statistics database, named ‘‘FAO Fish STAT,’’ reflected theimprovements after the project, all fish catch data were summed up as a total in the database, and records of fishing effort we
Trang 2O R I G I N A L A R T I C L E Fisheries
Isolation and characterization of 16 polymorphic microsatellite
markers from Nibea albiflora
Chunyan Ma•Hongyu Ma •Lingbo Ma•
Keji Jiang•Haiyu Cui •Qunqun Ma
Received: 4 January 2011 / Accepted: 10 May 2011 / Published online: 11 June 2011
The Japanese Society of Fisheries Science 2011
Abstract Nibea albiflora is a commercially important
fish species in China Herein we report 16 novel
poly-morphic microsatellite markers in Nibea albiflora by using
the 50 anchored polymerase chain reaction (PCR)
tech-nique The characteristics of these loci were estimated by
using a sample of 30 individuals A total of 79 alleles were
detected with an average of 4.9 alleles per locus The
number of alleles per locus ranged from three to nine The
polymorphism information content (PIC) values for the 16
microsatellite loci ranged from 0.3131 to 0.7910 The
observed and expected heterozygosity per locus ranged
from 0.2333 to 1.000 and from 0.3452 to 0.8421, with an
average of 0.7248 and 0.6592, respectively Four loci
sig-nificantly deviated from Hardy–Weinberg equilibrium after
Bonferroni correction (P \ 0.0031), and no significant
linkage disequilibrium between pairs of loci was found
This study provides useful information for studies on
genetic diversity and structure, construction of genetic
linkage maps of N albiflora, and effective management of
this fish resource
Keywords Nibea albiflora Microsatellite markers
Polymorphism 50 Anchored PCR
IntroductionNibea albiflora is mainly distributed in northwest PacificOcean, including southern Japan and East China Sea [1].Because of its good taste and valuable nutrient content,
N albiflora is commercially fished in China However, thewild resource of N albiflora has decreased sharply underthe pressure of overfishing and pollution Genetic diversity
in a given species is closely related to evolutionarypotentiality, and genetic variation is a basic prerequisite forliving organisms to cope with uncertainty in the environ-ment [2] Therefore, it is of significant importance toanalyze the genetic structure, divergence, and geneticdiversity in N albiflora to reveal its genetic background
To date, research on genetic diversity and populationstructure in N albiflora has been carried out by usingamplified fragment length polymorphism (AFLP), mito-chondrial DNA, and isozyme analyses [3 5]
Microsatellites are short tandem-repeat DNAs withlength of 1–6 bp, widely existing along the eukaryoticgenome [6,7] Microsatellite markers are popular geneticmarkers for use in molecular phylogeography and popu-lation genetics studies because of their high polymorphism,ease of genotyping, and codominant inheritance [8] Iso-lation of microsatellite markers has been carried out inmany fish species [9, 10] Furthermore, more and morepolymorphic markers have been applied in fishery science[11–13] To date, few microsatellite markers have beenreported for this important fish species [14] Lack of suf-ficient microsatellite markers has hindered the evaluation
of population genetic structure in N albiflora The 50anchored PCR technique is considered a rapid and eco-nomical protocol for isolation of microsatellite markers[15–17] It permits genomic amplification with only onespecific primer and produces libraries with highly enriched
C Ma H Ma L Ma (&) K Jiang H Cui Q Ma
Key Lab of Marine and Estuarine Fisheries Resources and
Ecology, Ministry of Agriculture, East China Sea Fisheries
Research Institute, Chinese Academy of Fisheries Sciences,
Jungong Road 300, Shanghai 200090, China
e-mail: malingbo@vip.sina.com
H Cui Q Ma
College of Fisheries and Life Science,
Shanghai Ocean University,
Shanghai 201306, China
DOI 10.1007/s12562-011-0376-1
Trang 3single-locus microsatellites [15] In the present study, we
isolated 16 novel polymorphic microsatellite markers in
N albiflora using the 50 anchored PCR technique
Materials and methods
Sample collection and DNA extraction
A total of 30 individuals of N albiflora were collected
from Zhejiang Province, China Genomic DNA was
extracted from muscle tissue using traditional phenol–
chloroform extraction protocols as described by Ma et al
[18] DNA was adjusted to 100 ng/ll concentration and
stored at -20C until use
50 Anchored primer design and PCR
50 Anchored primers were designed as follows: the seven
nucleotides at 50in the primers form the ‘‘anchor,’’ and the
repeat parts of the primers can anneal to microsatellite loci in
genomic DNA The sequences of four degenerate primers
were KKDBDBD(AC)6, KKHBHBH(AG)6, KKVRVRV
(CT)6, and KKRVRVR(GT)6, where K = G/T, D = G/A/
T, B = G/T/C, H = A/C/T, V = A/C/G, and R = A/G
The primers were synthesized by Sangon Company
(Shanghai)
PCR amplification was performed in total reaction
vol-ume of 25 ll containing 2.0 mM MgCl2, 0.2 mM dNTP
mix, 0.2 lM each primer, 1 U Taq DNA polymerase
(TaKaRa), 19 PCR buffer, approximately 100 ng template
DNA, and deionized water The cycling parameters were as
follows: initial denaturation of 5 min at 94C, followed by
35 cycles of 45 s at 94C, 45 s at annealing temperature,
and 45 s at 72C, and then final extra extension at 72C for
5 min Amplification products were separated on 1.5%
agarose gels (TaKaRa) and visualized by ultraviolet (UV)
light
Isolation of microsatellite markers
After being reclaimed, DNA fragments ranging from
200 bp to 750 bp were ligated with pMD19-T vector
(TaKaRa) and then transformed into Escherichia coli
DH5a cells (TianGen Biotech Co., Ltd.) The positive
clones were identified by PCR with vector-specific primers
PCR amplification was performed in 25 ll reaction volume
containing 2.0 mM MgCl2, 0.2 mM dNTP mix, 0.2 lM
each primer, 1 U Taq DNA polymerase (TaKaRa), 19
PCR buffer, and 1 ll bacteria cultured overnight The
cycling parameters were initial denaturation of 5 min at
94C, followed by 35 cycles of 45 s at 94C, 45 s at 55C,
and 45 s at 72C, and final extra extension at 72C for
5 min The PCR products were separated on 1.5% rose gels (TaKaRa) After being identified, the positiveclones were randomly selected for sequencing using anABI3730XL sequencer (Applied Biosystems)
aga-Microsatellite sequences were searched using HUNTER 1.3 software [19] Microsatellite primers weredesigned using Primer Premier 5.0 software (http://www
for primer design were set as follows: primer length 19–25nucleotides, size of PCR product 100–350 bp, and annealingtemperature 50–65C
PCR amplification and polymorphism assessmentThe polymorphisms of microsatellite primers were tested
in 30 individuals of N albiflora PCR amplification wasperformed in 25 ll volume containing 19 PCR buffer,0.4 lM each primer, 0.2 mM dNTP mix, 1 U Taq poly-merase (TaKaRa), and 50 ng template DNA After dena-turation for 5 min at 94C, amplification proceeded for 35cycles [94C for 30 s, annealing temperature for each pair
of primers (Table1) for 40 s, 72C for 45 s] and a finalstep at 72C for 5 min The PCR products were separated
on a 6% denaturing polyacrylamide gel and visualized bysilver staining The ranges of allele size were determined
by referring to the pBR322/MspI marker (TianGen BiotechCo., Ltd.)
Data analysisGenetic diversity indexes including observed number ofalleles (Na), observed (HO) and expected heterozygosity(HE), and polymorphism information content (PIC).Chi-square tests for Hardy–Weinberg equilibrium (HWE)were calculated using POPGENE version 1.31 software
for all multiple tests were corrected by the sequential ferroni procedure [21] The null allele frequency was esti-mated by MICRO-CHECKER version 2.2.3 software [22]
Bon-Results
In this study, microsatellites were isolated using fouranchored primers After recovery, the PCR products (sizeranging from 200 to 750 bp) were ligated into pMD19-Tvector and transferred into DH5a competent cells A total
of 105 recombinant clones were tested, and 87 positiveclones were randomly selected to be sequenced using anABI Prism 3730 automated DNA sequencer Of the 87sequences, 63 contained microsatellite repeats Only 24primer pairs could be designed using Primer Premier 5.0software, as the remaining ones were too close to the
Trang 5flanking region of the sequences The polymorphism of
these primers was assessed using 30 individuals of
N albiflora Although the PCR conditions were optimized,
eight pairs of primers had either amplified single PCR
products of the expected size or smears In total, we
iso-lated 16 novel polymorphic microsatellite markers in
N albiflora
A total of 79 alleles were identified in 30 individuals
Allele size was between 101 and 318 bp The number of
alleles per locus ranged from three to nine, with an average
of 4.9 The PIC values for the 16 microsatellite loci ranged
from 0.3131 to 0.7910 The observed and expected
heter-ozygosity per locus ranged from 0.2333 to 1.000 and from
0.3452 to 0.8421, with an average of 0.7248 and 0.6592,
respectively
Significant deviation from Hardy–Weinberg equilibrium
at four microsatellite loci (Niba3, Niba9, Niba10, and
Niba13) was detected after Bonferroni correction
(P \ 0.0031), and the MICRO-CHECKER analysis showed
no evidence for scoring error or technical or statistical
arti-facts No significant genotypic linkage disequilibrium (LD)
was found between all pairs of these 16 loci after Bonferroni
correction (P [ 0.0031) None of these 16 sequences were
similar to any of the sequences in GenBank by a homology
search using the BLASTn program
Discussion
As one of most valuable fish species in China, N albiflora
is of economical importance in fishing, but it has been
overexploited and its natural resource has severely
declined; the fisheries resource of N albiflora is at a rather
critical level In recent years, artificial breeding and cage
culture were carried out in Fujian and Zhejiang Provinces,
China [5] For the purpose of developing rational strategies
to protect the genetic resources and utilize valuable
resources sustainable, it is of significant importance to
analyze the genetic diversity in N albiflora to reveal its
genetic background Some reports are available on
popu-lation structure and genetic background of N albiflora
using different genetic marker technique [3 5]
Microsat-ellite markers have many advantages for understanding
population genetics To date, only 13 polymorphic
micro-satellite markers have been reported in N albiflora The 50
anchored PCR technique offers a number of advantages
such as more polymorphism than those from nonanchored
primers and reduction of the cost of microsatellite
dis-covery [15] In the present study, the 16 novel polymorphic
microsatellite markers isolated in N albiflora by the 50
anchored PCR technique are different from those markers
developed by fast isolation by AFLP of sequences
con-taining repeats (FIASCO) method [14] Evaluating the
variability in a sample of 30 individuals, all loci showedconsiderable variation in the Zhejiang population
Gene heterozygosity is thought to be a good criterion toassess the genetic diversity of organisms The averageobserved heterozygosity of N albiflora was 0.7248 inZhejiang population Compared with the heterozygosity ofother species such as Epinephelus awoara (HO= 0.598)and Verasper moseri (HO= 0.60) [23, 24], the heterozy-gosity in our study was higher However, given the dam-aged resource and population structure, we should protectthe genetic diversity of this species by decreasing envi-ronmental pollution and controlling fishing effort Signifi-cant deviation from Hardy–Weinberg equilibrium at fourmicrosatellite loci was detected after Bonferroni correction(P \ 0.0031), which may be due to the small sample size
or the presence of null alleles [22]
Polymorphism information content (PIC) is considered as
a measure of the usefulness of a molecular marker [25].According to the grades, twelve loci were shown to be highlyinformative (PIC [ 0.5), only four locus were shown to beintermediate informative (0.25 \ PIC \ 0.5), and no lociwere shown to be low informative (PIC \ 0.25)
Study on population structure is very important forsuccessful and sustainable management of fish resources.Determination of population genetic structure providesessential information to underpin resource recovery andaid in delineating and monitoring populations for fisherymanagement [4] Molecular genetic techniques can offermore direct evidence to identify and delineate fish stockstructure than phenotypic or behavioral characteristicscan show [26] As a popular genetic marker, microsat-ellites have been used successfully to understand thestructure of fish species [24, 27] These 16 loci willprovide useful information for studies on genetic diver-sity and structure, construction of genetic linkage maps
of N albiflora, and effective management of this fishresource
Acknowledgments This study was supported by National Profit Institutes (East China Sea Fisheries Research Institute) (2008M04).
