traditional tagging capture-recapture method a significantly higher probability of
recapture was obtained by using the radio and acoustic telemetry (Brousseau et al
2004) While a trial experiment on the movement of adult horseshoe crabs by using an
the behaviour and ecology of the adult horseshoe crabs Behaviour of adult horseshoe
crabs out of the water was previously studied particularly for the spawning behaviour
613 In-depth Studies on Phylogenetic and Geographical Genetic Variations in Juvenile
different populations of horseshoe crabs in Hong Kong waters The present data showed
that the inter-specific variations of both 18S and 28S rDNA sequence were larger than
the intra-specific variations with the percentage of pair-wise difference being lt 1 and
speciation of T tridentatus and C rotundicauda This also applied to the differentiation
between juveniles of T tridentatus with one or three spines Other genetic markers such
as the 18-28S ITS rDNA which not only provides information on speciation but also
214
some indications of population genetics can be adopted for further studies of the
population genetics in horseshoe crabs For the differentiation of closely related species
the mitochondrial DNA (mtDNA) can be used to indicate the overall genetic
differences Because of the energy-producing reactions mtDNA carries out
mitochondria mutate their DNA at a rapid rate and are less able to ldquoproofreadrdquo these
errors than genes in nuclear DNA Hence mtDNA has a high mutation rate and serves
as a perfect tool for spotting genetic differences between individuals of closely related
species and within a species In fact the mt AT-rich region DNA sequences had been
used for the determination of the subdivision of T tridentatus at three localities
Kinmen Tiexianwei and Dongwei in Taiwan (Yang et al 2007)
To examine the geographical variations in genetic diversity of the same species of
horseshoe crabs among different regions in Asia-Pacific Region a geographical genetic
study is suggested Individuals of horseshoe crabs can be collected from different
countries such as Japan Indonesia Malaysia mainland China and Hong Kong and the
genetic diversity among different populations can be examined and compared using the
method of amplified fragment length polymorphism (AFLP) and DNA (COI) genomic
sequence (Pierce et al 2000) AFLP is a highly sensitive method for detecting
polymorhism in DNA There are many advantages of AFLP over other marker
technologies including randomly amplified polymorphic DNA (RAPD) restriction
fragment length polymorphism (RFLP) and microsatellites AFLP not only has higher
reproducibility resolution and sensitivity at the whole genome level compared to other
techniques but the capability to amplify between 50 and 100 fragments at one time In
addition no prior sequence information is needed for amplification (Vos et al 1995
Meudt and Clarke 2007) As a result AFLP has become widely used for the
identification of genetic variations in strains or closely related species of plants fungi
215
animals and bacteria and population genetic studies DNA (COI) genomic sequence
had been used to survey the intraspecific differences of the populations of L
polyphemus in Delaware Bay and Chesapeake Bay (Pierce et al 2000) Hence further
investigations in the spatial variations of the genetic make up of horseshoe crabs in local
habitats by AFLP and DNA (COI) genomic sequence are suggested
614 Risk Assessment of Horseshoe Crabs
Concerns about the possible impact of heavy metal pollution upon horseshoe crabs
have been raised (Chiu and Morton 1999a Morton and Lee 2003) Chiu and Morton
(1999a) showed that the concentrations of cadmium and lead in sediments at Ha Pak
Nai were comparable to those in polluted bays in Tolo and Victoria Harbours Although
previous studies have shown that horseshoe crab embryos and larvae are highly tolerant
to heavy metals (Botton et al 1998) and organic contaminants such as TBT (Botton et
al 1999) heavy metals or organic pollutants would be accumulated in the body and
even sequestrated from the female during egg formation (Kannan et al 1995 Burger
1997) A high toxicity of heavy metals including cadmium lead manganese and
selenium and tetrodotoxin was detected in the eggs of L polyphemus and C
rotundicauda in the wild (Burger 1997 Burger et al 2002 Ngy et al 2007) while a
high toxic level of copper (2070 μg g-1 wet weight) and zinc (2890 μg g-1 wet weight)
was detected in the eggs of T tridentatus (unpublished data) High heavy metal levels
