (but less so than oocyte stage I), and the nucleus began to be apparent
with few or many pale spots in the lipid vesicles within. In mature active females, the oocyte cells
contained vitellin globules, which stained moderately with eosin. The oocytes were double in size in
the late mature stage, and the cell walls thickened. The vitellogenic oocytes become hydrated in the
post-spawning stage. Male stage features were found in the gonads of larger coral trout specimens,
some inactive primary stage oocytes 1/2 were recovered in this stage. Spermatocytes began to fill the
lumen in the mature stage. After spawning, there were empty spaces in the lumen of male coral trout
gonads (Figure 3C,D). The bisexual phase was recorded in both small and large leopard coral trout size
classes. Mature bisexual gonads contained stage 2 spermatocytes combined with a high abundance of
stage 1/2, 3 oocytes (Figure 3F). This condition is likely to be related to the transition stage from female
to male, indicating that this species may be protogynous.
Sexual patterns, sex ratio, and approximate mean size of sexual maturation in the leopard coral
trout were determined from the limited samples (n = 85) observed in this study (Figure 4A). The
smallest sampled female with inactive phase gonads measured 24 cm TL and the smallest mature, ripe
female measured 27 cm TL. The smallest mature, ripe male measured 36 cm TL. Coral trout in the
smaller size classes, but with mature gonad development stages, were mostly from the Kapoposang
MTP. For both male and female leopard coral trout, mature active (ripe) gonads were first observed in
fish caught during October and continued until December, with the largest proportion of mature active
fish observed in November (around 40% of all samples with mature gonads). Inactive females and
males (mature, resting) were most common in December (50% of all mature, resting samples). In the
leopard coral trout sampled, most inactive female gonads (immature and resting) were found in large
individuals (42–51 cm TL). The histological data indicate that males have two pathway developments:
direct from juvenile phase to male, or via sex change from adult female. These observations indicate
diandric protogyny; however, due to the relatively small sample size, further sampling is needed to
confirm whether this is indeed the case.
Animals 2019, 9, 643 9 of 18

3.1.2. Squaretail Coraltrout (Plectropomus areolatus)

The number of squaretail coral trout gonad samples (N = 24) was low, but covered a wide range
of size classes, enabling many stages of gonad development to be observed (Figure 4B). Inactive
female gonads contained the primary 1/2 and 3 stage oocytes. Mature active ripe female gonads
contained a high abundance of vitellogenic oocytes; post-ovulatory follicles were observed at this stage,
indicating spawning within the previous 24 h (Figure 2C). Based on the interviews with fishermen,
this species aggregates to spawn during daylight hours, beginning around noon and peaking in the
late afternoon, from around 4 p.m. until dusk. The length of the smallest reproductively inactive
female was 21 cm TL, and that of the smallest mature, ripe female was 33 cm TL. The smallest mature,
ripe male measured 42 cm TL. There was no evidence of bisexuality at any gonad development stage,
even in the larger-sized fish. The peak season based on gonad maturity was in December (50% of all
mature gonad samples) for both males and females. The sexual pattern for this species is unresolved.
Therefore, further research is urgently needed.

3.1.3. Camouflage grouper (Epinephelus polyphekadion)

Reproductively inactive, but clearly female and male, camouflage grouper were observed (n = 22)
in the small size class (27–31 cm) TL (Figure 4C). The smallest reproductively immature female was
21 cm TL in length, while the smallest mature active female measured 34 cm TL. The smallest immature
male was 32 cm TL, and the smallest mature active male was 34 cm TL. This indicates that sexual
differentiation tends to occur before the onset of gonad maturation, and sex reversal is unlikely.
Although the number of fish sampled in this study was relatively low, the presence of immature
males (and females) in the smaller size range (32–39 cm TL) and mature, resting female and males
in substantially the same (larger) size range (34–80 cm TL for females and 34–55 cm TL for males)
provides supporting evidence for an absence of sex reversal in this species. The highest percentage of
mature fish (male and female, 50%) was observed in January.