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Trang 7O R I G I N A L A R T I C L E Fisheries
Data mining and stock assessment of fisheries resources
in Tonle Sap Lake, Cambodia
Kazuhiro Enomoto•Satoshi Ishikawa•
Mina Hori•Hort Sitha•Srun Lim Song•
Nao Thuok•Hisashi Kurokura
Received: 17 January 2011 / Accepted: 25 May 2011 / Published online: 25 June 2011
Ó The Japanese Society of Fisheries Science 2011
Abstract The potential of catch per unit effort (CPUE)
analysis based on statistics of local fisheries in Tonle Sap
Lake in Cambodia was evaluated The fishery statistics
system was improved through a cooperative project
con-ducted by the Department of Fisheries and the Mekong River
Commission between 1994 and 2000, especially in the seven
provinces adjacent to Tonle Sap Lake However, the fisheries
statistics were not effectively utilized for sustainable stock
management After the cooperative project, fish catch data
sorted by species or species group were collected at the
province level in the seven provinces Another recent project
also revealed the numbers of fishing gears that operated in the
seven provinces The CPUEs of ten species in Kampong
Thom Province—including Channa micropeltes and
Cir-rhinus spp.—could be calculated from 1994 to 2007, because
these are caught solely using bamboo fence systems or
bar-rages CPUE analysis clarified that stocks of high-price
fishes such as Ch micropeltes, Hampala spp., and
Pangasius spp have deteriorated while those of relativelylow-price fishes such as Cirrhinus spp., Cirrhinus microl-epis, Cyclocheilichthys enoplos, and Channa striata haveincreased in recent decades
Keywords Cambodian inland fisheries CPUE Stock assessment Tonle Sap Lake
IntroductionInland fishes are vital as both food and income resourcesfor Cambodians More than 81.5% of per-capita animalprotein intake in Cambodia is supplied from fishery prod-ucts [1], and approximately 85% of total fish catch comesfrom inland fisheries [2] In addition, a recent studyrevealed that small-scale fishing in Cambodia is quite animportant income source for rural people [3] Almost allpeople have some fishery-related activities, especiallyaround Tonle Sap Lake [4] Recently, several reportsspeculated about the possibility of exhaustion of fisheryresources in the lake, and a relationship between stock
of fishery resource and hydrological fluctuations in theMekong water system was insinuated [5] However, thosereports and discussions were not based on fluctuations offishery resources but depended only on the trend of totalfish catch or correlation analysis between total fish catch byseveral fishing methods and hydrological records [6] TheCambodian fisheries authorities recognized the importance
of stock assessment, and in order to obtain an accuratestatus of the fish stock, the government, the Food andAgriculture Organization (FAO), and the Mekong RiverCommission (MRC) conducted a fisheries statisticsimprovement program in the seven provinces adjacent toTonle Sap Lake from 1994 to 2000 Although the FAO
K Enomoto H Kurokura (&)
Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
e-mail: akrkrh@mail.ecc.u-tokyo.ac.jp
S Ishikawa
School of Marine Science and Technology,
Tokai University, 3-20-1 Orido, Shimizu-ku,
Shizuoka, Shizuoka 424-8610, Japan
M Hori
Graduate School of Kuroshio Science, Kochi University,
2-5-1 Akebono, Kochi 780-8520, Japan
H Sitha S L Song N Thuok
Fisheries Adminisitration, Ministry of Agriculture Forestry
and Fisheries, 186 Preah Norodom Blvd, 582 Phnom Penh,
Kingdom of Cambodia
DOI 10.1007/s12562-011-0378-z
Trang 8statistics database, named ‘‘FAO Fish STAT,’’ reflected the
improvements after the project, all fish catch data were
summed up as a total in the database, and records of fishing
effort were not available Catch data sorted by species or
species groups might have been collected under the project
at the local level [7 9], but these detailed data were not
utilized Even as the statistics improvement project was
underway, the government and the MRC implemented
another project to collect information on Cambodia’s
fishing gear [10] This project revealed the existence of a
strong link between the type of fishing gear and the target
species Therefore, stock assessment using the catch per
unit effort (CPUE) method is possible for the species for
which records of both catch volume and fishing effort are
available from several local fisheries management
organi-zations The survey reported in this paper comprises two
steps: data mining of fishing records in provinces around
Tonle Sap Lake, and CPUE analysis and evaluation of the
present stock of each species
Materials and methods
Data mining
Domestic fisheries statistics data were officially collected
by the Department of Fisheries of the Government of
Cambodia [DOF, currently called the Fisheries
Adminis-tration (FiA)] and compiled as Fisheries Statistics Year
Books We collected as many yearbooks as possible from
the headquarters of the DOF in Phnom Penh in February
2005 In addition, we gathered information from the
cur-rent fisheries statistics data collection system through
interviews with government officers in charge at the DOF
We conducted field surveys in the five provinces directly
connecting with Tonle Sap Lake, namely Siem Reap,
Kampong Thom, Kampong Chhnang, Pursat, and
Battan-bang (Fig.1) from February to April 2005 In the
collab-orative project by the DOF and MRC [7 9], the provincial
government had collected monthly catch and effort data on
large-scale fishing, i.e., commercially operated fisheries, in
demarcated areas called fishing ‘‘lots.’’ We tried to gather
the actual primary records of fish catch and effort data for
each fishing lot, which had been collected by the provincial
governments Simultaneously, we also asked the provincial
governments about the current data collection systems
conducted by them, including the person who reported the
catch amount of each species, the person who described the
record, and how the accuracy of the reports was confirmed
Simultaneously, the relationship between the local
classi-fication of fish and scientific classiclassi-fication was confirmed
by direct interview using picture books and fish samples
Then, taxonomic group classifications written in Khmer
were translated to species or species groups according tothe contemporary system on the basis of published fieldguides and biological reviews [11–13] The species com-position of each fishing gear’s yield was examined on thebasis of outcomes of the MRC project compiled in theInformation of Major Fishing Gears in Cambodia [14] Asthe large-scale fishing season extends from the previousOctober to May of the current year, all annual data werecalculated by summing up the monthly data for the sameperiod
CPUE analysis and evaluation of present stock
of each speciesBefore CPUE analyses, the correlation between percent-ages of catch volume in a lot to the total catch volume inthe province in a year and number of fishing gears, size ofthe gears, and duration of season of the fishing method inthe year in the lot were analyzed, and the catch effort datawith the highest correlation to the contribution of eachspecies were selected as parameters of fishing effort Weselected Kampong Thom Province as the target area forCPUE analysis because of the existence of reliable rawdata CPUEs of Barbodes gonionotus, Channa micropeltes,Channa striata, Cirrhinus microlepis, Cirrhinus spp.,Cyclocheilichthys enoplos, Hampala spp., Osteochilusmelanopleurus, and Pangasius spp., in lots no 3, 4, and 5,were calculated by dividing the catch volume of eachfishing year (ton) by the total length of the bamboo fencesystems (km) in the lot as an index of fishing effort Thebamboo fence system is a huge barrier made from bambooalong the fringe of flooding forest For migratory fish
Fig 1 Five provinces around Tonle Sap Lake and location of fishing lots B Battanbang, C Kampong Chhnang, P Pursat, S Siem Reap,
T Kampong Thom
Trang 9species such as Cirrhinus spp and Trichogaster microlepis,
additional CPUEs were performed in lots 1, 2, and 7 using
the maximum width of the water current (m) where the
barrages (set nets) were set in the river A barrage is a
setting net with a bag net and wing net, being set across the
flow of the river or canal between both shores The target
species for each fishing gear were selected on the basis of
catch amount The species caught in analyzable amounts
by each gear were selected for CPUE analysis Among
them, Cirrhinus spp is a target species for both bamboo
fence and barrage This is because analyzable amounts of
Cirrhinus spp are also caught by bamboo fence due to their
huge biomass, although they are migratory species mainly
caught by barrage
Statistical analysis
The distribution pattern of the species among the lots in
Kampong Thom Province were categorized by cluster
analysis In the analysis, the Bray–Curtis similarity
coef-ficient was used as the similarity index, and the
neighbor-joining method was used for clustering of species The fish
groups were divided at 85% similarity level In the
corre-lation analysis for clarification of CPUE trends, fishes were
divided at 0.05 risk level without Bonferroni correction
Results
Data mining
Although the raw data were not compiled in provincial
offices, species-wise annual fish catch data from each
fishing lot in five provinces around Tonle Sap Lake were
reported in the annual year books from 1995 to 1997, and
the records of total catch of each species in all fishing lots
in 1998 existed in all provinces Among them, onlyKampong Thom and Battanbang Provinces maintainedprimary fish catch records We could obtain further fishcatch data for 2000–2007 in Kampong Thom Provincefrom records kept in the provincial office The datarecorded catch volumes of each fish species in each fishinglot Subsequently, we could collect catch data for eachspecies in each of five provinces from 1995 to 1998 Thus,
we had catch data for each species in different fishing lots
in Kampong Thom Province from 1995 to 2007 except
1999 The precise numbers of fishing gears used for scale fishing in the fishing lots were also described in thedomestic fisheries statistics for more than 10 years Noconsistent increase or decrease in catch volumes of anyspecies and fixed ratio among catch volume of speciesthroughout the recorded period were noticed in any fishingrecord of the five provinces We selected ten species forwhich multiyear catch records were obtainable in the fiveprovinces The species names were written in Khmer, and
large-we estimated the scientific name of each species based oninterview surveys referring to several previous field guides[11,12] Their scientific names and local names in Khmerare shown in Table1 Among them, the fish called trey rielwas mainly Cirrhinus siamensis, and other species in thegenera Cirrhinus and Henicorhynchus were included intrey riel However, among Cirrhinus, Ci microlepis wasclassified as other species and called trey pruol by localpeople Therefore, we classified trey riel as Cirrhinus spp.and trey pruol as Ci microlepis Hampala spp (treykhmann in Khmer) mainly consisted of H dispar, althoughsmall amounts of H macrolepidota were included.Pangasius spp (trey pra in Khmer) included P hypoph-thalmus, P djambal, and other species in Pangasius Treyraws included several species in the genus Channa, such as
Table 1 Local and scientific
Trey chhdaur (diep: juvenile) Channa micropeltes Trey raws (ptuok: juvenile) Channa striata Trey pruol (kralang: juvenile) Cirrhinus microlepis
(Ci siamensis, other species in Cirrhius and Henicorhynchus except Ci microlepis)
H dispar, H macrolepidota)
(P hypophthalms, P djambal, others)
Trang 10Ch marulia and Ch striata It was mentioned in the field
guide [12] that the most common snakehead in Cambodia
was Ch striata, and a review [11] reported that Ch striata
was distributed mainly in the lake We also confirmed the
scientific name of trey raws in Tonle Sap Lake as
Ch striata through interviews with DOF staff For these
reasons, we assumed that trey raw in the statistics records
of these provinces mainly included Ch striata Figure2
presents the interannual fluctuation in catch from 1995 to
2007 in five provinces The figure shows the sum of catch
amount of the above-mentioned ten species, and the catch
amount of Cirrhinus spp., Channa micropeltes, Chana
striata, and Trichogaster microlepis The total catch weight
in Kampong Chhnang Province was several times higher
than that in other provinces The catch volumes of
Cir-rhinus spp were prominently higher than those of other
species, especially in Kampong Chhnang and Kampong
Thom Provinces The catch volume of Ch micropeltes was
high before 1998 and decreasing after 1999 Chana striata
and Trichogaster microlepis were distributed mainly inBattanbang Province
In Kampong Thom Province, there are seven fishinglots The geographical features of the lots are differentfrom each other (Fig.3) The lots could be roughly dividedinto two types according to their geographical features.Lots no 1, 2, and 7 are located in large rivers or near theriver mouth We categorized this type as river-type lots Onthe contrary, lots no 3, 4, and 5 are located not in rivers but
in flooding areas in the high-water season We categorizedthis type as lake-type lots Lot no 6 is of intermediate type,located in both river and flooding areas Two types offishing gear were used in these fishing lots, namely bamboofence and barrage Bamboo fence is a long barrier madewith bamboo along the flooding area, and fish are caughtinside the barrier when water depth decreases Barrage is aset net used in the river with a bag net and wings across theflow Bamboo fences were used in fishing lots 3, 4, 5, and
6, and barrages were used in lots 1, 2, 6, and 7 Therefore,fish in lots 3, 4, and 5 were caught solely by bamboofences, and in lots 1, 2, and 7 they were solely caught bybarrages
The annual catch of the seven fishing lots had beenapproximately stable for 10 years from 1995, fluctuatingfrom 2437 to 4434 tons; significantly increasing ordecreasing trends were not observed However, the main fishspecies caught showed significant variation In particular,the catch of Cirrhinus spp (before 1997 this genus wascategorized as Henicorhynchus [15]) had increased duringthe period, with several fluctuations, whereas that of