may increase the mortality rate and abnormality of the embryos Some previous studies
showed that heavy metals had sublethal effects on the horseshoe crab embryos larvae
and juveniles including defective embryos inhibition of limb regeneration delay of
molting and abnormal eyes (Itow et al 1998a 1998b) Whether water pollution in
Hong Kong causes sublethal effects or even mortality in horseshoe crabs is unknown
and deserves further investigations
216
Therefore an ecological risk assessment (ERA) of various pollutants in particular
heavy metals to local horseshoe crab species is recommended ERA has been performed
for various organisms in Hong Kong including marine neogastropods Thais clavigera
and Thais luteostoma (Leung et al 2006) Indo-pacific humpback dolphin and Finless
porpoise (Hung et al 2004 2006) and Little egret and Black-crowned night heron
(Connell et la 2002 2003) For horseshoe crab species the probabilistic ERA would
be based on tissue burden of heavy metals Relationships between body burden of heavy
metals and sublethal effects such as defective embryos and inhibition of limb
regeneration could be established The ERA results would provide an essential
ldquobaselinerdquo for a long-term monitoring programme in Hong Kong and provide a
methodological framework for ERA of heavy metals in Southeast Asia An ERA of
heavy metals to horseshoe crabs in Hong Kong therefore is recommended to be done
with urgency
615 More Studies on Carcinoscorpius rotundicauda
C rotundicauda is found in coastal areas from the South China Sea to the Indian
subcontinent However information on C rotundicauda is scarce worldwide including
habitat characteristics and the ecology of the juveniles (Rao and Rao 1972 Sekiguchi
1988b Chatterji and Parulekar 1992) Some of the previous studies have suggested that
C rotundicauda go upstream during the rising tide from the river in the breeding season
(Rao and Rao 1972) In the Gulf of Siam egg clusters of C rotundicauda were also
recorded on muddy banks of rivers flowing through the mangroves (Chatterji and
Parulekar 1992) Sekiguchi (1988b) showed that the breeding environment for C
rotundicauda was quite different from that for the other three species with a preference
towards fresh or brackish waters (Sekiguchi 1988b) However in the present survey no
C rotundicauda was recorded in the additional 20 random quadrats (05 m times 05 m)
217
within the stream area at each sampling site Therefore more studies of the habitat
requirements of C rotundicauda should be conducted Nevertheless in this study
nursery grounds for this species were identified in Deep Bay area northeastern New
Territories and even on Lantau Island Detailed investigation on the hydrology and
sediment characteristics of these areas can provide more information on the habitat
preference of this species In addition although the living environments of T
tridentatus and C rotundicauda seem to be different (Sekiguchi et al 1977 Sekiguchi
1988b) they co-occurred on some of the shores A more in-depth study of their
microhabitat preferences should be conducted
616 Non-destructive Artificial Breeding Technique
Electrical stimulation has been regularly used in L polyphemus for artificial
breeding for over 20 years (Brown and Clapper 1981) It was mainly used in
experimental studies of the horseshoe crab eggs and larva such as developmental study
(Jegia and Costlow 1982 Laughlin 1983 Ehlinger and Tankersley 2004) and
toxicology test (Botton et al 1999) However it failed to apply to the Indo-Pacific
horseshoe crab species including T tridentatus (Sekiguchi 1988a) In the present study
an electric stimulus of 7 V and 1 mA ac was successful to stimulate the release of eggs
and sperm whereas 10 V was used in the study by Dong and Ng (1985) However the
electrical stimulation practice was not repeatable in this study Therefore a more
in-depth study on this method is needed especially with regard to the frequency of the
electrical stimulation and the most suitable sizes of adult horseshoe crabs to be used
Besides to avoid any harm caused by high voltages to the adults voltages ranged from
7 to 10 V are recommended for further investigations on this method In addition
applying a slight pressure anterior to the genital operculum is another possible
non-destructive method for egg collection (French 1979) However as only hundreds
218
of eggs could be collected by these two non-destructive gamete collection methods they
are more suitable for embryonic and larval study than massive population enhancement
