bioRxiv preprint doi: https://doi.org/10.1101/707349; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Feeding intensity and molecular prey identification of
2 the common long-armed octopus, Octopus minor
3 (Mollusca: Octopodidae) in the wild
4
1, 2 1, 3* 1 5 Qi-Kang Bo , Xiao-Dong Zheng , Zhi-Wei Chen
6
7 1 Institute of Evolution and Marine Biodiversity, Ocean University of China
8 2 Breeding station of Bohai Sea aquatic resources, Tianjin Fisheries Research Institute
9 3 Key Laboratory of Mariculture, Ministry of Education, Ocean University of China
10
11 *Corresponding Author
12 E-mail: [email protected] (X-D Z)
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14
15 Abstract
16 The common long-armed octopus, Octopus minor, is an important component
17 of systems and supports the local fisheries in the coastal areas of northern China.
18 And because of the overfishing, the national germplasm reserve was established as
19 conservation area at the Moon Lake for the genetic resource of O.minor in 2012. For
20 the fishery management and artificial breeding, especially for the management of
21 exclusive conservation reserves, its role in the ecosystem requires assessment.
22 Therefore, the feeding intensity of O. minor was studied from April to July 2014
23 when females reaching maturation, and prey composition was identified from
24 stomach contents using a DNA barcoding method. Of the 172 sampled octopuses, 66
25 had stomach contents that were nearly digested into pulp. Maximum feeding
26 intensity occurred during the month of April and the feeding intensity of the females
27 was greater than that of the males in April and May. A considerable overall
28 reduction of feeding intensity in both sexes occurred from April to July. A total of 8
29 species were identified as the prey of O. minor. Based on homology searches and
30 phylogenetic analysis, of 60 sequences, 30 matched with fish (50.00%, by number),
31 13 with crustaceans (21.66%), one with annelid (1.66%), one with nematode (1.66%)
32 and 15 with itself (25.00%). These results confirm that O. minor has habitual nature
33 of strong dietary preference, with Gobiidae families (62.79%, by number) being an
34 important prey during the time when females reach sexual maturation. From April to
35 July, the observed cannibalism showed an increasing trend.
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36 Introduction
37 The common long-armed octopus, Octopus minor (Sasaki 1920) is a benthic and
38 neritic octopod that has become a commercially important species in the north of
39 China and in South Korea [1, 2] and is identified to be furthermore ecologically
40 important as a generalist predator. Because of overfishing, a national germplasm
41 reserve was established at the Moon Lake (Fig 1) in 2012 to serve as conservation for
42 the genetic resource of O. minor. Moon Lake is a shallow (depth <3 m) lagoon with a
43 muddy, bivalve-covered bottom, and patches of sea grass growing in the subtidal zone,
44 which provides a good habitat for O. minor. Additionally, a program of artificial
45 propagation and release of O. minor has been launched in the north of China [2].
46
47 Fig 1. The location of the sampling site in Moon Lake and the dots show the
48 sampling site.
49 The five methods used to assess the natural feeding habits of marine animals
50 include the examination of prey remains from middens [3], direct observation [4],
51 stomach content analysis [5, 6], isotopic assessment [7, 8], trophic tracing [9] and
52 molecular prey identification [10-12]. In some studies, several different methods have
53 been used in combination [11, 13, 14].
54 A range of factors has made it difficult to determine the natural diet of O. minor;
55 the primary challenge that the oesophageal diameter of the octopus is physically
56 limited as it passes through the brain; therefore, the octopus’ beak bites small pieces
57 of tissue to swallow, avoiding the ingestion of hard skeletal material [11], and it is
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58 difficult to make an accurate identification of pray with lack of hard skeletal material.
59 Moreover, it is difficult to be collected on a large scale. O. minor burrow deep
60 (0.3-0.6 meter) interconnecting tunnels as nest in muddy marine bottom, leaving only
61 digging-holes and breathing-holes on the surface, into which it hides itself [15]; their
62 prey remains are left underground. Even if the prey remains were pushed out of the
63 nests, the light material would be easily removed by biotic and abiotic factors, while
64 the heavier material would be buried under the mud. Rapid digestion rates [16] and
65 external ingestion [17] make the stomach contents visually unidentifiable. These
66 specialized feeding strategies tend to bias data on prey species when morphological
67 analysis is used.