Ch micropeltes constantly diminished, except in 1999 Thecatch volumes of the seven fishing lots showed differentfluctuation patterns Fishing lot no 2 had the highest catchfor the 10 years except 1995 The catches of lots 1 and 3
Fig 2 Interannual fluctuation of catch amount in Battanbang (solid
squares), Kampong Chhmang (open triangles), Pursat (solid
trian-gles), Siem Reap (solid circle), and Kampong Thom Provinces (open
circles) The fluctuation is expressed as the sum of the catch amount
of 10 species and the catch amount of Cirrhinus spp., Channa
micropeltes, Channa striata, and Trichogaster microlepis
Fig 3 Location of lots in Kampong Thom Province The numbers in the figure indicate lots
Trang 11gradually declined during the 10 years For the 10 years
from 1994, the main fishing grounds for Cirrhinus spp
and Ch micropeltes had not changed Figure4 shows
the average share of each lot in the total catch volume of
each species in the province For statistical confirmation of
species categorization, we performed cluster analysis using
a similarity index (Bray–Curtis similarity coefficient),
including the result in the figure Based on cluster analysis,
fishes could be divided into three groups Group A
included O melanopleurs, Cy enoplos, Pangasius spp.,
and Ci microlepis, and group B included B gonionotus,
Hampala spp., Ch striata, and Ch micropeltes Cirrhinus
spp and T microlepis formed a clear cluster independent of
the other species (group C) The fishes in group A were
mainly caught in lake lots (lots 3, 4, and 5), and the fishes
in group C were mainly caught in river lots (lots 1, 2, and 7)
A large portion of group B fishes were caught in lot 6
(an intermediate-type lot)
CPUE analysis
Tables2 and 3 present the results of correlation analysis
between the ratio of each lot to the total annual catch of
each species and several annual catch effort records
Sig-nificant correlations (p \ 0.01) were observed between the
total length of bamboo fence in the lots and the share of the
lots in the annual catch volume of B gonionotus, Ch
mi-cropeltes, Cirrhinus spp., Hampala spp., and Pangasius
spp in lake lots For river lots, the contribution of each lot
to the total catch volume of Ci microlepis, Cirrhinus spp.,
Cy enoplos, and Pangasius spp had significant tions with maximum river width at the site of barragefisheries We calculated CPUEs for stocks of 9 speciesexcept T microlepis, namely B gonionotus, Ch micropeltes,
correla-Ch striata, Ci microlepiss, Cirrhinus spp., Cy enoplos,Hampala spp., Pangasius spp., and O melanopleurus inlake lots using the total length of bamboo fence in the lot asthe unit of catch effort, and implemented CPUE analysis forstock of Cirrhinus spp and T microlepis in river lots usingthe maximum river width as the unit of fishing effort CPUEanalysis showed different patterns of fluctuations amongthe species As the result of correlation analysis betweenyear and CPUE, four species (Ch striata, Ci microlepis,Cirrhinus spp., and B gonionotus) increased, two (Cy.enoplos and O melanopleurus) were stable, and three(Ch micropeltes, Hampala spp., and Pangasius spp.)decreased in lake lots Two species analyzed in river lotsincreased in CPUE at the risk rate of 5%
DiscussionFisheries statistics in Cambodia
In general, as fisheries statistics data for developingcountries contain ‘‘gray’’ data, they cannot be used forpractical stock assessment [16, 17] However, a coopera-tive project intended to improve fisheries statistics wasimplemented from 1994 to 1996 by the MRC and Cam-bodian DOF in the seven provinces around Tonle Sap Lake
Fig 4 Share of fishing lots in
Kampong Thom Province:
average shares of lots by catch
amount of each species (%C)
from 1995 to 2007 except 1999
in Kampong Thom Province
(right) and the result of cluster
analysis of similarity of catch
amount distribution of the
species (left) Bray–Curtis
similarity coefficient was used
as the similarity index The
component bar chart (right) and
diagram (left) are formed from
the share of lots in the total
amount of each species (%C) by
the Clark and Warwick method,
in which fish species can be
divided into three groups at 85%
similarity level Numbers in the
bar chart indicate lots
Trang 12[10] We did not find any suspicious trend in the data on ten
species in five provinces from 1995 to 1998 Kampong
Chhnang Province is located at the outlet of Tonle Sap
Lake to Tonle Sap River Battanbang Province is located
on the west coast of the lake and has broad flooding forest
areas All fish migrating to the Mekong River should pass
through Kampong Chhnang Province when the lake water
recedes The habitats of C striata are in sluggish or
standing water, such as in flooding forest areas [12]
Lamberts [18] reported that habitats with a lot of vegetation
cover that are not completely submerged during flooding
offer specific protection for T microlepis The significantly
highest total catch volume in Kampong Chhnang Province
and the inhomogeneous distribution of Ch striata and
T microlepis in Battanbang Province correspond to
pre-vious knowledge of fish behavior in this area We could not
find any conclusive evidence to discredit the data as bogus
The distribution pattern of catch volume was convincing,
and we can conclude that the project succeeded in makingsome progress in improving the quality of the statistics.After the project terminated, the Kampong Thom andBattanbang Province offices continued to collect fisheriesstatistics using the same system In Kampong ThomProvince, in particular, reliable catch data of large-scalefishing were collected until 2007, although 1999 data weremissing Fishery data of Kampong Thom Province wererecorded by fishing lot and sorted by species There wereseven lots in Kampong Thom Province: three in floodingareas (lake lots), three in water channels (river lots), andone of intermediate type Among the ten species analyzed,Cirrhinus spp and T microlepis were mainly caught inriver lots We concluded that O melanopleurs, Cy enop-los, Pangasius spp., and Ci microlepis could be classified
as lake-type species and Cirrhinus spp and T microlepis
as river-type species The local name of Cirrhinus spp istrey riel; the local people distinguish this fish from
Table 2 Catch volume records of ten species in five provinces around Tonle Sap Lake in 1995 (t)
Siem Reap Kampong Thom Battanbang Pursat Kampong Chhnang
Table 3 Catch volume records of ten species in five provinces around Tonle Sap Lake in 1998 (t)
Siem Reap Kampong Thom Battanbang Pursat Kampong Chhnang
Trang 13Ci microlepis by calling the latter trey pruol The fish trey
riel includes several fish species in the genus Cirrhinus,
though the greater part is Ci siamensis Ci siamensis is
well known for its migration between the floodplains
around Tonle Sap Lake and Mekong River They migrate
to Mekong River in the dry season T microlepis is not a
migratory species However, they migrate to flooding areas
in the early stage of flooding and return to the lakes with
the receding lake water [18] Cirrhinus spp and T
mi-crolepis were supposed to be caught during migration to
Mekong River by barrage when they return to the lake from
tributary flow
In the lake lots of Kampong Thom Province, the
cor-relation between total length of bamboo fence in a lot and
its contribution to the annual total catch volume in the
province was higher for B gonionotus, Ch micropeltes,
Cirrhinus spp., Hampala spp., and Pangasius spp
(Table4) Bamboo fences are set along fringes of flooding
forests in a lot, and fish are caught in the latter half of the
fishing season after the flooding water recedes Bamboo
fence is a traditional fishing gear, and little improvement in
its methods and fishing efficiency has been carried out
There has also been little change in the total length of
bamboo fencing in recent decades, although the number of
systems in a lot has changed as a result of fragmentation.Therefore, the total length of bamboo fencing in a lotexpresses the total fishing ground area covered by thesystems in the lot The possible duration of this fishingmethod is determined by the time the flooding water takes
to recede, and the fishing period cannot be used an index offishing effort
In river lots, significantly higher correlations existbetween the maximum width of the river at the site wherethe barrage fishery systems were set in the lot and thecontribution of each lot to the total catch volume of Cir-rhinus spp and Ci microlepis in a year (Table 5) Gener-ally, a number of barrages are set in a river or channel Theriver and channel function as catchment systems of flood-ing water while the water recedes Large portions of waterare thus filtered through the barrage in the river or channelmore than once The depths of rivers and channels do notdiffer from each other significantly, while the river widthincreases due to the confluence of water from the flood-plain As a result, the river downstream is wider thanupstream The maximum river width of the river at thebarrage site at the lowest position of the river can be used
as an index of the total volume of water filtered by allbarrages in the river Barrage is also a traditional fishingTable 4 Correlations between
the ratio of each lot to the total
annual catch weight of each
species and several indexes of
fishing effort in a year in lake
lots of Kampong Thom
Duration of fishing period
Table 5 Correlations between
the ratio of each lot to the total
annual catch weight of each
species and several indexes of
fishing effort in a year in river
lots of Kampong Thom
Duration of fishing period
Trang 14gear in Cambodia, and little improvement to this fishing
method has been carried out in recent history Therefore,
significant correlations existed in the maximum river width
and catch of widely and densely distributed fish species
such as Cirrhinus spp and Ci microlepis The catch
vol-umes of these species had negative correlations with the
duration of fishing period It would be very interesting to
clarify the mechanism or background of this negative
correlation, though we do not have any reliable information
to explain the background We presume that the following
two explanations could be possible: First, barrage fishery is
performed mainly during the first half of the fishing season
when the lake water level decreases rapidly with the
recession of water Fish are caught more efficiently when
the water level decreases in a shorter period Larger
vol-umes of fish can be caught in shorter fishing periods
Second, fishers close the fishing period earlier when the
catch is better However, we need more detailed field
observations of barrage fisheries to clarify the background
The results of CPUE analysis indicate that major fishspecies had been replaced and resource conditions haddeteriorated qualitatively, though there was little change inthe total fish resources in Tonle Sap Lake When wecompared the species that decreased and increased inCPUE, we found that all the species that decreased werelarge-size carnivorous fish with higher market value.Among them, Ch micropeltes and Pangasius spp arecultured species Large amounts of Ch micropeltes andPangasius spp fingerlings had been caught and sold asseed for aquaculture before aquaculture was regulated in
2004 The slight increases in CPUE after 2004 of bothspecies may possibly be attributed to the effect of regula-tion, and the increase of CPUE in these species will beshown by future surveys Most of the species that increased
in CPUE are omnivorous fish, being good prey for Channaand Pangasius (Figs 5,6)
From these characteristics of fish species, we presumethat the possible mechanisms of species replacement areoverfishing of valuable fish for direct consumption,
Fig 5 CPUE analysis of fish species in lake lots, namely lot number 3 (diamonds), lot number 4 (squares), and lot number 5 (triangles), in Kampong Thom Province
Trang 15aquaculture, and reduction of predators and competitors.
We cannot conclude the relevancy of this presumption only
from the present data Further research to estimate the
predation pressure of Channa and Pangasius and to
understand competition among fish species is urgently
required to establish adequate fish resource management in
Tonle Sap Lake
One of the most urgent issues relating to fish resources
in Tonle Sap Lake is the hydrological impacts caused by
water resource management of Mekong River Tonle Sap
Lake is a unique lake, whose area fluctuates depending on
the flow volume of Mekong River Tonle Sap Lake accepts
large amounts of water from the Mekong River in rainy
season through Tonle Sap River as a natural control basin,
and discharges water to Mekong River in dry season It is
said that hydropower is fundamental for regional
devel-opment of the Mekong Basin Every country around the
Mekong River and some international and regional
orga-nizations have agreed on the construction of new dams for
hydropower on the Mekong River Currently, a total of
fourteen plants are under construction or are ongoing [19,
20] Flood control and irrigation are also important for
improving agricultural production and human life
How-ever, these artificial alterations of the river system will lead
to huge changes in the hydrologic circumstances of the
Mekong River System and area of floodplain around Tonle
Sap Lake These changes would cause further damage to
fishery resources, because many fish adjust their biological
features to the unique hydrologic circumstances of the
Mekong River System [21–23] Unfortunately we could
not find any catch volume data for 1999 on any species in
any province The year was an extremely dry year We
cannot conclude from the present study whether the water
level of the lake has a strong impact on all fish species in
Tonle Sap Lake It is expected that future studies of fish
stock conditions in Tonle Sap Lake will clarify the
hydrological impact on each fish species
Acknowledgments We are grateful to the staffs of Fisheries Administration of Cambodia and Inland Fisheries Research and Development Institute of Cambodia, Phnom Penh who participated in the field survey for data collection We thank I McTaggart for proofreading the manuscript of the earlier version of this paper This study was supported in part by the research project ‘‘Water man- agement system of the Mekong River’’ under the Core Research for Evolutional Science and Technology by the Japan Science and Technology Agency and the 21st Century COE Program ‘‘Biodiver- sity and Ecosystem Restoration Research Project’’ from the Ministry
of Education, Culture, Sports, Science, and Technology.