practices
Apart from the direct extraction and electrical stimulation induction of spawning
in the laboratory is another possible alternative which is non-destructive with more
gametes collected (Chen et al 2004) By simulating the flood tide and substratum of
the natural spawning ground of the horseshoe crabs adult can be induced to spawn
during flood tide in the laboratory (Chen et al 2004) In a study by Chen et al (2004)
sand grain size ranged from 04 to 18 mm corresponding to medium to very coarse
sand were collected from the natural spawning ground as bedding and the horseshoe
crab mating pairs were induced to spawn during spring tides with a maximum of 10000
eggs being laid in a 5-hour period As this method can collect more gametes a
feasibility study of this method for local horseshoe crabs should be conducted
617 Further Study on the Requirements for Juvenile Rearing
Artificial insemination and breeding in the laboratory is a viable option to enhance
the horseshoe population in the natural environment By maintaining the artificially
fertilized eggs in the optimal environmental conditions (eg appropriate temperature
and salinity) the survival rate of the hatched trilobites can be enhanced
Laboratory-reared juveniles can be released to the field after they have grown to certain
size (may be ge 2 years old) to reduce predation risk Although this study has
successfully fertilized the eggs in vitro and larvae could be reared at least up to the
fourth instar in laboratory conditions mass mortality of the juveniles becomes an
obstacle in the mass production of the juveniles for the release purpose As the reasons
for the mass mortality are unknown further studies should be conducted to enhance the
219
survivorship with regard to the rearing conditions including formulation of the best diet
improvement of water quality and optimization of environmental conditions
62 Proposed Conservation Measures
The present study showed an obvious decline in the juvenile horseshoe crabs from
2002 to 2005 Comparing with the results obtained by Morton and Lee (2003) the
population has dropped by 80-96 (Section 2) Further decline in the wild population is
expected if no immediate conservation measures are taken considering the urbanization
and rapid development of infrastructure projects in the vicinity of these shores in Hong
Kong and the Pearl River Delta Hence a species specific conservation strategy should
be planned and implemented before this living fossil disappears in Hong Kong
A conservation strategy should contain eight elements including species
concerned habitats problems conservation actions monitoring strategy review
interagency coordination and public involvement (ODFW 2006) For the former two
elements they aim at gathering information on the distribution and abundance of the
species and describing the locations and relative conditions of key habitats essential to
the conservation of the species (ODFW 2006) These two elements have been achieved
by Chiu and Morton (1999a) Morton and Lee (2003) and the present study (Section 2
and 3) by providing comprehensive baseline information on the local horseshoe crabs
including morphology taxonomy growth behaviour ecology and their key habitats
The present study has also identified the ldquoproblemsrdquo which may adversely affect the
species or their habitats and identified information required to improve the situation
(ODFW 2006) For example Section 4 identified and evaluated the impact of human
exploitation on the horseshoe crab populations through market surveys on the sale of
220
horseshoe crabs in Hong Kong while Section 2 identified possible impacts of habitat
reclamation and pollution on local horseshoe crabs
This section suggests necessary conservation actions for the horseshoe crabs and
their associated habitats with monitoring measures for evaluating and reviewing the
effectiveness of the proposed conservation actions To formulate a successful and
comprehensive conservation strategy co-operation with international regional and state
agencies and engagement of the public are also suggested (Walls et al 2002 ODFW
2006)
621 Re-introduction Programme
Introduction of captive breeding for animal conservation has increased markedly in
the last decade To compensate for the loss through natural mortality and increase the
wild population a re-introduction programme for juvenile horseshoe crabs may be a
feasible conservation approach with a great potential in Hong Kong China and Taiwan
