Octopus Dofleini Research Articles

Design of experimental food patches to measure foraging intensity for octopus: a case study with the giant Pacific octopusEnteroctopus dofleini

ABSTRACTIn a study of the foraging behaviour of the giant Pacific octopus Enteroctopus dofleini, we designed two types of experimental food patches to measure habitat preferences and perceptions of predation risk. The first patch successfully measured giving-up densities (GUDs), confirmed by octopus prey presence and higher foraging at sites with historically greater octopus presence. However, nontarget foragers also foraged on these experimental food patches. Our second floating patch design successfully excluded nontarget species from subtidal patches, and from intertidal patches at high tide, but allowed for foraging by E. dofleini. The second design successfully measured GUDs and suggested that octopus preferred foraging in a subtidal habitat compared to an intertidal habitat. We ascribe the higher GUD in the intertidal habitat to its higher predation risk relative to the subtidal habitat. The second patch design seems well suited for E. dofleini and, in conjunction with a camera system, could be used to provide behavioural indicators of the octopus's abundance, perceptions of habitat quality and predation risk.

Разработка функциональных композиций сухих концентратов морепродуктов и исследование их свойств

Introduction. Nutrition has a significant impact on human health, performance, well-being, and lifespan. The research objective was to develop formulations for functional compositions of seafood cryoconcentrates.
 Study objects and methods. The study featured concentrates obtained by cryotechnology from carcasses of Pacific squid (Todarodes pacificus), muscular shell of cucumaria (Cucumaria japonica), scallop mantle (Mizuhopecten yessoensis), octopus skin (Octopus dofleini), milt of Pacific herring (Pacific herring), and Japanese kelp (Laminaria japonica), as well as their multicomponent mixes. The content of biologically active substances was determined by the spectrophotometric method. The safety and biological value were biotested on Tetrahymena pyriformis.
 Results and discussion. The dry seafood concentrates had a high content of protein, minerals, and biologically active components, i.e. hexosamines, triterpene glycosides, carotenoids, nucleic acids, etc. The seafood cryoconcentrate mixes were divided into fortifying, restoring, immunomodulating, and mineral. Their chemical composition varied greatly, depending on the functional aim of the mix. For instance, the protein content varied from 28.8 to 57.6%, lipids – from 3.1 to 6.9%, carbohydrates – from 15.0 to 35.9%, minerals – from 10.9 to 22.9%, and hexosamines – from 2.2 to 5.6%. The experiments made it possible to substantiate the composition of the dry mixes. The compilation principle was based on the chemical profile of the raw material, as well as on the choice of biologically active substances. All the dry seafood concentrates showed a high relative biological value. However, the values differed, depending on the type of raw material: from 65.5% for the scallop mantle sample to 86.3% for the octopus skin sample. The difference could be explained by the chemical composition of the cryopowders and their availability to ciliate enzymes. 
 Conclusion. The research resulted in formulations and functional mixes of dry seafood concentrates obtained by cryotechnology. The dry seafood concentrates can be used both independently and as functional compositions. The developed mixes demonstrated rather high content of animal proteins, minerals, and biologically active substance, e.g. hexosamines, triterpene glycosides, carotenoids, nucleic acids, etc. Their functional compositions proved to have excellent sensory properties; they are biologically safe for human consumption and have a high relative biological value.

The Use of a Species-Specific Health and Welfare Assessment Tool for the Giant Pacific Octopus, Enteroctopus dofleini

ABSTRACT Cephalopods are increasingly viewed as sentient animals that require the same welfare consideration as their vertebrate counterparts. In this study, an observational welfare assessment tool developed by the EU Directive was revised to be species-specific for the giant Pacific octopus, Enteroctopus dofleini. This E. dofleini health and welfare assessment tool includes categories assessing E. dofleini external appearance, behavior, and clinical signs of stress and disease. These categories are scored in severity from 1 to 4, allowing a quantitative perspective on health observations. Six facilities used the health and welfare assessment tool to evaluate E. dofleini until the animal was humanely euthanized or died naturally. Results showed an irreversible upward trend in scores for feeding behavior and response to stimulus beginning 4 weeks prior to death, with significant changes in health and welfare scores between 4 weeks and the final week prior to death. This suggests that upward trends in these two variables predict death within 3–4 weeks. Highly variable results between individuals for other categories indicate that a quantitative tool can help assess health and welfare at the individual level.