68 Food is an important factor to the extent that it governs growth, fecundity and
69 migratory movements. An understanding of the relationship between octopus species
70 and their favorite food items helps to locate potential feeding grounds, which may, in
71 turn, be helpful for the exploitation of these resources. The extent of the variation in
72 feeding must be taken into account when studying the diet of a generalist predator.
73 Octopods are opportunistic and their diets are often affected by the abundance of
74 available prey [3, 18]. The species, size and type of their diet in the wild are often
75 dominated by many factors such as maturity stage, seasonal variation [19], the body
76 size which reflects the prey ability [3, 14] and benthic assemblages which determine
77 prey availability [20]. In addition, what should be taken into account is that octopods
78 exhibit strong dietary preferences when given the same opportunity to different diets
79 items [18, 21-24].
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80 Few studies have focused on the ecology of O. minor, whose diet is roughly
81 known to include crustaceans, molluscs, polychaetes and fish [25, 26]. Investigation
82 into the natural diet of this species during sexual maturation, are of more research
83 value, and can provide trophic relationship information for fishery management,
84 especially for the management of exclusive conservation reserves. It also provides a
85 pray reference for aquaculture and artificial breeding of this commercially important
86 specie. In this study, we applied the method of molecular prey identification, DNA
87 barcoding method, to identify natural prey of O. minor based on stomach contents.
88 Materials and methods
89 Specimen collection
90 All the analyses have been carried out using freshly dead specimens collected
91 from fishermen of the Mashan Group Co. Ltd. No use of live animals has been
92 required for this study and no specific permissions were needed for the sampling
93 activities in all of the investigated areas because our species of interest is
94 commercially harvested (not endangered nor protected) and it was caught in areas
95 where fishing is allowed. One hundred and seventy-two O. minor specimens were
96 captured using traps placed during the evenings and collected at dawn from Moon
97 Lake (122º35’E, 37º21’N, Fig 1) from April to July 2014. The same number of males
98 and females were collected each month. All specimens were brought to the laboratory
99 where their stomach contents were removed. Each stomach was opened and the
100 contents were flushed into cryogenic vials. The stomach contents, potential residue of
101 prey, were weighed and then preserved in 70% ethanol at -20℃ for later DNA
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102 analysis [27]. Stomach content samples were numbered monthly, AP01 to AP19,
103 MA01 to MA13, JN01 to JN19 and JL01 to JL15 from April to July, respectively. To
104 avoid potential contaminants (e.g., blood and tissue attached to the stomach from the
105 predator), the exterior surface of each stomach was washed with sterile, distilled water
106 before removing the stomach contents [28]. The dorsal mantle length (ML), the
107 distance between the posterior midpoint of the mantle and the midpoint of the eyes,
108 and wet body weight (W) were also measured. This basic information and data were
109 showed in Table 1. Feeding intensity during the study months was determined based
110 on the degree of fullness of the stomach. The condition of feeding activity was
111 determined from observations of the degree of stomach distension as described by
112 Pillay (1952) [29]. Octopuses with stomachs that were gorged, full, ¾full, ½full were
113 considered to have been actively feeding (AF), while stomach ¼full, little and empty
114 were considered to denote poor feeding activity (PAF). The percentage of octopus in
115 the AF and PAF condition in both sexes for each month was calculated.
116
117 Table 1. The sample number, stomach content specimens number (Num.),
118 mantle length (ML, ±SD) and total weight (W, ±SD) of O. minor specimens
119 collected each month from Moon Lake
Sample Sample Female Male Num. date number ML (cm) W (g) ML (cm) W(g)
20/4/2014 52 8.68±0.95 120.47±24.99 8.60±0.89 135.65±28.26 19
14/5/2014 40 8.95±1.86 117.29±37.42 8.87±0.71 128.68±25.71 13
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20/6/2014 40 10.55±1.14 192.46±40.28 10.30±1.09 202.88±42.66 19
16/7/2014 40 9.99±1.23 172.95±37.96 9.57±0.89 200.35±36.80 15
120
121
122 DNA extraction and sequence acquisition
123 Stomach contents were evenly ground in a homogenizer. Duplicate 20-40 mg
124 samples of mixed tissue items were subsampled from each stomach content and
125 genomic DNA was then isolated by standard phenol-chloroform purification
126 procedures. A fragment of the mitochondrial cytochrome oxidase Ⅰ (COⅠ) gene
127 was amplified using the universal primers [30].