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agricul-5 Van Zalinge NP, Thuok N, Nuov S (2001) Status of Cambodian inland capture fisheries sector with special reference to the Tonle Sap Lake In: Van Zalinge NP et al (eds) Cambodia fisheries technical paper series volume 3 Inland fisheries Research and Development Institute, Phnom Penh, pp 10–17
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0.00 0.05 0.10
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
0 1 2 3
Fig 6 CPUE analysis of fish
species in river lots, namely lot
number 1 (diamonds), lot
number 2 (squares), and lot
number 7 (triangles), in
Kampong Thom Province
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Trang 17O R I G I N A L A R T I C L E Fisheries
Spatial and seasonal variation in a target fishery for spotted eagle
ray Aetobatus narinari in the southern Gulf of Mexico
Elizabeth Cuevas-Zimbro´n•Juan Carlos Pe´rez-Jime´nez •
Iva´n Me´ndez-Loeza
Received: 15 October 2010 / Accepted: 6 July 2011 / Published online: 5 August 2011
Ó The Japanese Society of Fisheries Science 2011
Abstract The target fishery for the spotted eagle ray
Aetobatus narinari in the southern Gulf of Mexico is little
known The landings of four small-scale vessels at two
fishing localities were sampled and fishermen were
inter-viewed in 2009 Rays landed at Campeche [mean ±
stan-dard deviation (SD) 1204 ± 225.3 mm disc width (DW)],
fished at 30–50 km from the shore, were larger than rays
landed at Seybaplaya (924 ± 206.5 mm DW), fished at
8–15 km from the shore Ray catches were male biased off
Campeche and female biased off Seybaplaya Catch rate
off Campeche was 6.6 (±4.9) rays per vessel trip and off
Seybaplaya was 3.0 (±2.9) rays per vessel trip Fishermen
stated that catches of A narinari are positively influenced
by winter cold fronts, turbidity, low sea temperature, and
new moon phase, and negatively influenced by the
pres-ence of cownose rays Rhinoptera bonasus Spatial
varia-tion in size composivaria-tion, and sex and maturity ratios of
A narinari were evident between sites Catch rates of
A narinari varied with individual fisherman and
season-ally between months with winter cold fronts versus warmer
months Fishermen reported a general decline in catches of
A narinari over recent decades in this region
Keywords Spotted eagle ray Artisanal fishery Landingtrends Fleet dynamics Gulf of Mexico
IntroductionThe spotted eagle ray Aetobatus narinari is a large-sizedray (230 cm disc width, DW) distributed in tropical andwarm-temperate coastal areas of all oceans [1] Recentmolecular studies have revealed that genetic exchangeamong ocean basins is highly restricted and that A nari-nari should be considered to be a species complex ratherthan a cosmopolitan species [2] In the Indo-West Pacific,for example, Aetobatus ocellatus has been reported toreplace A narinari A major revision of the A narinaricomplex is needed to delineate species and determinepopulation structure [3]
In the western Atlantic, A narinari is distributed fromNorth Carolina, USA to southern Brazil, including the Gulf
of Mexico [1] It is usually found in coastal waters, eitheralone or in large schools, where it can be caught withdiverse fishing gears [1] In Mexican coastal towns alongthe southern Gulf of Mexico (mainly in the Bay of Cam-peche), A narinari is traditionally used for human con-sumption [4]
Low reproductive rates (1–4 pups annually) combinedwith intense and unregulated inshore exploitation have led
to the A narinari species complex being listed as ‘‘nearthreatened’’ globally and ‘‘vulnerable’’ in Southeast Asia[5] In the USA, this species is fully protected in the State
of Florida by state law as a risk-averse conservationaction [5]
Mexican fisheries for elasmobranchs have been aged since 2007 by the Mexican Official Standard NOM-029-PESC-2006, Responsible Fisheries of Sharks and
man-E Cuevas-Zimbro´n J C Pe´rez-Jime´nez (&)
I Me´ndez-Loeza
Laboratorio de Pesquerı´as Artesanales, Departamento de
Aprovechamiento y Manejo de Recursos Acua´ticos, ECOSUR,
Unidad Campeche, Calle 10 X 61 No.264, Col Centro, 24000
Campeche, Campeche, Mexico
Trang 18Rays, Specifications for their Use [6] However, these
fisheries are poorly documented, and catch information for
the nation’s extensive artisanal fisheries is particularly
limited [7, 8] Available information from the Gulf of
Mexico indicates that landings of batoids (rays) have been
declining since the late 1990s [9], which suggests that more
information and management are needed A target fishery
for A narinari currently exists only in Campeche and
Yucata´n States in Mexican waters of the gulf, where it is
one of the few target fisheries for elasmobranchs on the
Mexican Atlantic (Gulf of Mexico and Caribbean Sea)
coast
Along the coast of Campeche State, A narinari is
tar-geted by an artisanal fleet of 22 small boats and is the
second most landed batoid species after the southern
stingray Dasyatis americana (Pe´rez-Jime´nez et al unpubl
data; Mexican Official Fishery Statistics, SAGARPA)
Between 1998 and 2008, this fleet landed an average ± SD
of 40.6 ± 21.5 tons of A narinari per year (Mexican
Official Fishery Statistics, SAGARPA) More detailed
information on this fishery, including allocation of fishing
effort, catch rates, and composition of landings, is not
available
The objectives of this study were to: (1) describe the
past and present target fishery for A narinari off the coast
of the State of Campeche in the southern Gulf of Mexico;
and (2) determine the size, sex, and maturity composition
of A narinari as they relate to individual fisherman,
sea-son, and capture location
Materials and methods
Description of the fishery and fishermen perceptions
A total of 10 fishermen, each with 10–60 years of
experi-ence fishing for A narinari in the State of Campeche, were
interviewed following the methods described by
Arce-Ibarra and Charles [10] Fishermen were asked about their
perceptions of catches through decades, gear
characteris-tics, seasonality of catches, commercialization (prices,
market, and local consumption), and the effects of
envi-ronmental factors on catch rates of A narinari Another 12
fishermen who targeted A narinari were contacted but
rejected the interview
Catch composition
Rays were provided by four fishermen during the 2009
fishing season: one fisherman from the locality of
Cam-peche fished a wide area in front of and northwest of its
locality, 30–50 km from shore at 8–12 m depths (Fig.1),
and three fishermen from the locality of Seybaplaya fished
a narrow area off their locality, 8–15 km from shore at6–8 m depths (Fig.1) The fishery for A narinari typicallyextends from January to July Between August andDecember, fishing effort is switched to target the Mexicanfour-eyed octopus, Octopus maya In 2009, however,fisherman from the locality of Campeche targeted A nar-inari only from January to April because of low catches
To catch the rays, fishermen used small fiberglass board-motored boats 7–7.6 m in length The fishermanfrom Campeche made trips of 1–3 days, set fishing gear for
out-12 h during the night (around 1800–0600 hours), and used30.5 cm stretched mesh, drift nets made from silk Thethree fishermen from Seybaplaya made 1-day trips, set for
19 h (around 1100–0600 hours), and used 36.5 cm ched mesh, silk bottom-fixed nets
stret-Details on the size, sex, and maturity status wereobtained from landed specimens Disc width (DW) of rayswas measured between the tips of the widest portion of thepectoral fins [1] Females and males were classified asjuvenile or adult following opportunistic macroscopicassessment of reproductive organs Calcification of clasp-ers was used to determine maturity for males followingClark and von Schmidt [11] Claspers of adult males (size
at maturity 1070–1280 mm DW) exceeded the posterioredge of the pelvic fins, presented hardened internal
Fig 1 Fishing areas for the spotted eagle ray Aetobatus narinari off the locality of Campeche (a) and off the locality of Seybaplaya (b) in the Bay of Campeche, southern Gulf of Mexico
Trang 19structure, and could be flexed and rotated toward the
anterior part without bending
First ovulation was the criterion used to determine
maturity for females The diameter of oocytes of the largest
cohort and the left uterus width were measured In the
uterus, the length and vascularization of trophonemata
were recorded and the uterus was examined to determine
the presence of embryos or uterine eggs or uterine milk
(histotrophe) that would be indicative of recent parturition
or abortion
Females were classified as adult when pregnant, or when
nonpregnant if: (a) they were not dissected but had length
equal to or larger than the smallest adult female that was
dissected and analyzed (1550 mm DW, oocytes 11 mm and
uterus 106 mm width), or (b) they were dissected and had a
wide uterus (55–126 mm width), well-developed and
vascu-larized trophonemata ([5 mm in length), and occasionally
large amounts of uterine milk (post partum condition)
Size- and sex-specific variation in catch composition
was evaluated between fishing locations and month of
capture Analysis of variance (ANOVA) was used to test
for size differences of rays caught by fishermen A factorial
ANOVA procedure was used to test for size differences per
month and sex of rays landed at the locality of Campeche
from January to April, and at the locality of Seybaplaya
from February to July To test for size differences between
sexes in the overall sample, a t test was used The data met
statistical assumptions of normality and homogeneity of
variances [12] Chi-square procedures were used to test for
sex frequency difference per vessel trip, and contingency
tables were used to test for independence between sex and
months [12]
Catch rates (fishing season 2009)
Catch rate was characterized as the number of rays caught
per vessel trip To test for differences in the number of rays
in vessel trips off Seybaplaya, an ANOVA procedure was
used Factors in the model included individual fisherman,
winter cold fronts, and lunar phases Data were square-root
transformed for analysis, and the transformed data met the
statistical assumptions for the ANOVA procedure [12]
According to fishermen’s perceptions, other environmental
variables such as sea surface temperature, turbidity, and
local abundance of cownose rays Rhinoptera bonasus can
affect catch rates of A narinari These variables were not
included in the factorial model, however, because data
were not available
Lunar cycle was determined by using tidal prediction
tables provided by the Mexican Navy for the Campeche
Station The winter cold front season in this area is
char-acterized by strong winds blowing north to south over the
Gulf of Mexico [13] In 2008–2009, this season extended
from October 2008 to March 2009 according to the NationalMeteorological Service of Mexico Based on this informa-tion, the fishing season of A narinari was divided into twoperiods: (a) a winter cold front season from January to March(with sea surface temperatures of 23.9–24.8°C), and (b) awarmer season from April to July (with sea surface temper-atures of 24.6–28.7°C); sea surface temperatures wereobtained from the Environmental Research Division,Southwest Fisheries Science Center, NOAA
ResultsDescription of the fishery and fishermen perceptionsThe fishery for A narinari in the State of Campeche began
at least 100 years ago A 70-year-old, third-generationfisherman recalled during his interview that approximately
40 years ago A narinari was targeted from small woodensailing boats using harpoons in this region
Fishermen noted a decline in catches of rays over timedespite an increase in fishing power, such as use of out-board-motored boats and large nets More than 10 yearsago, catches were reported to range from 20 to 90 rays pervessel trip, while currently, fishermen consider 8–20 raysper vessel trip to be a good catch (Table1) From January
to July 2009, the highest catches recorded off Campecheand off Seybaplaya were 17 and 22 rays per vessel trip,respectively
According to fishermen, catches of A narinari havedeclined because of: (1) a general increase in fishing effortfor all species, (2) overexploitation of this species, (3)overexploitation of some of its supposed main food (mol-lusks including the west Indian chank Turbinella angulata,perverse whelk Busycon perversum, fighting conchStrombus pugilis, and milk conch Strombus costatus), and(4) increases in the population of R bonasus, resulting incompetition for food and space
Aetobatus narinari meat is sold fresh in local marketsand also is salted and dried for local consumption By-catch elasmobranchs in this fishery were (in order ofimportance): R bonasus, D americana, Caribbean whip-tail stingray Himantura schmardae, bull shark Carcharhi-nus leucas, nurse shark Ginglymostoma cirratum, Atlanticsharpnose shark Rhizoprionodon terraenovae, and bon-nethead Sphyrna tiburo
Catch composition
A total of 896 A narinari were recorded from fisherylandings during 2009 Measurements and other biologicaldetails were obtained from a subset (85%) of the totallandings Of the 761 specimens directly examined, 157
Trang 20were sampled from Campeche (52 females and 105 males)
and 604 from Seybaplaya (383 females and 221 males)
The size composition of A narinari differed between
fishing locations (Table2) The size range of females
landed at Campeche was 580–1860 mm DW and for males
745–1500 mm DW (Fig.2a) Females in fishery landings
from Seybaplaya ranged between 540 and 2020 mm DW
and males were 440–1300 mm DW (Fig.2b) The rays
landed by the fisherman from Campeche were significantly
larger than those landed by the fishermen from Seybaplaya
(ANOVA, P \ 0.0001; Tukey test, P \ 0.001 for all
comparisons) The size of the rays landed at Seybaplaya
did not differ significantly among fishermen (Tukey tests,
P = 0.69–0.98)
The size and sex composition of A narinari differedsignificantly among months In Campeche, rays caught inApril were larger than rays caught in January and February(ANOVA, P \ 0.01; Tukey tests, P = 0.006–0.014; Fig.3),
Table 1 Fishermen’s
perceptions of good and low
catches (in number of rays per
vessel trip) in the past versus the
present, and years in the fishery,
In the present
In the past ([10 years)
In the present
Table 2 Comparison of fishing methods and catch composition of A.