started the re-introduction program for horseshoe crabs in the past few years In Taiwan
more than 10000 individuals of second-instar juveniles produced artificially in the
laboratory were released in the protected area in Kinmen in 2002 (Chen and Yeh 2005)
while about 40000 larvae were released to the wild in Xiamen China in 2004 Some
people may query the effectiveness and feasibility of the re-introduction programme as
the juveniles may be carried away from the shore by waves or eaten by fish or other
organisms However Chen et al (2006) showed that the re-introduction of juvenile
horseshoe crabs to sandymuddy shores is feasible as the sediment layer on the shores
would protect the juveniles from predators and waves
221
In Hong Kong studies are being carried out to improve the rearing technique for
juvenile horseshoe crabs Dr Paul Shin and Dr S G Cheung of the City University of
Hong Kong have been collaborating with the Ocean Park Hong Kong since October
2006 Being a world class aquarium the Ocean Park has provided expert advice and
technical help on laboratory rearing technique and skills and hopefully the survival of
the juveniles can be improved (Lai 2007)
622 Ban on Fishing and Possession of Horseshoe Crabs in Hong Kong
Apart from enhancing the wild population by re-introduction Hong Kong
government should also protect the local horseshoe crabs through legislation As the
threat of horseshoe crabs associated with human consumption is still high legislation
should be drawn up to ban the fishery and sale of the horseshoe crabs in Hong Kong In
Taiwan a ban on the catch of horseshoe crabs was made in the protected area in
Kinmen from 2000 to 2004 to avoid any human exploitation of the horseshoe crab
populations in the first four years after the establishment of the protected area (Chen and
Yeh 2005) Regional governments in China including Ningbo and Xiamen have drawn
up legislation to ban the catch of horseshoe crabs in the regional waters (Cheung and
Fan 2006) while USA has restricted the catch of horseshoe crabs by a fishing permit
system (Eagle 2001)
In addition public education should be introduced to increase the public awareness
on the life history and fragile status of this animal so as to reduce the fishing pressure
More importantly efforts should be made to place Asia-Pacific horseshoe crabs on the
endangered species list of CITES through international consensus so that the imports
and exports of these animals can be better managed to further reduce human
consumption and fishing pressure especially in this part of the world
222
623 Designation of Protected Areas
Apart from human exploitation the decline in horseshoe crab populations may also
be due to human activities like water pollution and degradation and loss of habitats
Among the potential threats scientists generally agree that habitat destruction is
currently the primary lethal agent for most of the endangered species (Wilcove et al
1998) In Hong Kong several developmental projects adjacent to the horseshoe crab
key nursery grounds have been planned and undergone environmental impact
assessments in the past few months One of them involved the construction of a golf
course and some estates adjacent to Pak Nai one of the key nursery grounds for the two
local horseshoe crab species (Fulland International Limited 2007) Another project is
the proposed Tuen Mun - Chek Lap Kok Link which may cause adverse impacts on the
water quality of the nursery grounds of the horseshoe crabs including Tai Ho Tung
Chung and San Tau (Highways Department 2007) If these environmental threats are
not lessened it is meaningless to breed and release the juveniles into the wild as they
cannot survive in a deteriorating environment (HKDCS 2007)
Protected areas for horseshoe crabs have been set up in various countries including
Taiwan China and Japan Early in 1928 T tridentatus has been protected with an
establishment of the largest horseshoe crab protected area in the Oe-hama Beach in
Japan (Botton 2001) In Taiwan a size of 800 ha protected area was established in
Kinmen in 1999 (Chen and Yeh 2005) while several horseshoe crab special reserves
have been set up in Pingtan Fujian China (Wang et al 2002) In Hong Kong
important nursery shores such as Shui Hau Wan and San Tau should be designated as
Sites for Special Scientific Interest (SSSIs) andor Special Conservation Area to prevent
future developments at these shores Although the designation of horseshoe crab
protected areas was proposed as early as 1999 by Professor Morton (Chiu and Morton