128 Each polymerase chain reaction (PCR) was carried out in 50-µL volumes
129 containing 2U Taq DNA polymerase (Takara Co.), about 60 ng template DNA, 0.2
130 mM dNTPs, 0.25 µM of each primer, 2 mM MgCl2 and 1×PCR buffer. The PCR
131 amplification was performed on a GeneAmp® 9700 PCR System (Applied
132 Biosystems). Cycling conditions consisted of an initial denaturation at 94℃ for 3 min,
133 followed by 35 cycles of: denaturation at 94℃ for 1 min, annealing at 50℃ for 1 min,
134 extension at 72℃ for 1 min, and a final step of 5 min at 72℃.
135 Amplification products were confirmed by 1.5% TBE agarose gel
136 electrophoresis stained with ethidium bromide. The cleaned product was prepared for
137 sequencing using the BigDye Terminator Cycle Sequencing Kit (ver.3.1, Applied
138 Biosystems) and sequenced bidirectionally using an ABI PRISM 3730 (Applied
139 Biosystems) automatic sequencer. PCR products producing multiple bands indicated
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140 that more than one prey species were present. Those PCR products were cloned using
141 the TOPO TA Cloning Kit (Invitrogen). Eight colonies per sample were selected for
142 colony PCR amplification and sequencing using the primers M13 (forward):
143 GTAAAACGACGGCCAG, andM13 (reverse): CAGGAAACAGCTATGAC.
144 DNA analyses and statistical analysis
145 Sequences of COⅠ were assembled and edited separately using DNASTAR
146 software (DNASTAR, Inc.), and then aligned with CLUSTAL_X 1.81 using the
147 default settings [31]. Sequences were considered to be part of the same ‘‘operational
148 taxonomic unit’’ (OTU), if there was less than a 1% sequence divergence, allowing
149 for the intraspecific variation and Taq polymerase errors [11, 13].
150 All of the obtained sequences were identified using the Identification System
151 (IDS) in the Barcode of Life Database (BOLD, www.boldsystems.org) and the Basic
152 Local Alignment Search Tool (BLAST) query algorithm in GenBank to establish
153 whenever possible the identification of the ingested material. The maximum
154 likelihood (ML) method was chosen to infer evolutionary history. Bootstrap
155 probabilities with 1,000 replications were calculated to assess reliability on each node
156 of the ML tree. Sequence divergence calculation and evolutionary analyses were
157 conducted in MEGA 6 software based on the Kimura 2-parameter model (K2P) [32].
158 The ML tree contained all of the sequences obtained from the stomach contents,
159 together with the closest matches to each sequence that were downloaded from BOLD
160 databases and GenBank. The criteria to assign identification to at the species level
161 required the sequence similarity display >98% in the BOLD database and BLAST [11]
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162 and, if not, identification was restricted to the highest taxonomic lineage supported by
163 bootstrap probabilities higher than 70% in the consensus tree [14, 15]. The AF and
164 PAF differences the between sexes were verified by the Chi-squared fit test (2).
165 Statistical analyses were performed using IBM SPSS statistics version 20 (IBM,
166 Chicago, IL, USA). To corroborate that the number of analyzed stomachs was
167 adequate for diet description, a cumulative prey curve was generated using the
168 Estimate S Version 8.2 based on the prey identified [33]. The number of samples was
169 assumed to be sufficient to describe the diet when the curve approaches the
170 asymptote.
171
172 Results
173 Feeding intensity
174 Of the 172 octopus individuals, 66 had contents in their stomachs (Table 1) and
175 feeding intensity of O. minor varied to an extent in respect to the months, and more
176 than half of the stomachs were found to be empty (Table 2). There was significant
177 difference in the feeding activity between July and other months (p<0.05) and no
178 difference among the first three months (p>0.05). However, the AF percentage of
179 octopus from April is greater than that of other months, meaning the maximum
180 feeding intensity occurred during the month of April. A considerable overall reduction
181 in the intensity of feeding for both sexes occurred from April to July (Table 2). The
182 feeding intensity of the females is greater than that of the males based on the
183 percentage of AF and PAF between the sexes in each month, and in April and May
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184 the feeding intensity of the females was significantly greater than that of the males
185 (Table 3). That is, compared to the males, more female individuals were actively
186 feeding.