Thermal properties of commercial hydrobionts’ tissues in the freezing process

The paper describes changes in thermal properties in the process of freezing of marine raw materials. The study objects were the skin of giant octopus (Octopus dofleini L.), pallium of Pacific squid (Todarodes pacificus L.), milt of Pacific herring (Clupea pallasii L .), a nd muscle t issue of Japanese c ucumaria (Cucumaria japonica L.). The mathematical relations of the studied thermal parameters allowing the calculation of specific heat capacity, thermal conductivity coefficient and tissue density of the studied objects in the process of freezing were obtained. It was found that the change in the total specific heat capacity during the freezing of all the objects under study was of the same type: first, this figure increases due to the intensive ice formation in the tissues of hydrobionts, and then decreases due to a significant decrease in the content of the liquid aqueous phase. The values of the total specific heat capacity before the freezing of seafood were determined (kJ/kg·K): 4.26 for squid, 3.58 for milt of Pacific herring, 3.66 for octopus skin, and 3.95 for the shell of cucumaria. It was revealed that an increase in the amount of frozen out water decreased the density of samples of frozen raw materials. This was due to the high (77.4–88.9%) content of water, turning into ice, which has a lower density index. The values of hydrobionts’ tissue density before freezing were obtained ( 0 ρ , kg/m3): 1226.74 for squid, 1209.6 for milt of Pacific herring, 1128.55 for octopus skin, and 031.26 for shell of cucumaria. It was established that the thermal conductivity of the hydrobiont tissue samples in the process of freezing increased with the growth of the proportion of frozen out water contained, approaching the thermal conductivity of ice. The calculated values of thermal conductivity coefficient of seafood tissue prior to freezing equal (W/m·K): 0.52 for squid, 0.47 for milt of Pacific herring, 0.63 for octopus skin, and 0.53 for cucumaria. The obtained thermal characteristics values of the objects studied are recommended for use in technical and technological calculations of aquatic biological resources cooling treatment processes.

Escape Openings Reduce the Catch of Small North Pacific Giant Octopus, Enteroctopus Dofleini from Fishing Basket, Aomori, Japan

Escape Openings Reduce the Catch of Small North Pacific Giant Octopus, Enteroctopus Dofleini from Fishing Basket, Aomori, Japan

Urbanization-related distribution patterns and habitat-use by the marine mesopredator, giant Pacific octopus (Enteroctopus dofleini)

Urbanization is a process that heavily alters marine and terrestrial environments, though terrestrial urban ecosystems have been studied far more intensively. Terrestrial studies suggest that urbanization can facilitate mesopredators by enhancing food and shelter resources and reducing predation pressure from apex consumers. This in turn has considerable consequences for ecological communities. We evaluated spatial distribution patterns and habitat-use of the marine mesopredator, giant Pacific octopus (Enteroctopus dofleini), relative to terrestrial urbanization intensity in Puget Sound, Washington, USA. Using field surveys and citizen-contributed data for E. dofleini, we examined whether: (1) Distribution was related to urbanization, (2) Abundance was related to the extent of benthic anthropogenic debris, and (3) Diet differed as a function of urbanization and den cover. Our results suggest that urbanization impacts may differ with depth. Mixed-effects logistic regression model estimates for the probability of occurrence increased with urbanization in deep-water (> 24 m), and decreased with urbanization in shallow water (< 18 m). Accompanying field surveys indicated that E. dofleini abundance was correlated with the number of benthic anthropogenic debris items, and that E. dofleini diets were not affected by urbanization intensity or den cover. Though E. dofleini may be synanthropic within certain urban environments, the mechanisms driving this pattern likely differ from those affecting common urban mesopredators on land, with den provisioning from man-made structures being more important than altered food resources.

