METHOD FOR CULTIVATING PARALARVAE OF THE COMMON OCTOPUS, OCTOPUS VULGARIS

Abstract

Method for cultivating paralarvae of the common octopus, Octopusvulgaris up to the settlement thereof (benthic phase), based on a diet of prey containing the caprellids Pfttisica marina and CaprelIa equilibra and/or gammarids of the genus Jassa spp. Factors such as light conditions, water renewal and temperature, inter alia, are optimized to obtain maximum paralarvae survival.

Claims

1. A method for cultivating paralarvae of the common octopus, Octopus vulgaris, placed in a culture tank containing water, said method comprising adding a prey selected from: gammarideas of the type Jassa spp., caprelids of the types Phtisica spp. and/or Caprella spp. or combinations thereof to said culture tank.

2. The method according to claim 1, wherein the gammarideas of the type Jassa spp. are selected from the group consisting of Jassa falcata and Jassa marmorata.

3. The method according to claim 1, wherein the caprelids of the type Phtisica spp. are Phtisica marina and the caprelids of the type Caprella spp. are Caprella equilibra.

4. The method according to claim 1, wherein from day 1 of life to 20 to 30 days of life of the paralarvae, gammarideas of between 1 and 4 mm are added, from this moment and up to 50 to 70 days of life, gammarideas of between 2 and 8 mm in length and caprelids of between 4 and 30 mm in length are added, from 50 to 70 days and up to settlement of the paralarvae only gammarideas of between 1 and 4 mm in length are administered.

5. The method according to claim 1, wherein the gammarideas are added from a day between 1 and 10 days of life of the paralarvae up to settlement of the same, while the caprelids are added from 20 to 30 days of life and up to settlement of the same.

6. The method according to claim 1, wherein the gammarideas are added from the first day of life of the paralarvae up to their settlement and the caprelids are added from day 10 of life up to their settlement.

7. The method according to claim 1, wherein both the gammarideas and caprelids are added from day 30 of life of the paralarvae up to their settlement.

8. The method according to claim 1, wherein when combinations of gammarideas and caprelids are added, between 1 and 10% of caprelids are administered with respect to the total number of prey and between 90 and 99% of gammarideas with respect to the total number of prey.

9. The method according to claim 8, wherein 5% of caprelids and 95% of gammarideas are administered with respect to the total number of prey.

10. The method according to claim 1, wherein the density of paralarvae in the culture tank is between 3 and 7 paralarvae/liter.

11. The method according to claim 1, wherein a total of between 3 and 5 prey per paralarva per day are supplied to the culture tank.

12. The method according to claim 1, wherein the culture tanks used between the first day of life and up to 50 to 70 days of life have a truncated cone shape and are black and from 50 to 70 days, the paralarvae are transferred to another flat-bottomed, gray culture tank.

13. The method according to claim 12, wherein the density of paralarvae in the flat-bottomed, gray culture tank will be from 0.1 to 0.5 larvae per liter.

14. The method according to claim 1, wherein the culture tank is artificially illuminated by means of a light placed on the edge of the tank.

15. The method according to claim 14, wherein intensity levels of the light supplied vary between 1077 and 1436 W/m.sup.2 for the first 10 to 15 days of life of the paralarvae; between 517 and 1077 W/m.sup.2 from 10 to 15 days of life and up to 50 to 70 days of life, from this moment and up to settlement, the light is reduced to a range between 69 and 517 W/m.sup.2.

16. The method according to claim 1, wherein the water in the culture tank is renewed as follows: no renewal of the water of any kind is carried out in the tank during the two first days, renewal subsequently starts at a flowrate of between 4 and 10% of the total volume of the tank each hour such that 20% is renewed daily for the first 5 days and from there it increases up to 100% daily at 10 to 15 days, this renewal is maintained at 100% up to 30 days and from there, the renewal is left open permanently 24 hours per day which represents a daily renewal of 200% approximately, this renewal percentage is maintained until the paralarvae are settled.

17. The method according to claim 1, wherein a mixture of microalgae Isochrysis spp. and Nannochioropsis spp. is also added to the tank until a concentration of the same of between 0.7 and 1.510.sup.6 cls/mL is obtained at the time of said addition.

18. The method according to claim 17, wherein the mixture of microalgae is added once per day.

19. The method according to claim 1, wherein the oxygen concentration in the water of the culture tank is greater than 5.5 mg/l.

20. The method according to claim 1, wherein the salinity of the water in the culture tank is from 35 to 36 g/l.

21. The method according to claim 1, wherein the temperature of the water in the culture tank is between 18 and 22 C.

22. The method according to claim 1, wherein the bottom of the culture tank is maintained without siphoning.

23. The method according to claim 1, wherein the prey are added to the culture tank from a storage tank of the same by means of siphoning the walls and bottom of the storage tank of the prey or by means of a concentrator using the outlet tube of the water of the cultivation itself such that a net is placed on said tube which allows prey to enter and a smaller net is placed at the bottom of the tube (which keeps them inside the tube) and once the prey are in the interior part of the tube, this tube is removed and the prey are transferred to a recipient in order to add them to the paralarvae tank.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIG. 1: shows a photo of a paralarva recently hatched from a common octopus, O. vulgaris, where its characteristic physiognomy can be seen.

[0054] FIG. 2: shows photos of exemplary adults Jassa falcata (A) and Jassa marmorata (B).

[0055] FIG. 3: shows photos of exemplary adults Phtisica marina, male and female (A) and Caprella equilibra, male and female (B).