187
188 Table 2. The distribution of stomach with different degree of distension and the
189 percentage of octopus with active feeding activity (AF) from April to July. Data
190 in the same column having different superscripted letters are significantly
191 different (p<0.05).
Stomach status (%) Feeding activity (%)
Month Gorge Full ¾Full ½Full ¼Full Little Empty AF d
Apr. 5.77 5.77 5.77 13.46 1.92 3.85 63.46 30.81a
May. 6.00 16.00 5.00 2.50 3.00 0.00 67.50 29.50a
Jun. 5.00 7.50 7.50 10.00 17.50 0.00 52.50 30.00a
Jul. 0.00 0.00 5.00 5.00 17.50 10.00 62.50 10.00b
192
193 Table 3. The percentage of octopus with active feeding activity (AF) and poor
194 feeding activity (PAF) in both sexes from April to July, including the difference
195 of feeding intensity between females and males in each month by Chi square fit
196 test.
Feeding Female Male Level of
activity (%) AF PAF AF PAF significance
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Apr. 50.00 50.00 11.54 88.46 **
May. 45.00 55.00 15.00 85.00 *
Jun. 35.00 65.00 25.00 75.00 NS
Jul. 20.00 80.00 5.00 95.00 NS
197 *, P<0.05; **, P<0.01; NS, not significant
198
199 Molecular prey identification
200 All stomach contents were nearly digested into pulp and as a result, the prey
201 items were impossible to visually identify. A total of 59 stomach contents yielded
202 amplifiable DNA and 60 sequences were obtained, ranging from 500bp to 708 bp
203 (Table 4). All sequences were submitted to GenBank (Accession numbers
204 MK688462-MK688521). Six OTUs were established, with a maximum sequence
205 divergence of 0.1%. Of the 60 sequences, 59 clones showed similarities higher than
206 98% to reference sequences, allowing identification at species level. Only one clone
207 displayed 97.56% similarity to reference sequences, and it was assigned to the
208 Diopatra genus level (Fig 2). One stomach contained two kinds of prey, and the
209 remaining 58 samples contained only one kind. The duplicate parallel PCR products
210 from the contents of each stomach yielded the same sequences.
211
212 Table 4. Pray DNA detected in wild O. minor by cloning the COⅠ fragment gene,
213 including number per month and min-max length of sequences, GenBank
214 Accession numbers and Sequence ID of closest matches, percentages of similarity
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215 obtained from BLAST and BOLD.
216
Ac. Min-max Similarity Pray Order Family Species Number or . length % DNA Seq. ID of Seq.
Acanthogobius JX679022, Perciformes Gobiidae 99.85,100 OUT1 500-700 flavimanus GU440207
Acanthogobius JQ738534, Perciformes Gobiidae 99.85,100 OUT2 640-697 hasta HQ536245
Tridentiger JX679061, MA07， Perciformes Gobiidae 100 684 bifasciatus HQ536534 MA11
Pholis FOJS101-1 Perciformes Pholidae 100 OUT3 556-694 crassispina 3
Charybdis HM237602, Decapoda Protunidae 100 OUT4 633-687 japonica HM237597
Alpheus Decapoda Alpheilae 100 HM180433 JU19 620 brevicristatus
Stomatopod Oratosquilla Squillidae 100 HM180739 OUT5 572-587 a oratoria
Eunicida Onuphidae Diopatra sp. 97.56 FJ428842 JU18 682
Hysterothylaciu Ascaridida Anisakidae 98.91 FJ907319 JU04 679 m aduncum
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Octopodida HQ638215 Octopoda Octopus minor 99.66,100 OUT6 562 e NC015896
217
218 Fig 2. Maximum likelihood tree (ML tree) for all sequences obtained from
219 stomach contents and the closest matches of each sequence that were
220 downloaded from BOLD databases and GenBank, using MEGA 6 software
221 based on K2p model
222
223 Of the 60 sequences, 30 matched with fish (50.00%, by number), 13 with crustaceans
224 (21.66%), one with annelid (1.66%), one with nematode (1.66%) and 15 with its own
225 species (25.00%). A total of 8 species were identified as prey of O. minor (Table 2).