narinari between one fisherman from Campeche and three fishermen
from Seybaplaya The mean and standard deviation is used to describe
the size of rays, and the range is used to describe distances from shore
and depths
Fisherman from Campeche
Fishermen from Seybaplaya
Females [ males Females \ males
Sex ratio Male biased (1:0.5) Female biased (1:0.6)
Maturity stages
(males)
Mainly adults (68%)
Mainly juveniles (85%) Maturity stages
(females)
Mainly juveniles (69%)
Mainly juveniles (98%)
Fig 2 Size composition of the spotted eagle ray Aetobatus narinari:
a off Campeche (n = 157) and b off Seybaplaya (n = 604)
Trang 21whereas there were no differences for the rest of the
com-parisons (Tukey tests, P = 0.16–0.99) At Seybaplaya, rays
landed in May were smaller than those landed in all other
months, with the exception of April (ANOVA, P \ 0.01;
Tukey tests, P = 0.001–0.035; Fig.3), whereas there were
no differences for the rest of the comparisons (Tukey tests,
P = 0.21–0.99) No significant difference was detected in
the interaction between month and sex at either fishing
location (ANOVA tests, P = 0.10–0.39)
The mean size of females and males differed
signifi-cantly between fishing location In Campeche, females
were larger than males (ANOVA test, P \ 0.01) However,
among rays landed at Seybaplaya, females were
signifi-cantly smaller than males (ANOVA test, P \ 0.0001)
(Table2) Overall, females were significantly larger (t test,
P\ 0.0001) than males
The sex composition of A narinari landings differed
between fishing locations Catches from Campeche were
dominated by males (105 males and 52 females), with a
ratio of 0.5:1 females to males (chi-square test, P \ 0.001)
The observed sex frequency was independent of the month
of capture (independence test, P = 0.053) Landings from
Seybaplaya, however, were female biased (383 females
and 221 males) with a ratio of 1.0:0.6 females to males
(chi-square test, P \ 0.0001) Sex frequency within the
landings was found to be dependent on the month of
cap-ture (independence test, P \ 0.01) Females were more
frequently landed in March, with 27% recorded in this
month, 20% in May, and 18% in June, and less frequently
captured in February (10%) and July (11%) Males were
more frequently landed in March (28%) and July (19%),
and less frequently in April (7%)
Assessments of maturity status indicated that the
land-ings at both locations were dominated by juveniles At
Campeche, most males were adult and the majority of
females were determined to be juveniles (females: 16adults, 36 juveniles; males: 71 adults, 34 juveniles;Table2) Rays of both sexes were predominately juveniles
in the landings at Seybaplaya (females: 378 juveniles, 5adults; males: 188 juveniles, 32 adults; Table2)
Catch rates
A total of 195 rays were caught off Campeche in 30 vesseltrips (mean ± SD, 6.6 ± 4.9 rays per vessel trip) (Fig.4a).With regard to average monthly catch rates, the highestaverage catch rate was recorded in January (7.8 ± 2.9 raysper vessel trip) Average monthly catch rates for February,March, and April were 5.8 ± 2.0, 6.6 ± 7.2, and 6.2 ± 7.5rays per vessel trip, respectively Eight vessel trips weremade per month, with the exception of April, in which onlysix trips were made
In the fishing area off Seybaplaya, a total of 701 rayswere recorded in 250 vessel trips (3.0 ± 2.9) (Fig.4b) Thehighest catch rate (7.6 ± 4.4) and fewest number of trips(n = 14) were recorded in February The second highest
Fig 3 Monthly mean disc width of spotted eagle rays Aetobatus
narinari caught off Campeche and off Seybaplaya (mean ± 95%
confidence interval)
Fig 4 Time series of the catch rate of the spotted eagle ray Aetobatus narinari: a fisherman from Campeche, and b fishermen from Seybaplaya The dotted line represents the end of the fishing season at Campeche and the beginning of the fishing season at Seybaplaya
Trang 22catch rate was recorded in March (5.3 ± 3.3) The lowest
catch rates were recorded in June and July (1.9 ± 1.9 and
1.4 ± 1.1, respectively) during a period in which the largest
number of vessel trips were made (58 and 56, respectively)
The number of rays caught per vessel trip off Seybaplaya
was statistically different among individual fishermen
(ANOVA test, P \ 0.01), lunar phases (ANOVA test,
P\ 0.01), and between the winter cold front season and the
warmer season (ANOVA test, P \ 0.0001) Fisherman 1 had
higher catch rates (3.6 ± 3.6) than fishermen 2 and 3
(2.3 ± 2.6 and 2.3 ± 2.7, respectively) During the full
(3.3 ± 3.2) and waning (3.3 ± 3.9) phases, more rays were
caught per vessel trip than during the new phase (2.0 ± 2.6),
and the number of rays caught in waxing phase (2.7 ± 2.5)
was not different from any other phase During the winter cold
front season, with relatively low sea surface temperatures of
23.9–24.8°C, more rays were caught per vessel trip
(5.6 ± 3.8) than during the warmer season (2.0 ± 2.3) with
its higher sea surface temperatures of 24.6–28.7°C
Based on surveys with fishermen, catch rates of
A narinari are positively influenced by the winter cold
front season, turbidity, low sea temperature, and new moon
phase (Table3) Presence of R bonasus is also perceived
by the fishermen to negatively influence the catch rate of
A narinari, especially for fishermen who operate more
than 5 km from shore, such as in the localities of
Campe-che and Seybaplaya
Discussion
Mexican elasmobranch fisheries of the Gulf of Mexico use
multiple gear types and are multispecific [14, 15], and
fisheries of batoids are often opportunistic off the MexicanPacific coast [7, 8] as occurs off the Mexican Atlanticcoast The fishery for A narinari in the southern Gulf ofMexico is one of the few targeted batoid fisheries inMexican waters of the Atlantic coast or in any other part ofthe Gulf of Mexico This is mainly due to the traditionalfood consumption of this species in the State of Campeche,where A narinari meat commands one of the highest pri-ces among all elasmobranchs in Mexico (US $4.10–$5.80per kg)
According to fishermen, catches of A narinari havedeclined over recent decades, but a lack of data (time series
of catch and effort) makes it impossible to confirm thistrend Declines in A narinari populations due to overfishinghave been suggested previously [5,16] Other factors maycontribute to potential declines; for example, fishermenfrom the State of Campeche believe that declines of thisspecies have been a result of increased fishing pressure on allmarine resources, declines in some of its potential prey(mollusks), and increased populations of R bonasus.Baqueiro-Ca´rdenas et al [17] documented that, starting in
1984, mollusk populations off Seybaplaya began to decline
in response to increasing exploitation which included anexpansion of the fishery to greater depths
The fishermen surveyed in this study ceased fishingoperations when large schools of R bonasus were detected
in the fishing area These large schools sometimesdestroyed their nets They perceive that the local popula-tion of R bonasus has increased in the last decade.Increases in mesoconsumer elasmobranchs, such as
R bonasus in the northwestern Atlantic, have been linked
to declines in populations of apex predatory sharks [18].This and other possible ecological connections should beexplored in future studies in the southern Gulf of Mexico.Allocation of target fishing effort for spotted eagle ray isprobably related to consumption preferences of peoplefrom the localities of Campeche and Seybaplaya, affectingthe catch composition Fisherman from the locality ofCampeche tend to target large rays, in a wide area withmultiday trips, due to the larger rays’ high price in theCampeche fish market (approx US $4.50 per kg) Fisher-men from Seybaplaya targeted small and medium-sizedrays close to the shore, because of local size preferencesand prices (approx US $5.00 per kg) Additional fishermanfrom Campeche not directly surveyed as part of this studyalso targeted A narinari in an extended area Fishermenfrom Seybaplaya who were not included in this study alsoreported a similar pattern of allocating fishing effort closer
to shore as those included in our survey However, analternative explanation is that fishermen probably are usingtraditional fishing areas, and markets and consumptionpreferences in both localities were developed based on thesize of rays landed there
Table 3 Fishermen’s perceptions of the effects of environmental
factors on the catch rate of A narinari
5 fishermen: the moon cycle has no effect on catch
rate, as they only fished in highly turbid areas
Rhinoptera
bonasus
7 fishermen: presence of R bonasus negatively
influences catch rate of A narinari
3 fishermen: R bonasus has no effect on catch rate;
they only fished very close to the shore (2–3 km
from shore)
Trang 23Fishing gear characteristics, such as mesh size and
gillnet location in the water column, probably do not
contribute to the observed differences in size composition
between fishing locations Rays caught with the smallest
mesh size nets (30.5 cm, off Campeche) were larger than
the rays caught with the largest mesh size nets (36.5 cm,
off Seybaplaya) In addition, despite the fact that sexual
dimorphism exists in A narinari with females growing
larger than males [19; also observed in this study], male
rays caught offshore of Campeche were larger than females
caught close to shore near Seybaplaya Because
mylioba-tiform rays are often entangled in nets by their tail spine
and are therefore vulnerable to a broad range of mesh sizes,
considerably less size selectivity is observed in fisheries for
these species [20] Furthermore, mainly juvenile rays of
both sexes were caught off Seybaplaya, where 85% of
males and 98% of females were juveniles, whereas adult
rays were more predominant off Campeche, where 68% of
males and 31% of females were adults The size range of
A narinari observed in this study is therefore likely to be
representative of spatial variation in the size composition in
the region during the study period
In addition to differences in allocation of fishing effort,
potential size and/or sex segregation of A narinari also
could affect the composition of the catches Segregation by
size and sex is a common feature of elasmobranch
popu-lations [21,22] Although landings of A narinari consisted
of both sexes, significant bias in the sex composition was
evident Catches off Campeche were male biased, whereas
those sampled from Seybaplaya were strongly female
biased Differences in the size composition of rays between
fishing areas also suggest spatial segregation of size classes
in the region Larger rays were caught further from shore
(off Campeche) and smaller rays closer to shore (off
Sey-baplaya) A similar pattern has been observed for A
nar-inari in Puerto Rico (Dubick JD, unpubl data, 2000)
Catch rates off Seybaplaya differ for individual
fisher-man and are influenced by the winter cold front season
(with low sea temperatures 23.9–24.8°C) The first result
underscores the importance of considering the variation in
fishing success of individual fishermen when making
comparisons of catch rates between fishing areas or years
There is no literature specifically on the influence of winter
cold fronts on the catch rates of elasmobranch species
However, Heupel et al [23] found that juvenile blacktip
sharks Carcharhinus limbatus sensed the approach of a
tropical storm, moved out of their shallow nursery area to
deeper waters in response, and then returned to the
pro-tection of the nursery after the storm’s passage In this
study, our data corroborate the fishermen’s perception of
the positive influence of winter cold fronts on the catch rate
of A narinari Fishermen stop fishing for safety reasons
when winter cold front events occur (durations of
2–3 days) because of high winds ([30 km/h) They ceive that catch rates of elasmobranch and teleost speciesincrease when they resume fishing, because these speciesmove towards the shore during periods of high, cold winds.Abundance and distribution of elasmobranchs areinfluenced by water temperature [24, 25], and catches of
per-A narinari off Seybaplaya decline in warmer months(April–July) Silliman and Gruber [26] found that A nar-inari move to deeper waters around Bimini, Bahamas, inlate spring, and the rays return to shallow waters at the end
of summer These authors suggest that temperature is theprimary causal factor This is in agreement with the anal-ysis of catches in the present study and the perception offishermen, who believe that A narinari moves offshore inwarm months, around April, and moves back inshore whentemperatures begin to decrease, around October
According to fishermen, turbidity is another factor thatpositively influences the catch rate of A narinari, and theyprefer to set their nets in turbid areas when fishing for therays Hueter et al [27] selected study areas for juvenileshark tagging in Mexico based on the experience of fish-ermen from Yalahau Lagoon (northeastern Yucata´n Pen-insula), who recommended the use of turbid areas tomaximize catches of C limbatus in gillnets
Future studies using fishery-independent approachescould help determine the influence of temperature [24,25],lunar cycle [28], and turbidity on the catch rates of
A narinari, and use of satellite tags could provide mation on the influence of winter cold fronts on thebehavior of this species
infor-Because of the global near-threatened status of A inari [5] and the fact that it is the target species in thisspecialized Mexican fishery, we strongly recommend thatharvest of A narinari in the Mexican Atlantic be managed
nar-by use of a species-specific fishing permit rather than themultispecific permit for all elasmobranch species Fisher-men should provide records of fishing trips and catches bymeans of a logbook system, as established in NOM-029-PESC-2006 [6], to generate time series of catch and effortdata for analysis of fluctuations in catch per unit effort.Future analyses of Mexican elasmobranch fisheries shouldcarefully consider the spatial allocation of fishing effort as
it may strongly influence the size and sex composition oflandings as well as the proportion of juvenile and adultindividuals within catches Fisherman identity (individualvariability) likewise must be considered in comparisons ofcatch rates between areas or years, because differences can
be influenced by this factor [29]
If the perceptions of fishermen are correct and catches of
A narinari have declined over past decades, the threatened status determined by the IUCN [5] is valid inthis region However, it is important to consider that theincidence of adult rays is relatively low in this fishery (5%
Trang 24near-and 32% of females near-and males, respectively, were adult
rays) To better evaluate the status of this population and
determine the most appropriate management measures,
development of a demographic stage-based model is
rec-ommended Age and longevity data are lacking for this
species, and a stage-based model based on size classes
would assess the potential impacts of fishing pressure on
various size classes on the overall population growth rate
[30] of A narinari in this region
Acknowledgments We thank the fishermen of Campeche State who
allowed us to analyze their landings and who provided us information
about their fishing activities Thanks are due to J Ortega-Puch for
valuable field assistance Special thanks are extended to R.E Hueter,
W.D Smith, and M Heithaus, whose valuable comments, edits, and
recommendations considerably improved this manuscript Funding
for this project was provided by ECOSUR.