3.2. Mean Size at Sexual Maturity

Length–frequency histograms of various maturity stages by sex (Figure 4.) showed that the
mature gonad development phases dominated in the grouper size-classes represented in the study
sample. In leopard coral trout and squaretail coral trout, male gonads (testes) were more common in
the larger size classes. In contrast, camouflage grouper males were also common in the smaller size
classes, with a similar size distribution to females. Based on the fitted regression curve for each of
these three species, the total length at 50% sexual maturation (Lm50 ) varied between species (Table 3).

Table 3. Mean length at first sexual maturity of three grouper species from different areas in Eastern
Indonesia, based on gonad samples collected during this study.

Species Name Sampling Area Lm50 (TL, cm) Estimated Weight (g) No. of Samples
Kapoposang MTP 31.56 404.4 34
Plectropomus Takabonerate NP 37.18 670.3 14
leopardus Karas Islands 40.35 723.9 31
Kei Island 47.78 1150.3 5
Plectropomus Takabonerate NP 37.80 826.9 9
areolatus Wakatobi NP 40.20 948.4 15
Epinephelus Takabonerate NP 37.48 885.9 15
polyphekadion Wakatobi NP 40.90 1058.4 5
MTP, Marine; Tourism Park. NP; National Park; TL, Total Length.

Based on the fishers’ interviews, the history of grouper fishing for the LRFT differs between the
sampling sites, with some beginning to be exploited earlier than others. Hong Kong traders have been
buying live reef fish caught in the Makassar Strait since the 1980s, including the Kapoposang Marine
 Lm50 (TL, Estimated No. of
Species Name Sampling Area
cm) Weight (g) Samples
Kapoposang MTP 31.56 404.4 34
Plectropomus Takabonerate NP 37.18 670.3 14
leopardus
Animals 2019, 9, 643 Karas Islands 40.35 723.9 31 10 of 18
Kei Island 47.78 1150.3 5
Plectropomus Takabonerate NP 37.80 826.9 9
Tourism Park (MTP) site, followed by expansion to the Takabonerate National Park (NP) area and the
areolatus Wakatobi NP 40.20 948.4 15
Wakatobi (not yet a national park) around 1984. The trade continued to expand eastwards in Indonesia,
reachingEpinephelus Takabonerate
West Papua (Karas Islands) and NP 37.48Moluccas (Kei885.9
the Southeast 15 Each area
Islands) in the 1990s.
polyphekadion Wakatobi NP 40.90 1058.4 5
has specific dominant grouper species; for example, the LRFT in Kapoposang and Kei is dominated by
MTP,
leopard coral trout, while inMarine; Tourismand
the Wakatobi Park.Takabonerate
NP; National Park; TL,the
areas, Total Length.
main species fished and traded
are the squaretail coral trout and camouflage grouper. Table 3 shows that fish at the most extended
Based on the fitted regression curve applied to all specimens of each species (all sites pooled),
places have been extracted, related to a smaller size at maturity.
the mean total lengths at 50% sexual maturation were as follows: for leopard coral trout Lm50 = 37.7
Based on the fitted regression curve applied to all specimens of each species (all sites pooled), the
cm TL; for squaretail coral trout Lm50 = 36.6 cm TL; and for camouflage grouper Lm50 = 37.0 cm TL
mean lengths at 50% sexual maturation were as follows: for leopard coral trout Lm50 = 37.7 cm TL;
total 5).
(Figure
for squaretail coral trout Lm50 = 36.6 cm TL; and for camouflage grouper Lm50 = 37.0 cm TL (Figure 5).

100
100
90 A B
90
80
80
70
70
60
% mature

% mature
60
50 50
40 40
30 30
20 20
10 10
0 0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 0 5 10 15 20 25 30 35 40 45 50 55
Animals 2019, 9, 643 12 of 20
Upper limit of size class (TL cm) Upper limit of size class (TL cm)

100
90 C
80
70
60
% mature

50
40
30
20
10
0
0 5 10 15 20 25 30 35 40 45 50 55 60
Upper limit of size class (TL cm)

Figure 5. Length
Figure at 50%
5. Length maturity
at 50% (Lm(Lm
maturity 50 ) 50
estimated
) estimated forfor
leopard
leopardcoral trout
coral (A),
trout squaretail
(A), coral
squaretail coraltrout
trout(B),
and(B),
camouflage grouper grouper
and camouflage (C) from (C)
Eastern
fromIndonesia during theduring
Eastern Indonesia spawning season (n =
the spawning 76). Percentages
season (n = 76).
reflect the proportion
Percentages reflect of
themature females
proportion (mature
of mature resting,
females ripe, and
(mature post-spawning)
resting, per size class.per
ripe, and post-spawning) The
size class.
function = 100/1
is: y The + e−1(Xmid−X0.5)/d)
function is: y = 100/1 + e. −1(Xmid−X0.5)/d).