226 When considering the importance of prey to O. minor by number (%N), and ignoring
227 the nematode and its own species as prey, the families with the highest %N were
228 Gobiidae (61.36%), comprising Acanthogobius flavimanus (40.91%), Acanthogobius
229 hasta(15.91%) and Tridentiger bifasciatus (4.55%); Protunidae (18.18%), comprising
230 the one species Charybdis japonica; Pholidae (6.82%), comprising one species Pholis
231 crassispina; and Squillidae (9.09%), comprising one species, Oratosquilla oratoria.
232 Alpheilae and Onuphidae, accounted for same percentage, 2.27%, respectively
233 comprising one species each, Alpheus brevicristatus and Diopatra sp. The occurrence
234 frequency of Family of prey identified in the stomachs of O. minor caught in the
235 Moon Lake was showed in Fig 3.
236
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237 Fig 3. Diet of O. minor in Swan lake using frequency of occurrence of prey items
238 for stomach content analysis by DNA Barcoding from April to July.
239
240 The number of prey species consumed by O. minor in a month was 2-5 from April to
241 July, and in June, the prey diversity was the most abundant, with 5 species. A.
242 flavimanus and C. japonica appeared with the highest occurring frequency during 3
243 months, followed by A. hasta. Cannibalism was found in 15 individuals, and the
244 cannibalism occurred 2, 3, 4 and 6 cases respectively from April to July, showing an
245 increasing trend. The cumulative prey curve approached the asymptote showing that
246 59 stomachs were adequate to describe the diet of this species (Fig 4).
247
248 Fig 4. The species-accumulation cures. The slopes curve rapidly approached
249 asymptotes, indicating that there were enough stomach content specimens
250 collected to detect most prey species.
251
252 Discussion
253 Based on our recent studies, the male octopus reaches maturity in April,
254 meanwhile, the females are at maturing stage when the female octopus begins egg
255 production actively, which mainly involves the synthesis of vitellus. At this stage the
256 female octopuses use energy directly from food, with no storage reserves being
257 transferred from organs to the gonads [34, 35]. Hence, it is unsurprising then that the
258 feeding intensity of the female was greater than that of the male. However, the
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259 difference in feeding intensity between females and males was subsequently
260 indistinguishable as a considerable overall feeding intensity reduction occured in
261 both sexes caused by the listlessness of the male after mating and of the female after
262 spawn-movement [2, 36].
263 One advantage of molecular methods is that when morphological methods are
264 ineffective, sufficient DNA can be recovered by successful DNA amplifications [11,
265 12]. This can be achieved in poor quality samples as well because it requires only a
266 small amount of tissue for DNA extraction. Moreover, Meusnier et al. (2008) have
267 demonstrated that DNA barcoding could identify species with fragments as short as
268 100 bp with at least 90% efficiency [37]. The read lengths of the DNA fragments
269 obtained in this study was 500-700 bp, and the relative large read lengths strengthen
270 the accuracy of the identification. Only one clone was assigned to the genius level on
271 the basis of their supported topographical position on the bootstrap consensus tree
272 (Figure 2).
273 One intriguing discovery we found was that almost all of the stomach contents
274 (58 out of 59) contained only one kind of prey. In consideration of stochastic
275 sampling errors when subsampling stomach contents for DNA extraction and lack of
276 detection of some prey species arising from low-concentration DNA to PCRs [13],
277 duplicate subsamples with 20-40mg material each were used in this study. However,
278 the same sequences were obtained for duplicate subsamples showed that it was
279 unlikely that the lack of detection of diet composition was due to the procedural
280 errors. In addition, the universal COⅠ primers used in this study are able to amplify
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281 COⅠ gene fragments from 11 invertebrate phyla and fish [30], as shown in previous
282 studies [38, 39]. A strong dietary preference probably played a decisive role in this
283 result. When presented with the same opportunity to different diets, O. minor makes
284 a strong preference, which has been observed in other similar octopods, such as O.