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Trang 25O R I G I N A L A R T I C L E Biology
Immunoreactive changes in pituitary FSH and LH cells
during seasonal reproductive and spawning cycles of female
chub mackerel Scomber japonicus
Mitsuo Nyuji•Tetsuro Shiraishi •Sethu Selvaraj•Vu Van In•
Hajime Kitano•Akihiko Yamaguchi•Kumiko Okamoto•
Shizumasa Onoue•Akio Shimizu•Michiya Matsuyama
Received: 22 January 2011 / Accepted: 1 June 2011 / Published online: 21 June 2011
Ó The Japanese Society of Fisheries Science 2011
Abstract The physiological functions of pituitary
gonadotropins (GtHs) are well established in higher
ver-tebrates, whereas those in teleosts are still poorly
under-stood To describe the role of GtHs during gonadal
development of female chub mackerel Scomber japonicus,
changes in follicle-stimulating hormone (FSH) and
lutein-izing hormone (LH) cells were investigated
immunohisto-chemically during the seasonal reproductive and spawning
cycles FSH and LH cells were identified in the different
cell types of the proximal pars distalis (PPD); FSH cells
were located in the central PPD, whereas LH cells were
localized along the border of the pars intermedia To
examine changes in FSH and LH cells, the percentage of
FSH or LH cell-occupying area in the PPD was evaluated
and represented as FSHb-immunoreactive (ir) or LHb-ir
levels, respectively FSHb-ir levels increased significantly
from immature to the completion of vitellogenesis, whereas
LHb-ir levels were maintained at high levels from early
vitellogenesis to post-spawning During the spawningcycle, which consisted of four stages from just afterspawning to the next oocyte maturation, both FSHb-ir andLHb-ir levels showed no significant changes among dif-ferent stages; however, LHb-ir levels remained relativelyhigh, and FSHb-ir levels were constantly low These resultssuggest that both FSH and LH may be involved in vitel-logenesis and LH may act at final oocyte maturation infemale chub mackerel, although the role of FSH during thespawning cycle is still unclear
Keywords Follicle-stimulating hormone Luteinizinghormone Chub mackerel Vitellogenesis Final oocytematuration Immunohistochemistry
Introduction
In fish, as in other vertebrates, gonadal development andsexual maturation are regulated by various endocrine hor-mones in the brain–pituitary–gonadal (BPG) axis A vari-ety of environmental factors and physiological informationintegrated in the brain are transported to the pituitarythrough secretion of neurohormones such as gonadotropin-releasing hormone (GnRH) GnRH produced in the brainregulates the release of two kinds of gonadotropins (GtHs),follicle-stimulating hormone (FSH) and luteinizing hor-mone (LH), from the pituitary [1, 2] GtHs, which areheterodimeric glycoproteins composed of a common asubunit and a specific b subunit, control gametogenesis,final oocyte maturation (FOM), and ovulation/spermiation
in the gonad through biosynthesis of gonadal steroid mones [3 5] The physiological functions of both GtHs arewell established in higher vertebrates; however, those inteleosts are only poorly understood
hor-M Nyuji T Shiraishi S Selvaraj V Van In H Kitano
A Yamaguchi M Matsuyama ( &)
Laboratory of Marine Biology, Faculty of Agriculture,
Kyushu University, Fukuoka 812-8581, Japan
e-mail: rinya_m@agr.kyushu-u.ac.jp
K Okamoto S Onoue
Oita Prefectural Agriculture, Forestry and Fisheries Research
Center, Saiki 879-2602, Japan
A Shimizu
National Research Institute of Fisheries Science,
Fisheries Research Agency, Yokohama 236-8648, Japan
Present Address:
V Van In
National Broodstock Center for Mariculture in Northern
Vietnam, Haiphong, Vietnam
DOI 10.1007/s12562-011-0380-5
Trang 26Plasma levels of FSH and LH can be measured only in
salmonids, which are single spawners with a
group-syn-chronous type of ovarian development In addition to these
plasma GtH profiles, experiments investigating features
such as steroidogenic capacity and vitellogenin uptake
have also indicated that FSH regulates early phases of
gametogenesis, whereas LH is responsible for final
matu-ration processes [5 8] In contrast, in nonsalmonid fishes,
including multiple-spawning perciform species with
asyn-chronous type of ovarian development, the functional roles
of FSH and LH are not well understood In nonsalmonids,
adequate methods for measuring plasma levels of GtHs
have not been established, mainly due to the difficulty of
purifying GtHs, especially FSH Recently, homologous
immunoassays for measuring plasma levels of FSH and LH
have been developed using recombinant GtHs in Nile
tilapia Oreochromis niloticus [9] However, seasonal
pro-files of FSH and LH in other perciform species have yet to
be subjected to gene expression analysis [10] In addition,
some in vitro experiments using purified GtHs have been
performed in a limited number of nonsalmonid fish species,
but the results show that the function of each GtH is
pre-sumably different depending on species and gender [11–
Specific antisera raised against synthetic fragment
pep-tides of mummichog Fundulus heteroclitus b subunit have
recently been developed and successfully used to recognize
GtH cells in the pituitary of numerous acanthopterygian
fishes [16,17] Using these antibodies, the changes in FSH
and LH cells during the reproductive cycle have been
examined immunohistochemically in the mummichog [18]
and Japanese flounder Paralichthys olivaceus [19] These
reports demonstrate that immunohistochemical study is
also suitable for investigating seasonal profiles of GtHs in
multiple-spawning fishes
Japanese chub mackerel Scomber japonicus is widely
distributed in temperate and subtropical waters of the
Pacific Ocean This species is one of the most important
commercially exploited fish in Japan Due to unreliable and
unpredictable wild catches, aquaculture of chub mackerel
commenced recently in southwestern Japan using young or
adult fish caught from the wild [20, 21] Captive chub
mackerel reared in sea pens and outdoor tanks undergo
normal spermatogenesis and vitellogenesis [20] However,
female fish fail to undergo FOM and ovulation after
completion of vitellogenesis (April–June) [22] We have
recently developed an experimental system for chub
mackerel using GnRHa, in which FOM and ovulation
could be controlled in captivity at any time for a period of
3 months during the spawning season [23] This
experi-mental system facilitates fish sampling at different gonadal
stages to elucidate the role of key endocrine hormones
regulating seasonal reproductive and spawning cycles
In the present study, we investigated the tive changes in FSH and LH cells during seasonal repro-ductive and spawning cycles in captive female chubmackerel to understand the roles of FSH and LH duringovarian growth and maturation
immunoreac-Materials and methodsAnimals
For experiments on the seasonal reproductive cycle, about
350 fish (2? years old) were caught offshore of Oita fecture and maintained in sea cages belonging to the OitaPrefectural Agriculture, Forestry and Fisheries ResearchCenter from November 2004 Body weight (BW) and forklength were 321 ± 9.0 g [mean ± standard error of themean (SEM), n = 15] and 292 ± 2.5 mm, respectively.Female fish with developing ovaries were collected threetimes, in November 2004 (11 fish), March (24 fish), andMay (19 fish) 2005 In early June 2005, chub mackerelreared in sea cages were transferred to the FisheryResearch Laboratory of Kyushu University in FukutsuCity, Fukuoka Prefecture and moved into a concrete tankwith running seawater The next day, after anesthetizingthe fish with 2-phenoxyethanol (200 ppm), females withoocytes greater than 600 lm in diameter and spermiatingmales were selected as described previously [22] andinjected intramuscularly with GnRHa (des Gly10-D-[Ala6]LHRH ethylamide; Sigma-Aldrich, St Louis, MO, USA)(400 lg/kg BW) combined with coconut oil [24] AfterGnRHa injection, 28 fish were divided into two groups(each group consisting of seven females and seven males)and kept in two 3000-l flow-through seawater tanks withrunning seawater under natural day length and watertemperature The fish were fed defrosted fish and acommercial dry pellet (Higashimaru Co Ltd., Tokyo,Japan) twice per week during the experiments Fertilizedeggs were first obtained from each tank 2 days afterGnRHa injection, and daily spawning continued for
Pre-14 days Fish in each tank were sacrificed at 20 and
75 days after GnRHa injection, or 5 and 60 days after lastspawning, respectively
For experiments during the spawning cycle, 26 femalesand 20 males collected on June 2007 were injected withGnRHa, divided into two groups, and maintained in the3000-l tanks in the same manner as described above Dailyspawning began 2 days after GnRHa injection and wasobserved between 2100 and 0100 hours, generally peaking
at around 2300 hours At 7–8 days after GnRHa injection,8–14 fish were sacrificed at 1200, 1800, 0000, and
0600 hours to collect fish with differently staged ovariesbased on the time course of FOM and ovulation and the
Trang 27degenerative post-ovulatory follicle process in chub
mackerel [23,25]
Ovarian histology and stages
The ovaries were excised, and small pieces of ovaries were
fixed in Bouin’s solution, dehydrated, and embedded in
Technovit resin (Kulzer, Wehrheim, Germany) Then,
4-lm-thick sections were cut and stained with 1% toluidine
blue solution for light microscopy
The ovarian developmental stages in the chub mackerel
were classified by the most advanced group of oocytes
according to Shiraishi et al [22], with slight modification
Ovarian stages of the seasonal reproductive cycle were
divided into six stages as follows: IM (immature), EV
(early vitellogenesis), LV (late vitellogenesis), SP
(spawning), PS (post-spawning), and RS (resting) stages(Fig.1) To analyze the spawning cycle, the SP stage wassubdivided into four groups as follows: GVM (germinalvesicle migration), HY (hydration), POV (post-ovulation),and LVsp(late vitellogenesis in spawning)
Immunohistochemistry of FSH and LH cellsPituitary glands were fixed in Bouin’s solution, dehydratedthrough a graded series of ethanol concentrations, andembedded in paraffin Three sets of successive sectionswere cut at [20 lm intervals, which is larger than the longdiameter of GtH cells (\15 lm) The thickness of eachsection was 4 lm Some of the deparaffinized pituitarysections from each set were stained with Masson’s tri-chrome to identify the pituitary cell types and to confirm
Peri-nucleolus Yolk vesicle Primary yolk Secondary yolk Tertiary yolk Atretic Peri-nucleolus
Seasonal reproductive cycle
Multiple spawning
Germinal vesicle breakdown (GVBD) Germinal vesicle
migration (GVM) Tertiary yolk
Final oocyte maturation (FOM)
POF Oocyte
HY GVM
post-ovulatory follicle (POF)
Fig 1 Schematic diagram of the chub mackerel ovarian stages
sampled during the seasonal reproductive and spawning cycles.
Seasonal reproductive cycle (top panel): ovarian stage and month of
sampling are shown on a black background with white letters.
Ovarian stages during the seasonal reproductive cycle were classified
based on the developmental stages of the most advanced oocytes: IM
(immature), oocytes at peri-nucleolus stage and yolk vesicle stage;
EV (early vitellogenesis), oocytes at primary and secondary yolk
stage; LV (late vitellogenesis), oocytes at tertiary yolk stage; SP
(spawning), ovaries at any stage shown in the spawning cycle as
described below; PS (post-spawning), atretic oocytes; RS (resting),
oocytes at peri-nucleolus stage Spawning cycle (bottom panel): ovarian stage and time of sampling are shown on a black background with white letters Ovaries collected during the spawning cycle were classified into four stages based on the developmental stages of the most advanced oocytes and the degenerative stages of the post- ovulatory follicles (POFs) [ 23 , 25 ] GVM (germinal vesicle migra- tion), oocyte at GVM stage; HY (hydration), hydrated and transparent oocytes after germinal vesicle breakdown (GVBD); POV (post- ovulation), oocytes at tertiary yolk stage with new POF (spawning
\6 h); LV sp (late vitellogenesis during spawning cycle), oocytes at the tertiary yolk stage without new POF
Trang 28that sections were cut through the third ventricle, which is
located around the midline Other sections for each set,
where the location of the third ventricle was confirmed,
were used for immunohistochemical staining to detect FSH
and LH cells
Immunohistochemistry was performed according to the
method of Shimizu et al [18] Briefly, primary antisera for
immunostaining were raised against conserved regions of
FSH and LH b subunit peptides from mummichog Fundulus
heteroclitus (Fh FSHb 50–60 and Fh LHb 91–106) [16]
Deparaffinized pituitary sections were treated with an
epitope-unmasking solution (Target Unmasking Fluid; Sanbio BV,
Uden, The Netherlands) before incubation with a blocking
reagent, 3% normal goat serum in 10 mM phosphate-buffered
saline (PBS) Then the sections were processed with avidin–
biotin–horseradish peroxidase (HRP) kit (ABC kit; Vector
Laboratories, Burlingame, CA, USA) Primary antisera were
diluted to 1:1000 (anti-Fh FSHb) or 1:2000 (anti-Fh LHb)
with ethylenediamine tetraacetic acid (EDTA)-PBS (PBS
containing 50 mM EDTA and 0.1% gelatin) Immunoreactive
cells were visualized using 0.05% 3,30-diaminobenzidine
(DAB) solution (Vector) The specificity of the
immuno-staining reactions was assessed in several adjacent sections by
treating them with PBS rather than primary antisera All
procedures were performed at room temperature
The immunoreactive (ir) levels of GtH cells were
calcu-lated as follows: FSHb-ir (or LHb-ir) levels = [FSH (or LH)
cells occupying area/proximal pars distalis (PPD)
area] 9 100, where the FSH cell or LH cell-occupying area,
and PPD area were calculated using the NIH Image software
program provided free of charge by the National Institutes of
Health (Bethesda, MD, USA) via the Internet (http://rsb.info
examined, and a mean value was calculated for each fish The
FSHb-ir (or LHb-ir) levels for different stages are presented as
the mean of 3–5 fish of the same stage
Statistics
All data are expressed as mean ± SEM Statistical
differ-ences in FSHb-ir (or LHb-ir) levels were determined by
one-way analysis of variance followed by Tukey’s
multi-ple-comparison test Significance differences were
accep-ted at P \ 0.05
Results
Pituitary structure and immunohistochemistry of GtH
cells
The overall structure of pituitaries stained with Masson’s
trichrome obtained from the fish at the EV stage is shown
in Fig.2 The pituitary could be separated into four clearregions: the rostral pars distalis (RPD), the PPD, the parsintermedia (PI), and the neurohypophysis (NH) The PI wasencircled by the PPD The NH connected with the dien-cephalon and extended into all three other regions
In the RPD, most cells stained red with Biebrich scarlet(Fig.3a) and appeared homologous to the lactotrophsdescribed in other teleosts Cells that stained faint blue withaniline blue were also partly observed in the dorsal part ofthe RPD These cells appeared to be thyrotrophs, asdescribed in other teleosts (Fig.3a) Cells staining red orred–purple were observed in the dorsal part of the PPD(Fig.3b) and appeared to be somatotrophs Most cells inthe PPD were stained dark or faint blue with aniline blue(Fig.3b) The majority of cells in the PI were stainedfaintly red–purple A few small clusters of cells stainingfaint blue were scattered in the PI along the external border
of the PPD (Fig.3c) The NH had granular neuronal cellsthat stained purple and penetrated the entire PI (Fig 3c).Three successive sections through the PPD of the femalechub mackerel pituitary at the EV stage and stained withMasson’s trichrome, anti-Fh FSHb, and anti-Fh LHb areshown in Fig.4 In the PPD, FSHb-ir cells likely corre-sponded to the cells stained dark blue with aniline blue(Fig.4a, b) In contrast, LHb-ir cells were observed asstaining faint blue with aniline blue in the PPD (Fig.4a, c).Small clusters of cells, stained faint blue with aniline blue
in the PI, also showed immunostaining with anti-LHb (datanot shown)
Fig 2 Sagittal section of female chub mackerel pituitary at the early vitellogenesis (EV) stage stained with Masson’s trichrome Anterior
is on the left RPD rostral pars distalis, PPD proximal pars distalis, PI pars intermedia, NH neurohypophysis, TV third ventricle
Trang 29Changes in GtH cells during the seasonal reproductive
and spawning cycles
Representative sections of female chub mackerel pituitaries
immunostained with anti-Fh FSHb and anti-Fh LHb during
the seasonal reproductive cycle are shown in Fig.5
FSHb-ir cells were mainly distributed in the central PPD, whereas
LHb-ir cells were distributed widely in the PPD,
particu-larly along the border of the PI The size of the pituitary
increased as gonadal development proceeded, and it was
largest at the LV stage (Fig.5c, i)
Changes in FSHb-ir and LHb-ir levels in the female
chub mackerel pituitary during the seasonal reproductive
and spawning cycles are shown in Figs.6 and7,
respec-tively As shown in Fig.7, no significant changes were
found during the spawning cycle for both FSHb-ir and
LHb-ir levels in the female chub mackerel pituitary
FSHb-ir levels were 14.6% (GVM), 16.4% (HY), 17.2% (POV),
and 12.3% (LVsp), and LHb-ir levels were 43.0% (GVM),
44.4% (HY), 39.9% (POV), and 44.0% (LVsp) Therefore,the mean values of the four stages for both FSHb-ir andLHb-ir levels were represented as SP-stage levels in theseasonal reproductive cycle (Fig.6)
FSHb-ir levels were lowest in fish at the IM stage(9.3%), increased slightly (15.1%) at EV, then peaked(30.8%) at LV (Fig 6) In spawning fish, the FSHb-irlevels were about half (15.1%, SP) of previous levels, andthe levels were maintained in the fish at post-spawning(15.9%, PS) and during the resting condition (14.6%, RS).Thus, FSHb-ir levels were constant during the seasonalreproductive cycle except during LV, when FSHb-ir levelsincreased significantly
LHb-ir levels were lowest, although relatively highcompared with FSH, in immature fish (18.6%, IM),increased through vitellogenesis (32.2%, EV; 45.3%, LV),and were highest in spawning fish (42.8%, SP) LHb-ir wasmaintained at high levels post-spawning (43.6%, PS) butdeclined drastically to low levels during the resting
Fig 3 Three different regions in female chub mackerel pituitary at
the early vitellogenesis (EV) stage stained with Masson’s trichrome.