ForFor squaretail
squaretail coral
coral trout,
trout, the
the lengthofoffish
length fishaggregating
aggregating to spawn
spawnduring
duringspawning
spawningseasons
seasons over
over
the the period
period 2005–2011
2005–2011 waswas confirmedbybythe
confirmed theunderwater
underwater census
census data
data from
fromknown
knownaggregating
aggregating sites in in
sites
the Wakatobi NP. In fact, several species of grouper were seen to aggregate at these sites,
the Wakatobi NP. In fact, several species of grouper were seen to aggregate at these sites, including including
Epinephelus
Epinephelus fuscoguttatus,
fuscoguttatus, E. E. polyphekadion,P.P.areolatus,
polyphekadion, areolatus,and
and Lutjanus
Lutjanus bohar.
bohar. The
Theonly
onlyspecies
speciesobserved
observed
which did not show any evidence of spawning behavior (such as aggression, courtship,and
which did not show any evidence of spawning behavior (such as aggression, courtship, andvisibly
visibly
gravid fish) was Epinephelus polyphekadion. The mean size of mature squaretail coral trout estimated
visually at the spawning sites ranged from 23 TL cm to 73 TL cm (Figure 6); the number of individual
fish counted in one survey ranged from 2–92 individuals (Figure 7) (Data from the Wakatobi National
Park Authority, 2017).

160
 size class. The function is: y = 100/1 + e−1(Xmid−X0.5)/d).

For squaretail coral trout, the length of fish aggregating to spawn during spawning seasons over
the period 2005–2011 was confirmed by the underwater census data from known aggregating sites in
the
Animals Wakatobi
2019, 9, 643 NP. In fact, several species of grouper were seen to aggregate at these sites, including 11 of 18
Epinephelus fuscoguttatus, E. polyphekadion, P. areolatus, and Lutjanus bohar. The only species observed
which did not show any evidence of spawning behavior (such as aggression, courtship, and visibly
gravid fish)fish)
gravid Epinephelus
was was polyphekadion.
Epinephelus polyphekadion. The
Themean
meansize
sizeofofmature
maturesquaretail
squaretail coral troutestimated
coral trout estimated
visually
visually at theatspawning
the spawning
sitessites ranged
ranged from
from 23 23
TLTLcmcm
toto7373TL
TLcmcm(Figure
(Figure6);
6); the
the number
number ofofindividual
individual
fish counted
fish counted in one
in one survey
survey ranged
ranged from
from 2–92
2–92 individuals(Figure
individuals (Figure7)
7)(Data
(Data from the
the Wakatobi
WakatobiNational
National
Park Authority,
Park Authority, 2017). 2017).

160
2005
140
No. of Plectropomus areolatus

2006
120
2007
100
2008
80
2009
60 2010
40 2011

20

0
23 28 33 38 43 48 53 58 63 68 73
Total length (cm)

Figure
Figure 6. Estimated
6. Estimated totaltotal length
length of Plectropomus
of Plectropomus areolatus
areolatus observed
observed at a at a known
known spawning
spawning aggregation
aggregation site
Animals 2019,
in Wakatobi 9, 643
site in Wakatobi Park (n =
NationalNational Park (n =Source:
1876). 1876). Source: Datathe
Data from from the Wakatobi
Wakatobi National
National Park Park 13
Authority,
Authority, of 20
2017.
2017.
180
160
No. of Plectropomus areolatus

140
120
100
80
60
40
20
0
9 11 2 5 8 10 12 2 4 6 9 11 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 11 4 10

2005 2006 2007 2008 2009 2010 2011

Years

Figure 7. Count
Figure of individual
7. Count Plectropomus
of individual areolatus
Plectropomus in a known
areolatus spawning
in a known aggregation
spawning site in site
aggregation Wakatobi
in
National Park (n = 1876). Source: Data from the Wakatobi National Park Authority, 2017.
Wakatobi National Park (n = 1876). Source: Data from the Wakatobi National Park Authority, 2017.