285 maorum [21], O. vulgaris [14] and Enteroctopus dofleini [18]. This situation has also
286 been observed in our previous studies, where when presented with the opportunity to
287 manila clams, the bivalve Potamocorbula laevis and the Asian shore crab, O. minor
288 frequently preyed on the Asian shore crab and rarely on the clams. Biandolino et al.
289 (2010) observed that only after octopus had voraciously fed on all available fish and
290 crabs, they then fed on mussels, as the last option [24]. Ambrose (1984) found that
291 the diet of O. bimaculatus in the field was influenced by food preferences, where
292 highly preferred prey (crabs, bivalves, sedentary grazers) were rare in the field,
293 however the most abundant species available, the snail Tegula eiseni Jordan, was the
294 least preferred [20]. The results showed that although there distributed a large of
295 mollusks, 15 molluscan species belonging to 14 families, in Moon Lake [40],
296 octopuses did not feed on mollusks. The diet of O. minor in the wild was also
297 influenced by food preferences. False negatives cannot be ruled out in this study.
298 PCR dropout is a common phenomenon in genotyping studies because PCRs are
299 known to amplify preferentially the DNA of a higher quality, and variable rates of
300 DNA degradation between different prey items may have biased PCR amplification
301 success [13]. The promise of high throughput methods in the future will only
302 improve this approach.
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303 The prey consisted of 8 species in this study, a relatively narrow dietary for
304 octopus species. However, previous studies suggested that at least 12 prey species
305 were consumed by similar octopus though examination of their middens or via
306 morphological analyses of stomach contents [5, 6, 41]. From our analysis, the slopes
307 of the saturation curves rapidly approached asymptotes, which indicate that
308 sufficient stomach content specimens were collected to capture the major prey items
309 (Fig 4). In this study we were interested in the months when the females were
310 maturing and the dietary range wound likely be greater than reported here if the O.
311 minor stomach contents had been sporadically collected from Moon Lake over a
312 longer period [6]. Fish, including Gobiidae and Pholidae, accounted for the vast
313 majority proportion (68.19%) of the prey. The benthic fish, Gobiidae, in the lake
314 where the octopuses were collected, appear to be an especially important food
315 supply for females during sexual maturation and they provided a substantial and
316 favorite food supply for the octopods. When the preferred diet does meet the feeding
317 needs, O. minor would not feed the other candidate items [17, 18, 22, 24, 32], which
318 is a possible reason for the reduced number of types of consumed prey.
319 The genetic data indicate that cannibalism may be a significant feeding strategy
320 in long-armed octopus, with long-armed octopus DNA being detected in 15 of 59
321 stomachs. We do not believe that the host DNA contaminated the stomach contents
322 in these cases, as suckers and tops of arms were present in the stomachs and the host
323 was sound with no wounds. Cannibalism in octopus has been observed in artificial
324 culture [26, 42] and wild research [3, 43]. In this study, the number of cannibalism
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325 cases increased from April to July.
326 Conclusions
327 The results confirm that although O. minor is a generalist and opportunistic
328 predator, it has strong dietary preferences in the wild. The discovery of the feeding
329 voracity of females during maturation provides a reference for aquaculture and
330 artificial breeding of this commercially important species, as well as accumulating
331 data for the rational resource exploitation of this kind animal. The DNA barcoding
332 method was shown to be successful in unraveling the feeding habits of wild O.
333 minor, which can simplify the field operation as stomach contents can be fixed by
334 the infiltration of ethanol in the field. This method shows higher taxonomic
335 resolution of the determination of prey items compared to traditional descriptions of
336 stomach contents. The results provide trophic relationship information for fishery
337 management, especially for the management of exclusive conservation reserves of O.
338 minor.
339 Acknowledgments
340 This study was supported by research grants from National Natural Science
341 Foundation of China (Nos. 31672257; 31172058) and Fundamental Research Funds
342 for the Central Universities(No.201822022); We are indebted to Mr. Cai B of
343 Mashan Group Co. Ltd (Shan-Dong Province, China) and Mr. Song MP for their
344 kind assistance in specimen collection.
345
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