a Rostral pars distalis (RPD), b proximal pars distalis (PPD), and
c pars intermedia (PI) Most cells in the RPD stained red, and some
stained faint blue (arrow) Cells in the PPD stained dark or faint blue and red-purple (arrow) Cells in the PI stained faint red-purple and faint blue (arrow)
Fig 4 Three adjacent sections through the central part of the
proximal pars distalis (PPD) of the female chub mackerel pituitary
stained with a Masson’s trichrome, b anti-Fundulus heteroclitus (Fh)
FSHb, and c anti-Fh LHb FSHb and LHb immunoreactivity was
localized in different cell types Cells stained dark blue by Masson’s trichrome staining are immunostained with anti-Fh FSHb (a, b), and those stained faint blue are immunostained with anti-Fh LHb (a, c)
Trang 30condition (18.2%, RS) Thus, changes in LHb-ir levels
showed a similar pattern to those of FSHb between IM and
LV but were maintained as high levels during and after
spawning In the RS, the LHb-ir level decreased to a value
(18.2%) similar to that of IM
DiscussionFOM and spawning in captive chub mackerel were induced
by treatment with GnRHa combined with coconut oil.When GnRHa is injected, it degrades within a few days[24] As spawning of chub mackerel in captivity generallycontinues over a month, injected GnRHa plays a role as atrigger for spawning, but a series of spawnings may beinduced by endogenous GnRH, which controls pituitaryGtH secretion Therefore, females collected at 7–8 daysafter GnRHa injection seemed to undergo natural FOM andspawning, which were regulated by the BPG axis.The present study clearly identified FSH and LH cells inthe pituitaries of chub mackerel Fh FSHb and Fh LHbimmunoreactivity were localized distinctly in separatecells, indicating that chub mackerel FSH and LH are pro-duced in different cell types, as in many other teleosts(bluefin tuna Thunnus thynnus [26], salmonid [27], mum-michog [28], pejerrey Odontesthes bonariensis [29]) Incontrast, co-localization of FSH and LH in the same cells
Fig 5 Representative sections of female chub mackerel pituitary
sampled at different ovarian stages during the reproductive cycle;
immunostained with anti-Fundulus heteroclitus (Fh) FSHb (a–f) and
anti-Fh LHb (g–l) a, g Immature (IM) stage; b, h early vitellogenesis
(EV) stage; c, i late vitellogenesis (LV) stage; d, j spawning (SP)
stage; e, k post-spawning (PS) stage; f, l resting (RS) stage
Fig 6 Changes in the FSHb-ir and LHb-ir levels in female chub mackerel pituitary during the seasonal reproductive cycle See Fig 1
for ovarian stages during seasonal reproductive cycle Bars represent mean ± SEM of 3–5 fish Bars with different superscripts are significantly different (P \ 0.05)
Trang 31was observed in the pituitaries of Mediterranean yellowtail
Seriola dumerilii [30], swamp-eel Monopterus albus [18],
and Nile tilapia [31] This co-localization has been ascribed
either to biological variation or to the possibility that
het-erologous antisera might be inappropriate to investigate the
occurrence of two types of gonadotrophs [31,32]
Both FSH and LH cells occupied the entire PPD in the
pituitaries of chub mackerel; however, FSH cells were
localized mainly in the central area of the PPD, whereas
LH cells were observed along the border of the PI
Fur-thermore, LH cells were also observed in a few small
clusters of cells in the PI along the external border of the
PPD FSHb- and LHb-ir cells corresponded to cells that
stained dark blue and faint blue with aniline blue in
Mas-son’s trichrome staining, respectively These distribution
patterns of FSH and LH cells and distinctive particolored
observation in the chub mackerel pituitary were strikingly
similar to those of the same scombrid fish, bluefin tuna
[26]
The roles of the two GtHs during reproductive events
have only been well examined in salmonids, in which a
comparison among plasma levels, biological activities, andreceptor levels of FSH and LH has been made It is wellknown that, in salmonids, FSH is involved in vitellogenesisand LH acts at FOM and ovulation [3] In the case offemale red seabream Pagrus major with asynchronousovarian development, pituitary FSHb messenger RNA(mRNA) levels are low throughout the reproductive cycle,whereas pituitary LHb mRNA levels are high from ovariandevelopment to the spawning season, unlike in salmonids[33] It has also been reported that only LH induces FOM
in vitro [13] Gen et al [34] suggested that red seabreamFSH has no important role during vitellogenesis and FOM,whereas LH may be involved in regulating both vitello-genesis and FOM In this study, both pituitary FSHb- andLHb-ir levels increased significantly from basal valuesduring pre-vitellogenesis (IM) to the end of vitellogenesis(LV) These results suggest that both FSH and LH may beinvolved in the first vitellogenesis before entering thespawning cycle in chub mackerel Both FSHb- and LHb-irlevels increased significantly during the first vitellogenesisprior to the spawning cycle in the Japanese flounder pitu-itary using the same antibodies (anti-Fh FSHb and anti-FhLHb) [19], indicating similar results to our study
FSHb-ir levels decreased in chub mackerel sampledduring the spawning cycle, whereas LHb-ir levels weremaintained at high levels During chub mackerel vitello-genesis, the second clutch of oocytes proceeds to developconcomitant with FOM of the first clutch, as shown inFig.1 Therefore, the results suggest that LH may beinvolved in vitellogenesis and FOM during the spawningcycle; however, the role of FSH during the spawning cycle
is unclear If FSH is involved in vitellogenesis in chubmackerel, it should be synthesized during the spawningcycle In Nile tilapia, a multiple spawner in whichhomologous immunoassays for both FSH and LH havebeen developed, clear peaks in both GtHs were observed inthe plasma of females at the vitellogenic phase 2–3 daysafter spawning [9] These increases in plasma FSH and LHshow that both FSH and LH may be involved in vitello-genesis in tilapia A second peak in LH and FSH levels wasevident just before the next spawning; the increase in LHindicates a role during FOM, and the increase in FSHprobably reflects the hormone profile during recruitment of
a new generation of follicles for the next cycle [9] In chubmackerel, the development of homologous FSH and LHimmunoassays will be necessary to verify the regulation ofpituitary GtHs during gametogenesis It has been shownthat pituitary contents of GtHs does not necessarily reflecttheir release [35]
In conclusion, we revealed that FSH and LH are duced in different cell types in the chub mackerel pituitary.The results suggest that LH may be involved in bothvitellogenesis and FOM in female chub mackerel, whereas
pro-Fig 7 Changes in FSHb-ir and LHb-ir levels in female chub
mackerel pituitary during the spawning cycle See Fig 1 for ovarian
stages during spawning cycle Bars represent mean ± SEM of 3–5
fish Bars with different superscripts are significantly different
(P \ 0.05)
Trang 32FSH may be involved in vitellogenesis before entering the
spawning cycle, but its role during the spawning cycle is
still unclear The experimental procedures in this study are
appropriate to the proposed objectives as they mimic the
events naturally occurring during the spawning cycle
Therefore, we expect the rearing system described herein to
be very useful for study of the regulation mechanism of
gametogenesis by the GtH system in the BPG axis of
multiple-spawning species
Acknowledgments We thank the students of the Laboratory of
Marine Biology, Kyushu University, for their kind help with this
study M.N is supported by a JSPS Research Fellowship for Young
Scientist This research work was supported by a grant for scientific
research (20380113 to M.M.) from the Ministry of Education,
Cul-ture, Sports, Science, and Technology (MEXT), and through a
sub-project on studies on the prediction and application of fish species
alternation (SUPRFISH) financed by the Agriculture, Forestry, and
Fisheries Research Council (AFFRC) of Japan, as part of the
Popu-lation Outbreak of Marine Life (POMAL) Project.