3.3. Spawning Seasons

3.3. Spawning Seasons
Based
Basedon on
thethe
gonad development
gonad developmentof the
of fish
the sampled, spawning
fish sampled, appears
spawning to begin
appears to in late September
begin in late
and continues until early April, for all three grouper species in this study (Figure 8).
September and continues until early April, for all three grouper species in this study (Figure 8).Ripe male
Ripeand
female leopard coral trout were found in the last quarter moon phase in both November
male and female leopard coral trout were found in the last quarter moon phase in both November and December.
Mature ripe female
and December. and male
Mature squaretail
ripe female coral squaretail
and male trout were found
coral troutinwere
the last
foundquarter
in themoon phase in
last quarter
January.
moon Mature
phase inmale and female
January. Maturecamouflage grouper
male and female were found
camouflage over several
grouper months,
were found and
over this was
several
themonths,
only species studied
and this withonly
was the indications of reproductive
species studied activity of
with indications in reproductive
April, aroundactivity
the new in moon.
April,
around the new moon.
 September and continues until early April, for all three grouper species in this study (Figure 8). Ripe
male and female leopard coral trout were found in the last quarter moon phase in both November
and December. Mature ripe female and male squaretail coral trout were found in the last quarter
moon phase in January. Mature male and female camouflage grouper were found over several
months, and this was the only species studied with indications of reproductive activity in April,
around
Animals the
2019, new moon.
9, 643 12 of 18

Figure8.8. Mature
Figure fish (mature
Mature fish (matureresting,
resting,ripe,
ripe,
andand post-spawning)
post-spawning) sampled
sampled June 2017–April
June 2017–April 2018, by2018,
bycollection
collectionmonth
monthandandmoon
moonphase:
phase:leopard
leopard coral trout (black); squaretail coral trout (red); andand
coral trout (black); squaretail coral trout (red);
camouflage grouper (green).
camouflage grouper (green).

These data were supported by interviews with fishermen (n = 228). According to the respondents,
leopard coral trout spawn during October to January; squaretail coral trout from September to May, and
camouflage grouper from October to April (Table 4). Data from the Wakatobi National Park Authority
monitoring show squaretail coral trout spawning around the new moon, from November–December
and March–May (Figure 8).

Table 4. Spawning aggregation periods according to fisher interviews: leopard coral trout, squaretail
coral trout, and camouflage grouper in Eastern Indonesia. The aggregation sites are not listed here to
protect the site.

Spawning Spawning Month No. of

Month/Location Respondents
P. leopardus P. areolatus E. polyphekadion
Kapoposang MTP Nov–Jan - - 30
Takabonerate NP - Sept–Feb Jan–Apr 100
Wakatobi NP - Nov–Dec, Feb–May Oct–Jan 48
Kei Islands Oct–Dec - - 24
Karas Islands - Oct–Feb Nov–Dec 26

3.4. Live Grouper Market Size

The size of live groupers in the LRFT is commonly recorded as weight rather than length. Data
on the weight of commercial catch from 2015–2016 show that weight per fish for P. leopardus ranged
from 213–6000 g (n = 1249) in 2015 and 500–2900 g (n = 1226) in 2016. The bodyweight of P. areolatus
ranged from 500–7086 g per fish in 2015, and 500–3300 g per fish in 2016, with a statistically significant
decrease in mean weight from 1083 g (n = 1140) in 2015 to 1046 g (n = 1042) in 2016 (T-test, d.f = 2136,
p < 0.025). Epinephelus polyphekadion is a relatively large species [24], and was on average significantly
larger than leopard coral trout and P. areolatus (Kruskal–Wallis, d.f. = 2, p < 0.000), with a mean weight
of 1170 g per fish (n = 799) and a range from 500 g up to 5350 g per fish in 2016 (Figure 9).
Animals 2019, 9, 643 13 of 18
Animals 2019, 9, 643 15 of 20