飼育下における未成熟ミズダコの成長率，摂餌率，餌料転換効率に及ぼす水温の影響

水温が上昇する夏季にミズダコが浅所から深所へ移動する要因を探るために，飼育下で未成熟ミズダコの日間成長率，日間摂餌率，餌料転換効率を水温別に調べた。その結果，日間成長率は10.0℃を頂点にして水温の上昇とともに低下し，20.0℃ではマイナスとなった。日間摂餌率は15.0℃以上で低下した。餌料転換効率は2.5-5.0℃で高く，水温の上昇とともに低下し，20.0℃ではマイナスとなった。したがって，夏季から春季にかけてミズダコは水温上昇に関連した生理的変化により，次第に深所へ移動すると考えられた。

Analysis of the drift speed and drift direction of fishing gear for pot drift fishing for &lt;i&gt;Enteroctopus dofleini&lt;/i&gt; by obtaining positional information

Analysis of the drift speed and drift direction of fishing gear for pot drift fishing for &lt;i&gt;Enteroctopus dofleini&lt;/i&gt; by obtaining positional information

Body Patterns of the Frilled Giant Pacific Octopus, a New Species of Octopus from Prince William Sound, AK

We tested whether body patterns distinguished two haplotypes of large octopus in Prince William Sound Alaska. Live octopuses were photographed in captivity and assigned to a morphotype based on whether longitudinal mantle folds (a characteristic body pattern feature of the giant Pacific octopus (GPO), Enteroctopus dofleini (Wulker 1910)) were present (the GPO morphotype, N = 14) or absent (a novel morphotype, N = 6). Novel morphotype octopuses were distinguished without exception from GPO morphotype octopuses by the presence of a lateral mantle frill and the absence of longitudinal mantle folds, ventral mantle texture below the lateral frill, and patch and groove patterning. Additional traits could be used in combination to reliably characterize the novel morphotype. The genetic haplotypes of these octopuses were determined from nucleotide sequence data from two microsatellite loci and a portion of the OCDE gene. The GPO morphotype was identified with E. dofleini based on the match of body pattern traits to published descriptions of that species and the match of its genetic haplotype to published sequences of E. dofleini. Novel morphotype body patterns did not match descriptions of any species from the eastern north Pacific, while its genetic haplotype matched that of a recently sequenced undescribed octopus. The GPO and novel morphotypes are sister clades, and body pattern traits reliably identified individuals to morphotype and haplotype. Body pattern traits can be used in field identification of live octopuses allowing population assessments, by-catch frequency estimates, and other studies of both octopus types. We offer the common name of the frilled giant Pacific octopus for the novel morphotype, and based on genetic and morphological data suggest this clade is a new species of large Pacific octopus in the genus Enteroctopus. A full species description remains to be done.

Diet of endangered Steller sea lions (Eumetopias jubatus) in the Aleutian Islands: new insights from DNA detections and bioenergetic reconstructions

The endangered western stock of Steller sea lion (Eumetopias jubatus (Schreber, 1776)) still declines in the western Aleutian Islands and accurate diet information is vital to test leading hypotheses. We undertook the first bioenergetic diet reconstruction using both molecular and hard part prey identifications from &gt;600 scats collected in March–April 2008 and 2012. Atka mackerel (Pleurogrammus monopterygius (Pallas, 1810)) remained a primary prey (17%–27% by energy), but large (mean 60 cm) Pacific cod (Gadus macrocephalus Tilesius, 1810) also emerged as important prey (20%–24%) in a more diverse diet than previously reported, with Cottidae and smooth lumpsucker (Aptocyclus ventricosus (Pallas, 1769)) also contributing ∼10%. DNA detections highlighted a potentially important and previously underestimated prey, giant Pacific octopus (Enteroctopus dofleini (Wülker, 1910) (diet contribution 2%–15%, dependent on prey size assumptions). Although 504 unique DNA identifications resulted in significant increases for cephalopods, Pacific cod, and smooth lumpsucker, hard part alone species rankings were similar to composite ones and bioenergetic species rankings similar to occurrence-based ones. Retention or regurgitation of large cephalopod beaks, the removal of large cod heads, and skeletal fragility of lumpsuckers may explain these differences. DNA identifications provide valuable comparative and complementary prey occurrence data for pinnipeds, but composite diet estimates are optimal.