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Trang 34O R I G I N A L A R T I C L E Biology
Reproductive characteristics of slipper lobster, cuttlefish
and squid species taken as byproduct in a tropical
prawn trawl fishery
Mark L Tonks•David A Milton•Gary C Fry
Received: 30 August 2010 / Accepted: 2 June 2011 / Published online: 28 June 2011
Ó The Japanese Society of Fisheries Science 2011
Abstract Reproductive characteristics relevant to
popu-lation sustainability were examined for eight abundant
invertebrate species caught as byproduct by the Northern
Prawn Fishery (NPF) in northern Australia Slipper lobsters
Thenus parindicus and Thenus australiensis differed in
their size at maturity, with T parindicus maturing at
smaller size Both species had similar reproductive
sea-sonality, with most recruitment early in the year (January–
March) Our estimates of carapace length (CL) at which
50% of females are mature (CL50) suggest that current
management regulations (minimum legal size 52 mm CL)
for Thenus are probably adequate for T parindicus, but
suboptimal for T australiensis However, T australiensis
only contributes a small proportion to the NPF Thenus
catch This species is likely to be protected as its preferred
habitat is coarse substrate and deeper water ([40 m),
which does not overlap greatly with the current commercial
trawl effort distribution Uroteuthis squid and Sepia
cut-tlefishes also varied in size at maturity and reproductive
seasonality Squid and cuttlefish populations are likely to
be underexploited based on historical catches Under
cur-rent fishing levels, squid stocks appear to be resilient to the
opportunistic targeting of spawning aggregations in similar
NPF regions over several years
Keywords Northern Prawn Fishery GSI
Spawning aggregation Fisheries management
Uroteuthis Sepia Thenus
IntroductionCommercial fishing in many countries is now regulated by
a complex combination of fisheries and environmentallegislation Markets and public perception have changed,with increasing expectation for fisheries to demonstratethat they are operating in an ecologically sustainablemanner [1, 2]; for example, the Australian FisheriesManagement Authority (AFMA) was required to assess allfederally managed fisheries for their compliance with theCommonwealth’s Environment Protection and BiodiversityConservation (EPBC) Act 1999 This act requires thatfisheries demonstrate that their impacts on target, byprod-uct, bycatch and threatened, endangered and protectedspecies are ecologically sustainable
The Northern Prawn Fishery (NPF) is Australia’s largestprawn trawl fishery This fishery was assessed under theEPBC Act in 2003 This assessment recommended thedevelopment and implementation of harvest strategies and
a spatial management system for all target and byproductspecies by 2008 For the NPF to demonstrate that they areoperating in an ecologically sustainable manner the fisheryhas to implement the AFMA harvest strategies for all targetand byproduct species This requires accurate species-specific life history information to better understand theimpacts of fishing on stocks While there is good biologicalunderstanding of the targeted penaeid prawns [3 5], verylittle is known of life history traits for the numerousbyproduct species caught in the NPF
Size at sexual maturity, seasonal reproductive tivity, spawning sites, fecundity and growth rates are some
produc-of the most important life history information needed forstock assessment and sustainable exploitation of marineanimals [6] It is important to understand the timing ofreproduction and recruitment of a fished population to
M L Tonks ( &) D A Milton G C Fry
CSIRO Marine and Atmospheric Research,
Ecosciences Precinct, G P O Box 2583, Brisbane,
QLD 4001, Australia
e-mail: mark.tonks@csiro.au
DOI 10.1007/s12562-011-0381-4
Trang 35produce management strategies that avoid overfishing [7];
for example, recruitment overfishing is of particular
con-cern for squid populations which consist of new recruits
annually [8] Currently the limited life history information
for byproduct species in the NPF makes it difficult to
develop management plans that ensure their sustainability
The NPF catches several byproduct groups, including
slipper lobsters (Thenus spp.—commonly known as bugs),
loliginid squids (Uroteuthis spp.) and sepiid cuttlefishes
(Sepia spp.) Input controls are the primary management
strategy used for this fishery These include limits on the
number of trawlers (52 since 2010), restrictions on gear
used (size, number and type of demersal nets) and spatial
and temporal restrictions on fishing operations The spatial
and temporal closures have been chosen based largely on
the biological characteristics of the main target prawn
species: the tiger prawns Penaeus esculentus and Penaeus
semisulcatus, and the banana prawns Penaeus merguiensis
and Penaeus indicus Management measures for some
byproduct also exist in the NPF For slipper lobsters
The-nus, there are two measures There is a minimum legal size
(MLS) of 75 mm carapace width (*52 mm carapace
length) and a prohibition on retaining egg-bearing females
[9] These restrictions are based on biological parameters
associated with yield optimization [10] Under these
restrictions, slipper lobsters should reach reproductive age
and spawn at least once prior to capture [10]
In contrast, management restrictions for squid are not
based on biological data; for example, the catch of squid is
currently limited to the total weight of prawns reported by
the fleet each year Since 2006, the AFMA has set an
annual 500 t interim limit reference point or ‘trigger limit’
for squid If this total catch is reached then a review of
management arrangements for squid will be conducted
There are no restrictions on the retention of cuttlefish
For the slipper lobsters Thenus, there have been few
detailed studies of their reproductive characteristics
Courtney [10] and Jones [11] summarised the most detailed
studies from north-eastern Australia These provided
information on growth, longevity, maturation, fecundity
and seasonal catch The taxonomy and reproductive
char-acteristics of the squid and cuttlefish species in northern
Australia are poorly known [12–14] Preliminary genetic
studies in the early 1990s have shown the Australian squid
taxa to be different from similar species in southeast Asia
[14] The two most abundant squid species, Uroteuthis
sp 3 and Uroteuthis sp 4, occur across northern Australia
[14]
This study was undertaken to: (1) identify size at
maturity and examine spatial and temporal variation in
reproductive condition for species in the three most
eco-nomically important byproduct groups caught in the NPF,
(2) describe some reproductive characteristics of a known
squid spawning aggregation and identify other possiblespawning aggregations based on commercial logbook catchrecords, (3) consider species-specific reproductive charac-teristics in relation to current management measures andfishing activity, in order to aid sustainability assessmentsand (4) discuss alternative management measures
Materials and methodsField sampling
Between August 2002 and July 2007, byproduct samples(slipper lobsters, squid and cuttlefish) were collectedaboard commercial prawn trawlers on fishery-independentprawn population monitoring surveys [15] These surveyswere generally undertaken twice a year, the first in Janu-ary–March (wet season) where approximately 200 sites aresampled and the second in June–August (dry season) whenapproximately 300 sites are sampled (Fig 1) Sampleswere also collected from additional surveys conductedfrom September to October in 2003 and October 2004 (dryseason) The sampling sites were allocated among sixgeographic regions based on commercial prawn trawlingeffort: Weipa, Karumba, Mornington, Vanderlins, southGroote and north Groote Within regions, the locations oftrawl sites were stratified by depth Each site was trawledfor a period of 30 min at 3.2 knots Trawls were conducted
at night to mirror commercial operating hours For sistency, vessels used twin ‘Florida Flyer’ nets, each with a12-fathom headrope, 2-1/4 inch diamond mesh and a co-dend of 120 meshes long and 150 meshes round (1-7/8 inchcodend mesh size) A straight bar top opening turtleexcluder device (TED) was used in each net After eachtrawl, all slipper lobsters (Thenus spp.) were identified tospecies, and total weights and numbers were recorded foreach net Up to 100 slipper lobsters of each species weremeasured (carapace length, CL in mm) per trawl, and theirsex and egg-bearing condition recorded All squid andcuttlefish were counted, weighed onboard and frozen forfurther laboratory analysis
con-Slipper lobster measurements
The length at sexual maturity (CL50) of female lobsters, ofboth species, was defined from the size at which theybecame egg-bearing It was estimated by fitting a logisticfunction with the equation
where y is the proportion of mature individuals bearing females) by carapace length, k is the parameter
Trang 36(egg-determining the slope of the maturity curve and CL50is the
estimated carapace length at 50% maturity (Fig.2)
Squid and cuttlefish measurements
We processed a proportion of trawls, stratifying by region,
season and year For the trawl catches examined, we
identified and dissected all squid and cuttlefish caught
Identification of squid and cuttlefish species was based on
taxonomic descriptions provided by [13] (cuttlefish) and
[14] (squid) Specimens were measured (dorsal mantle
length, ML in mm), weighed (±0.001 g) and sexed, and
gonads (ovary/testis) removed and weighed The
gonado-somatic index (GSI) was calculated with the following
formula:
GSI¼ ðgonad weight=ðbody weight gonad weightÞÞ
100:
ð2ÞBiological sampling and commercial catches of squid
aggregations
Anecdotal information from fishers and fleet masters from
NPF fishing companies suggested that most squid catches
reported in the commercial logbooks were taken fromspawning aggregations To verify the logbook records andassess the species composition and reproductive status ofsquids caught in large aggregations, we examined a 20-kgsubsample taken by an AFMA scientific observer onboard
a commercial trawler that found a large squid aggregation
in May 2007 The subsample was sent to the AustralianCommonwealth Scientific and Research Organisation(CSIRO) and processed in a similar manner to other sci-entific samples Commercial logbook records (availablefrom 1998 to 2007) were also examined to assess thespatial and temporal variation in catches of [400 kg ofsquid day-1 in order to assess the predictability of theseaggregations
Spatial and temporal variation in spawningThe proportion spawning of each species was estimatedfrom the number of sexually mature females with hydratedeggs (cephalopods) or egg-bearing (slipper lobsters) Forcephalopod species, size at 50% maturity was determined
by fitting a logistic model (Eq 1) to the mantle length andGSI data using the SAS NLIN procedure (Fig.3) Macro-scopic examination of the ovaries of females of each
Fig 1 Map of the Gulf of
Carpentaria, showing the
location of the
fishery-independent trawl survey sites
(filled circles) sampled for
byproduct life history studies
between 2002 and 2007
Trang 37species defined as mature by this criterion confirmed that
all individuals had enlarged and ripening eggs The size at
maturity was then used to determine the number of
potentially mature females in the sampled population of
each region (Karumba, Mornington, Vanderlins, north
Groote, south Groote, Weipa) and season (wet, January–
March; dry, June–October) over the 6-year sampling period
(2002–2007) The percentages of mature females of each
species in spawning condition were estimated by the
following criteria: (1) cuttlefish with hydrated eggs or GSI[2.5% and (2) squid with hydrated eggs or GSI [5%.Individuals for each cephalopod species with these GSIvalues were observed to have large hydrated yellow eggs
We assumed that this represented an indicator of spawningcondition For slipper lobsters we used the estimated size atmaturity (CL50) to determine the number of mature indi-viduals by region and season The proportion of spawningfemales of the mature population was then determined bythose that were egg-bearing
ResultsOverall catch
A total of 191,411 slipper lobsters, squid and cuttlefishwere collected over the 6 years (Table1) Of these, 13,693were identified to species and examined for GSI, sex andlength–weight relationships Slipper lobsters were readilyidentified in the field and represented the most numerousspecies examined Cuttlefish were the most abundantlycaught species group, but the need to examine theminternally for species identification reduced the numberidentified (Table1)
Seasonal spawning patternThe seasonal spawning pattern of slipper lobsters showed asimilar cycle for the two species (Fig.4) However, T par-indicus generally had a higher percentage of egg-bearingfemales, particularly during February and March (late wetseason) The percentage of egg-bearing females of both spe-cies was highest late in the dry season (August–October),when almost 50% of the mature female population were car-rying eggs Both species appear to have an extended spawningseason where egg-bearing females were detected through theyear (Fig.4) The exception to this was for T australiensis,where there were few females caught (n = 18) later in the wetseason (March) and none were egg-bearing
The spawning season for the squid and cuttlefish speciesalso appears to be extended for several species (Fig.5).Mean GSIs were higher later in the dry season (August–October) for both Uroteuthis species, particularly forUroteuthis sp 4, and Sepia smithi and S papuensis Theseasonal pattern of reproduction was less clear in the othertwo species of cuttlefish (S elliptica and S pharaonis),with a similar mean GSI throughout the year (Fig.5).There was evidence that spawning might also be occurring
in the wet season for some species (Uroteuthis sp 3, Sepiaelliptica, S smithi and S pharaonis) Unfortunately, nosamples were collected between April and June (dryseason)
Fig 2 Percentage of egg-bearing female Thenus slipper lobsters in
each size class from samples collected between 2002 and 2007 The
vertical dotted line is the minimum legal size that is allowed to be
retained in the NPF The dashed curve shows the mean logistic
regression best fit
Trang 38Commercial catches from the squid spawning
aggrega-tion subsampled in May 2007 were from west of
Mor-nington Island in the Gulf of Carpentaria where the water
depth ranged from 22 to 27 m (Fig.6) All squid examined
were identified as Uroteuthis sp 4 In total, 119 adult squid
weighing 7.3 kg were processed These comprised 57
males and 62 females (M/F ratio 0.92) Female GSIs varied
widely (Table2), with some specimens clearly spent andsuffering some mantle muscle degeneration Egg capsuleswere detected among the squid samples, and spermato-phore bulbs were evident in the buccal pouches of thefemales, indicating that mating and spawning were occur-ring at the same time There were two modal sizes of themales, with all specimens mature and dominant among the
Fig 3 Female gonadosomatic
index (%) of two species of
squid and four species of
cuttlefish caught in the Gulf of
Carpentaria from August 2002
to July 2007 The dashed
vertical line indicates the
estimated size at 50% maturity
determined by fitting a logistic
model
Trang 39larger size classes (Fig.7) Several other large catches of
squid were recorded in commercial logbooks in May near
Mornington Island (Fig.6) Large catches later in the year
(September–October) were also made further west, around
Groote Eylandt The occurrence of these aggregations
appears to be relatively predictable, as large catches were
made in the same area in May 2001 and 2002
Slipper lobster sexual maturity
The dorsal carapace length of mature female T parindicus
ranged from 34.5 to 81.5 mm, and from 52.7 to 89.5 mm
for T australiensis The mean estimated carapace length offemales at sexual maturity (CL50) was 52.0 ± 0.5 mm for
T parindicus (n = 15473) and 58.9 ± 0.5 mm for
T australiensis (n = 523) (Fig 2) The mean sizes atmaturity are described by the equations
at smaller size than Uroteuthis sp 4 Uroteuthis sp 3 assmall as 80 mm and Uroteuthis sp 4 as small as 100 mmhad hydrated yellow eggs and GSI C5% For cuttlefish, thelargest growing species, Sepia pharaonis and Sepia smithi,mature at C106 and C90 mm, respectively Both specieshowever had a few individuals as small as 80 mm with GSI[2% The most abundant species Sepia elliptica matures atC67 mm, which was similar to Sepia papuensis at C60 mm
Spatial distribution and relative abundanceThe commercial logbook catch of slipper lobsters wasspread throughout the entire Gulf of Carpentaria fished area(Fig.8) The mean retained catch rate was less than
15 kg day-1in most regions, but there were localised areaswhere more than 30 kg day-1 was recorded These weremostly in the south-eastern Gulf around Karumba and north
of Mornington Here, data from the fishery-independentprawn population monitoring surveys indicate that the
Table 1 Summary of total
number of byproduct specimens
collected in the Gulf of
Carpentaria and processed for
reproductive characteristics
from August 2002 to July 2007
a Species could not be
identified at sea
Byproduct group Common name Species Collected at sea Laboratory
processed
Slipper lobsters Mud bug Thenus parindicus 127,308 240
Reef bug Thenus australiensis 3,127 8
Cuttlefisha Ovalbone cuttlefish Sepia elliptica 6,981
Papuan cuttlefish Sepia papuensis 571 Pharaonis cuttlefish Sepia pharaonis 1,129 Smith’s cuttlefish Sepia smithi 1,553
Fig 4 Mean percentage ± 95% confidence limit of egg-bearing
females of two species of slipper lobster caught in the Gulf of
Carpentaria from August 2002 to July 2007
Trang 40commercial catch is likely to be almost exclusively T
par-indicus The survey data showed that egg-bearing female
slipper lobsters were caught in all regions However, the
proportion of both Thenus species that were egg-bearing
varied, mostly between seasons rather than spatially (Fig.8)
The spatial distribution of commercial squid catches was
more restricted than those of slipper lobster (Fig.9) Large
catches over 250 kg day-1 were reported from several
grids in the Vanderlins and Mornington Island regions Thefishery-independent survey data show that squid popula-tions were in spawning condition throughout most of theyear, with mature females representing a higher proportionduring the dry season in most regions (Fig.9)
The reported commercial catches of cuttlefish werealmost exclusively from the southern and western parts ofthe Gulf of Carpentaria, particularly around the Vanderlins
Fig 5 Mean monthly female
gonadosomatic index (%) of
two species of squid and four
species of cuttlefish caught in
the Gulf of Carpentaria from
August 2002 to July 2007