Figure
Figure9.9.Data
Dataon
onmean
mean weight
weight (g) of P. leopardus
of P. leopardus(n(n==2475),
2475),P.P.areolatus
areolatus
(n (n = 2182)
= 2182) andand E. polyphekadion
E. polyphekadion
= =779)
(n (n 779)caught
caughtand
andtraded
traded in the live
live reef
reeffish
fishtrade
trade(LRFT)
(LRFT)inin Eastern
Eastern Indonesia
Indonesia from
from (2015–2016).
(2015–2016).

Based on the length–weight relationship conversion of the first length maturity of groupers, the
4. Discussion
mean weights at which these three species reach the first maturity are 759 g for P. leopardus, 771 g for
P. 4.1. Sexual and
areolatus, Patterns
890 g for E. polyphekadion. Meanwhile the trade data show that the average sizes of
tradedEvidence
fish are 892.3
of sex g for P. leopardus;
transition (presence for P.gonad
975.3ofgmale areolatus; andin1051.3
tissue g for
the late E. polyphekadion.
female Thus,
stage of gonad
thedevelopment)
mean size at[29]
firstwas
maturity is lower than the average size of fish in the commercial catch.
observed in a leopard coral trout, TL = 42 cm (Figure 5), but was not found in
squaretail coral trout or camouflage grouper. Although the squaretail coral grouper males were in
4. the
Discussion
large size classes, the sample size was not sufficient to draw any strong conclusion; the sexual
pattern remains unresolved, but with strong suggestions of hermaphrodite protogyny [10]. The
4.1. Sexual Patterns
presences of small immature males in camouflage grouper indicate potential sexual differentiation
before gonad of
Evidence maturation; in other
sex transition words, of
(presence it male
seemsgonad
likely tissue
that theincamouflage grouper
the late female does
stage of not
gonad
undergo sex reversal.
development) [29] was The sex pattern
observed of the leopard
in a leopard coralTL
coral trout, = 42is cm
trout strongly
(Figuresuggested as diandric
5), but was not found
in protogyny
squaretail[33],
coralmeaning that
trout or male leopard
camouflage coral trout
grouper. can derive
Although the from immature
squaretail coralorgrouper
mature females.
males were
Most grouper species are known to change sex; the lower number of males compared
in the large size classes, the sample size was not sufficient to draw any strong conclusion; to females in
the sexual
this research
pattern remainsisunresolved,
consistent with the strong
but with sex reversal processes.
suggestions Considering protogyny
of hermaphrodite that this is theThe
[10]. firstpresences
such
of small immature males in camouflage grouper indicate potential sexual differentiation beforeand
research on these species in Eastern Indonesia, further research appears to be both warranted gonad
urgent.
maturation; in other words, it seems likely that the camouflage grouper does not undergo sex reversal.
The sex pattern of the leopard coral trout is strongly suggested as diandric protogyny [33], meaning
4.2. Spawning Period
that male leopard coral trout can derive from immature or mature females. Most grouper species are
knownMost groupers
to change sex;traded in thenumber
the lower LRFT are
of intrinsically
males comparedvulnerable to over-fishing,
to females due to is
in this research theconsistent
large
number
with of fish
the sex aggregating
reversal at predictable
processes. places
Considering and
that times
this [18].
is the This
first is known
such to be
research onathese
particularly
species in
high risk factor for three grouper species (P. leopardus, E. polyphekadion, and E. fuscoguttatus) [12]. In
Eastern Indonesia, further research appears to be both warranted and urgent.
this study, the spawning period of leopard coral trout lasted from October to January with a peak
season in November and December around the last quarter moon phased, similar to the spawning
Animals 2019, 9, 643 14 of 18