Delayed discard mortality of the giant Pacific octopus, Enteroctopus dofleini, in the Gulf of Alaska cod pot fishery

Abstract There is a lack of information available on octopuses in Alaskan waters and because of this lack of data, catch limits are set based on historical incidental catch rates or survey biomass estimates that are likely to be overly conservative. This issue is further exacerbated by the fact that catch accounting for octopuses assumes 100% mortality for all octopuses caught whether retained or discarded. We examined the delayed mortality of the giant Pacific octopus, Enteroctopus dofleini , captured in Pacific cod, Gadus microcephalus , pot fisheries in the Gulf of Alaska. Octopuses captured as bycatch during commercial fishing operations were held on the vessel and brought back to the laboratory to assess delayed mortality over a period of 21 days. The relationship between survivorship and injury presence, sex, maturity, weight, season of capture, air temperature, water depth, and pot soak time were examined using generalized linear models. In addition, the relationship between injury presence and the same parameters were examined. Survivorship ranged from 77.8% to 93.6% during each of three fishing seasons and was significantly related to injury presence. Injury presence ranged from 10.0% to 63.2% during each season and was related to season with an increase in injuries during the fall. These data indicate the conservative assumption of 100% bycatch mortality for octopus is not appropriate for some fisheries, in particular those utilizing pot gear.

Temperature- and size-dependent growth and movement of the North Pacific giant octopus (Enteroctopus dofleini) in the Bering Sea

ABSTRACTOctopus growth and movement occurs during all life stages and have implications for survival, food web dynamics and reproduction. From 2009 to 2011, 1714 North Pacific giant octopus (Enteroctopus dofleini) were tagged and recaptured in the eastern Bering Sea with visible implant elastomers to better understand the ecology of this data-poor species. Over this period, 246 of the tagged individuals were recaptured. In autumn, when temperatures were warmest, E. dofleini had higher growth rates and moved more than in the colder winter months. For both short- and long-term recaptures, small octopus grew faster than large octopus. Movement of octopus over short- and long-term periods was low for very small and very large octopus and high for median-sized octopus, which is likely to be a function of maturity status and reproductive activities. Approximately 80% of recaptures moved less than 2 km from the initial tagging location for time periods up to a year, suggesting long-term site fidelity. As temperature and size may be tightly linked to growth and movement rates for E. dofleini in the eastern Bering Sea, predicted climate warming will likely alter ecological processes for the species and impact their distribution.

北海道北部日本海沿岸のたこ箱漁場におけるミズダコの鉛直分布の季節変化

北海道北部日本海沿岸のたこ箱漁場におけるミズダコの鉛直分布の季節変化

Spatial and ontogenetic patterns of Pacific cod (Gadus macrocephalus Tilesius) predation on octopus in the eastern Bering Sea

In 2012, the North Pacific Fishery Management Council adopted a consumption-based stock assessment method to determine catch limits for the non-target, multi-species octopus complex in the Bering Sea-Aleutian Islands (BSAI) fishery management area. The method uses Pacific cod (Gadus macrocephalus) diet data as a basis for estimating octopus complex natural mortality and minimum biomass. To enhance understanding of the predator-prey interaction between Pacific cod and octopus, we examined patterns of octopus consumption by Pacific cod using long-term stomach contents data from the eastern Bering Sea continental shelf, a large, ecologically unique subarea of the BSAI. Generalized additive modeling of octopus presence/absence in Pacific cod diets revealed distinct spatial, ontogenetic and seasonal consumption patterns. Prey octopus frequency of occurrence (FO) generally increased with bottom depth, latitude and Pacific cod fork length, and FO in the southern BSAI was lower during winter and spring than during summer. Prey octopus FO patterns may reflect overall consumption patterns and likely indicate long-term distribution patterns of small-sized (<1 kg) octopus on the EBS shelf, although we could not visually distinguish between octopus species using prey remains. Multi-species beak length-to-body mass regressions developed from three octopus species allowed reasonable estimation of prey octopus mass, and we found Pacific cod fork length was positively correlated with prey octopus mass, suggesting predator-prey interactions are sensitive to predator and prey size composition. Pacific cod consumed octopus with estimated masses ranging from 0.000017 kg to 4.62 kg, while octopus taken during concurrent bottom trawl surveys range from 0.05 kg to greater than 25 kg. Based on our findings, we expect the consumption-based stock assessment underestimates BSAI octopus complex biomass because it cannot account for larger octopus, such as the 10–20 kg Enteroctopus dofleini which dominate incidental take in BSAI Pacific cod pot fishery.