4.2. Spawning Period

Most groupers traded in the LRFT are intrinsically vulnerable to over-fishing, due to the large
number of fish aggregating at predictable places and times [18]. This is known to be a particularly high
risk factor for three grouper species (P. leopardus, E. polyphekadion, and E. fuscoguttatus) [12]. In this
study, the spawning period of leopard coral trout lasted from October to January with a peak season in
November and December around the last quarter moon phased, similar to the spawning period in
Great Barrier Reef, Australia in September to December [33]. The spawning period of squaretail coral
grouper lasts from September to May in the last quarter near new moon, with a peak season during
January and February. This is similar to spawning patterns for this species reported from Komodo
National Park [13], Papua New Guinea [34] and Pohnpei, Micronesia [10]. The camouflage grouper
spawns for three consecutive months (February to April) in Pohnpei, Micronesia [24]. The gonad
development data show that Camouflage grouper seem to have a spawning peak in late January and
April during the last quarter moon phase, which has been known as a partial spawner [21,24]. Based
on the interviews with fishers (n = 174), camouflage groupers are occasionally seen in shallow water
first in the last quarter period, and followed by tiger grouper and squaretail coral trout around the
new moon.

4.3. The Minimum Size of Sexual Maturity

The live reef food fish trade (LRFFT) management in Indonesia needs to achieve sustainable
fisheries, preferably using simple management approaches that could be efficiently applied by
stakeholders [25]. Live fish destined for the LRFFT are generally weighed but only rarely is the length
measured. Therefore, the mean length at first maturity (Lm50 ) of the three economically important
groupers in this study (leopard coral trout, squaretail coral trout, and camouflage grouper) needed to
be converted to weight (using the L-W relationship based on samples from this study). The size of the
three studied grouper species was consistent with previous studies on the mean size at first sexual
maturity [10,24,33].
In this study in Eastern Indonesia, leopard coral grouper achieve 50% size of sexual maturity
(Lm50 ) at 37.7 TL cm, which is comparable to the finding in the Great Barrier Reef, Australia [33], where
the fish reached first sexual maturity at 35 cm TL. The first size at sexual maturity of squaretail coral
trout was the same with the squaretail coral trout found in Pohnpei, Micronesia [10] around 36.6 cm
TL. Our findings of the first length maturity are for camouflage grouper are bigger for camouflage
grouper compare with the conspecifics in Pohnpei, Micronesia [24], who reported that the first length
of maturity for the species was 27 cm TL smaller than found in Eastern Indonesia up to 36.95 cm
TL cm (Table 5). Based on the length–weight relationship at first length maturity of groupers, the
average weight of market size groupers tends to be above the weight at first maturity. Nonetheless, the
perception of size limits among live reef fish stakeholders needs to be adjusted to accommodate the
data on reproductive biology, with a minimum weight limit of 800 g for the two coral trout species and
at least 900 g for the camouflage grouper. An across the board size limit of 500 g is not appropriate
for these three grouper species, as at this size very few individuals will have had an opportunity to
reproduce. One challenge is that plate-sized (under 1000 g and preferably closer to 500 g) fish are most
highly-priced in the LRFFT, especially for groupers [35].

Table 5. Representative of length at first maturity (Lm50 ) of three groupers species.

Lm50 (cm)
Species Name Australia References
Indonesia Pohnpei
Plectropomus leopardus 37.7 - 35 [33]
Plectropomus areolatus 36.6 36.6 - [10]
Epinephelus polyphekadion 36.9 27 - [24]
Animals 2019, 9, 643 15 of 18

The fishery and trade stakeholders would like a single size limitation on wild-caught live groupers
in order to make the regulations relatively easy to comply with and also to enforce. However, in
Indonesia there are many grouper species, with different life histories and more importantly size at
sexual maturity. Some grouper species exploited in Indonesia mature well below the existing 500 g,
while others would not mature at 1000 g or more [9]. Thus, any legislation will be a compromise,
endeavoring to balance the biological requirements of the species exploited with the “art of the possible”
in terms of real-world fisheries management in a still developing country.
Lessons learned from the size regulations for the Napoleon wrasse Cheilinus undulatus are
instructive, and strike a cautionary note. Under Ministerial Regulation Number 37/KEPMEN-KP/2013,
the export of Napoleon wrasse is limited to fish in the 1000 g to 3000 g range. To date, this regulation
remains poorly, or indeed not, implemented [36]. There is a mismatch between the size allowed under
the regulation and the available fishery resources (C. undulatus population structure and dynamics).
Furthermore, size affects the profitability of the trade. These days, consumers in Hong Kong and other
LRFFT destination countries prefer plate-sized fish (one per person) rather than a large fish to share.
Consequently, for larger fish species, regulations based on fish reproduction will encounter market
preference resistance and thus be difficult to implement. For the three groupers in this study, the
average size of fish in the LRFFT in Eastern Indonesia is larger than the size at first sexual maturity,
which is a positive finding. However, combining the data on LRFFT fisheries history with the Lm50
data in Table 1 indicates that the size at first maturity is lower (now) in areas where the LRFFT has
been operating for longer. While it is not impossible that this could be a natural phenomenon or a
chance result (especially as the sampling size was relatively low), this empirical correlation could be
due to fishing pressure, and should be considered as a warning signal.

5. Conclusions
The exploitation of LRFFT species in Indonesia needs to be properly recorded, including volume
and composition (species and size classes). Such data are vital for sustainable fisheries management,
including assessing resources and management effectiveness. In a multi-species multi-scale fishery,
this is no easy task. In the meanwhile, as a measure suitable or data-limited fisheries such as the
LRFFT in groupers, size or weight limits are an attractive option [37]. While the current (unofficial and
partial) weight limit of 500 g could have a positive impact, especially for smaller sized grouper species,
the minimum weight for the three groupers species studied should be readjusted to 800 g per fish
for both dead (fresh/frozen) and live fishes. Such a regulation is urgently needed for grouper fishery
and trade sustainability, and would benefit both the wild fish stocks and the people who depend on
them for their livelihoods [4]. In view of the high value and volume of the trade in P. leopardus, as
well as the ease of recognizing this fish, it would be possible to begin with a specific regulation for
this species. As data on grouper size at first maturity and spawning periods improve in quantity and
quality, spatial and temporal closures (at spawning aggregation sites during peak spawning seasons)
are recommended. Further research on sex change is urgently needed, especially if there are concerns
that the rate of male removal may be too high and could potentially result in sperm limitation during
spawning due to low male abundance [38]. The change in utilization, from a no-take zone to tourism
zone (for dive activity), could potentially be an option for some permanent or temporary closures.
Where spawning locations are relatively safe, they could be attractive for visitors, and such a use could
contribute to the livelihoods of the surrounding community

Author Contributions: M.K. conceived the idea for the manuscript and contributed to writing and editing of the
paper and led the article. M.K., J.J. and N.N.K. critically revised the manuscript and added intellectual content.
M.K. reviewed and approved the final version of the manuscript to be published.
Funding: This research and the APC was funded by the Ministry of Research, Technology and Higher Education
of the Republic of Indonesia grant number [132/SP2H/LT/DRPM/IV/2017].
Acknowledgments: We would like to thank Yvonne Sadovy De Mitcheson for helping us to analyse the histological
data. Special thanks to Heru Purnomo for useful data. Many thanks also to local fishers, traders, Taka Bonerate
Animals 2019, 9, 643 16 of 18

National Park Authority, Wakatobi National Park Authority, Kapoposang Marine Tourism Park for their generous
help. Thanks to Mala, Marti, Adi Pranata Sopyan, Fajar Izas, and Salim who helped the author collect the gonad
samples. This research article is a part of a Ph.D. Thesis and partly financed by PMDSU scholarship program held
by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia with Grant Research
Contract No: 132/SP2H/LT/DRPM/IV/2017.
Conflicts of Interest: The authors declare no conflicts of interest.

Appendix A
Questionnaire for fishers
Name:
Address (resident/not resident):
Age:
Last education:

1. When the first time you catch live reef fish?

2. Where you go to fishing live groupers (please mark in this map and tell me what kind of fish you
will get there)?
3. Is the place the same as your first time fishing live groupers? If not, please mark where the first
time are you fishing live groupers?
4. Did you ever see the fish aggregation? What kind of fish? How depth and when they aggregate?
If any, please mark the place?
5. When do you see, the fish came to aggregate? Please tell us the month and moon phase?
6. Is there any missing place (a long time ago were many groupers there, and now the groupers
were gone), if any, please mark the place?

Additional information:

References
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