Characteristics and discard mortality of octopus bycatch in Alaska groundfish fisheries

Octopus are caught incidentally in several US federally-managed trawl, longline, and pot fisheries in Alaska. The majority caught are giant Pacific octopus Enteroctopus dofleini. Recent changes in fisheries management in Alaska have resulted in the creation of an octopus species complex with annual catch limits, leading to increased interest in management and catch accounting for this data-poor assemblage. This study characterized the incidental octopus catch in Alaska groundfish fisheries and the mortality rate of octopus caught and discarded at sea. Onboard fisheries observers collected data on octopus weight, sex, and condition at discard in a variety of Alaska groundfish fisheries from 2006 to 2011. A field study aboard a commercial pot-fishing vessel examined delayed mortality resulting from the capture process in giant Pacific octopus during routine pot fishing. Octopus incidental catch varied widely in size and condition at capture for various fishing gear types. Vessels fishing using pot gear captured larger octopus than vessels using longline or trawl gear. Initial condition at capture was best in pot gear, with over 90% of octopus discarded from pot vessels alive in excellent condition. Octopus taken in trawl gear had the highest immediate mortality rate, with 68–94% dead or injured at discard. Giant Pacific octopus held for 24–60h following pot capture showed no signs of delayed mortality or decline in condition. These results suggest that assuming 100% mortality of discarded octopus may overestimate fishing impacts.

RETROSPECTIVE REVIEW OF MORTALITY IN GIANT PACIFIC OCTOPUS (ENTEROCTOPUS DOFLEINI).

The giant Pacific octopus (Enteroctopus dofleini) is a popular exhibit species in public display aquaria, but information on health and disease is limited. This retrospective review evaluates time in collection and describes antemortem clinical signs and pathology of giant Pacific octopuses in an aquarium setting. Between March 2004 and December 2013, there were 19 mortalities: eight males, 10 females, and one individual whose sex was not recorded. Average time spent in collection for all octopuses was 375 ± 173 days (males 351 ± 148 days, females 410 ± 196 days). Ten (52.6%) of the octopuses were sexually mature at the time of death, six (31.6%) were not sexually mature, and reproductive status could not be determined in three octopuses (15.8%). Minimal changes were noted on gross necropsy but branchitis was histologically evident in 14 octopuses, often in conjunction with amoeboid or flagellate parasites. Senescence, parasitism, and husbandry were all important contributors to mortality and should be considered when caring for captive octopuses.

Evaluation of the 5′ end of the 16S rRNA gene as a DNA barcode marker for the Cephalopoda

The present study seeks to incorporate a highly variable DNA barcode marker, additional to the standard regions of the cytochrome oxidase I gene and the 3′ end of the 16S large ribosomal subunit (16S rRNA), for a more effective species-level identification among cephalopods. Thus, we evaluated whether the 5′ end region of the 16S rRNA gene can be suitable as a DNA barcode marker among these taxa. Using a novel primer set, 28 different species were evaluated based on the pairwise intra- and interspecific distance and neighbor-joining (NJ) analysis. Except for Enteroctopus dofleini, we were able to obtain the sequences for the remaining species which formed highly supported clusters in the NJ tree. Divergence at the interspecific level may indicate high genetic variation of the region being proposed as compared to the standard of 16S rRNA. In addition, two well-supported clades and high levels of divergence within Sthenoteuthis oualaniensis, Loliolus japonica and Sepia pharaonis suggest the occurrence of cryptic species. This study confirms the efficiency of the 5′ end region of the 16S rRNA gene as a DNA barcode marker which can be used along with the standard DNA markers in future studies.

Functional chondroitin sulfate from Enteroctopus dofleini containing a 3-O-sulfo glucuronic acid residue

There are several reports that chondroitin sulfate containing K-type units [GlcA (3S)-GalNAc (4S)] exhibiting similar levels of neurite outgrowth promoting activities as CS having high amounts of B-, D- and E-type disulfated disaccharides. Although CS containing K-type units possess important biological activities, there are only few sources, such as king crab cartilage, squid cartilage or sea cucumber. In this study, CS containing 13.9% of K-type units was found in octopus (Enteroctopus dofleini) cartilage using different substrate specificities of chondroitinases. The 2D NMR spectra showed cross-peaks assigned to protons on sugar ring of GlcA (3S), demonstrating the presence of K-type units in octopus CS. Furthermore, proportion of fucosylated disaccharide units in octopus CS was very low. Octopus CS showed high affinity for growth factors and stimulated neurite outgrowth of hippocampal neurons, similar to the activity of squid CS-E. These results strongly suggest that octopus cartilage is a rich source of CS-K and has important biological activities.

Multiple Paternity and Preliminary Population Genetics of Giant Pacific Octopuses, Enteroctopus dofleini, in Oregon, Washington and the Southeast Coast of Vancouver Island, BC

A total of 77 giant Pacific octopus, Enteroctopus dofleini, tissue samples were collected from the Oregon Coast (OR), Neah Bay Washington (NB), Puget Sound Washington (PS) and the southeast coast of Vancouver Island, British Columbia, Canada (BC) for genetic analyses. A suite of eight variable microsatellite markers developed from giant Pacific octopuses were amplified in these samples to determine population diversity, structure, relatedness and paternity. The majority of loci met Hardy-Weinberg equilibrium expectations within each population. We found moderate genetic diversity (average observed heterozygosity = 0.445, range = 0.307–0.515 and average expected heterozygosity = 0.567, range = 0.506–0.696) and moderate population structuring with distinct separation of groups (FST values ranged from 0.101 between BC and PS to 0.237 between BC and NB). Several egg strings from the BC population were collected from three female octopus dens for relatedness and paternity analyses. Results suggest strong support for multiple paternity within one egg clutch with progeny sired by between two to four males.

오징어류 종 판별을 위한 다중 유전자 검사법 개발 및 검증

본 연구에서는 오징어류에 해당하는 대왕오징어, 오징어, 문어, 한치 및 이를 이용한 가공식품에 대해서 분자생물학적 기법을 활용한 시험법을 검토하였다. 시료 중 원료성분 확인을 위하여 오징어류 4종에 대해 최적의 종 특이 프라이머를 디자인하였으며, 시료로부터 직접 genomic DNA를 추출하여 PCR을 실시하였다. PCR 수행과정에서 반응을 저해하는 염 성분을 제거하기 위하여 증류수를 이용하여 3~4회 세척 후 PCR을 실시한 결과, Single PCR의 경우 대왕오징어(552 bp), 오징어(463 bp), 문어(247 bp), 한치(354 bp)에 해당하는 종 특이적인 증폭산물을 확인하였으며, Multiplex PCR 의 경우 서로 다른 종 사이의 교차반응없이 동시다발적으로 증폭이 일어남을 확인할 수 있었다. 또한 이들 4종에 대해 PCR 민감도를 조사한 결과, 모두 약 <TEX>$0.1ng/{\mu}l$</TEX>의 농도까지 검출이 가능함을 확인하였으며 multiplex PCR의 경우 약 <TEX>$0.25ng/{\mu}l$</TEX>의 농도까지 검출이 가능함을 확인하였다. 이를 이용하여 오징어류가 함유된 수산물 가공식품 8건에 대해 적용성을 검토한 결과, 모든 시료에서 유효한 결과를 확인할 수 있었다. 따라서 본 연구에서 제작된 오징어류 4종에 대한 종 특이적 프라이머는 생물 상태뿐만 아니라 수산물 가공식품에 대해서도 이를 판별할 수 있어 식품안전관리에 활용할 수 있을 것으로 기대된다. In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.