[0056] FIG. 4: graphics showing the growth in mg dry weight of paralarvae of octopus in different experiments: 4A: a control group with paralarvae fed exclusively with Artemia sp. up to 40 days of life and an experimental group fed with Artemia sp. and zoea up to day 30 and subsequently with amphipods exclusively from day 31 to 50. The bars indicate the standard deviation. 4B: Paralarvae fed up to day 25 with a control treatment (Artemia sp.) and an experimental treatment with Artemia sp. up to 8 days and amphipods from 9 to 25 days old.

[0057] FIG. 5: diagram showing the culture tanks used in the invention. 5A: truncated cone-shaped tank used from the first day until the larvae enter the settlement pages (6 to 9 mg dry weight which usually occurs between 50 and 70 days of life). In the diagram is represented the water inlet (1) and light source (2), the central aeration (3), the central tube (4) provided with a filter and the exterior drain allowing the water height (5) to be regulated. 5B: Settlement tank where the larvae are transferred until reaching the settlement phase (above 9 mg dry weight). In the diagram is represented the water inlet (1) and light source (2), the refuges, both hanging and deposited on the bottom (3), the central tube provided with a filter (4) and exterior drain allowing the water height (5) to be regulated.

[0058] FIG. 6: Paralarvae in settlement phase. PVC tubes used as refuges are also observed in B).

EXAMPLES

[0059] The invention is illustrated by means of tests carried out by the inventors which reveal the effectiveness of the product of the invention.

Example 1: Octopus Larval Culture Test Known in the Prior Art (Garrido, D. et al. (2017). Aquaculture, 468, 558-568)

[0060] Experiment carried out in truncated cone-shaped tanks of 500 l volume, black walls and bottom, gentle to moderate aeration in a central position. Use of green water (phytoplankton) and a renewal of 150% per day during the entire experiment (30 days) with water filtered at 2 micron by means of cartridge filters. In terms of the light, a 36 W cold white fluorescent light is used, situated in the central upper part of the tank. A single intensity of 700 lux was used during the entire experiment. The food was based on Artemia sp. enriched with microalgae with respect to another enriched with a marine phospholipid. The Artemia sp. was supplied in 3 separate doses throughout the day. The survival at 30 days old was between 0.14 to 3.77%, while the growth rate showed an increase in dry weight of between 3.9 and 6.4% per day.

Example 2: Control Test: Common Octopus Larval Culture (1000 l Tank) Using Artemia Sp. Exclusively as Prey from Day 1 to Day 40 of Life of the Paralarvae

[0061] Experiment carried out in a 1000 l volume truncated cone-shaped tank, black walls and bottom, aeration in a central position and moderate, preventing the current created by the air bubbles impeding the paralarvae from moving freely through the tank (FIG. 5A). A density of 5 paralarvae/liter and values for the dissolved oxygen oscillated between 5.5 and 6.7 mg/I for a temperature range of 18.5 to 21.3 C. and a salinity of 35 g/I (for the entire experimental period). Green water was used up to 30 days, with a concentration of 10.sup.6 cls/mL of Nannochloropsis sp. and Isochrysis aff. galbana at the time of adding the microalgae. This method was the same as in the previous test (test of the prior art), but the modifications in the renewal in this second test affected its concentration. These differences consisted in maintaining the tank closed for the two first days, with maturation of the tank taking place (1.sup.st difference with the method of the prior art). Following this period, renewal starts (with water filtered at 1 micron with cartridge filters), starting with a renewal rate of 15 to 20% of the volume of the tank (10 ml/s for 5 hours) for the following 5 days, increasing the time gradually up to 100% renewal at 15 days. This percentage is maintained like this until day 30, from which continuous renewal (24 hours per day) is carried out which represents a renewal of around 200% per day and which is maintained until the tank is closed at 40 days of life (2.sup.nd difference). The outlet of the water was carried out by way of a central tube with a net of 300 microns and the level of the water is maintained owing to an exterior tube.

[0062] In terms of the light, a 35 W cold white fluorescent light was used over the edge of the tank (instead of a central position), causing a change in the light incidence angle over the surface and producing heterogeneous light conditions inside the water column (3.sup.rd difference). The intensity values used during the test remained within three intensity levels. The levels used go from 600 to 800 lux (1077 to 1436 W/m.sup.2) for the first 15 days of life and between 300 and 600 lux (517 to 1077 W/m.sup.2) for the period between 15 and 40 days of life (4th difference). There was a photoperiod of 14:10 (light:darkness) between las 8:00 and 22:00 from the first day of life to the end of the test.

[0063] The food during the entire test (40 days) was based on Artemia sp., fattened for 7 days with Isochrysis aft galbana and supplied to the paralarvae with a density of 0.5 individuals/ml, distributing the doses such that there was always prey available in the tank which gave a range of between 1 to 4 doses/day. It was decided to close the tank at 40 days due to the growth rate (GI) not increasing more than 4% per day, being maintained between 1 and 1.5 mg dry weight (see FIG. 4A). The survival at this age was from 65%.

Example 3: Common Octopus Larval Culture Test (1000 l Tank) Using Artemia Sp. and Crab Zoeas from Day 1 to Day 30 and with the Amphipods of the Present Invention from Day 31

[0064] This test was carried out simultaneously with the previous test and using the same culture conditions with the aim of comparing the effect of the different prey. In this case, Artemia sp. was complemented with crab zoeas (0.1 individuals/ml) up to 30 days old (however, given the difficulty in obtaining the zoeas, these could only be added to the tank in 10 of the 30 first days, which reduced the expected growth). From then, they started to be supplied exclusively with amphipods. On the one hand, the gammarideas of the type Jassa spp. (preferably J. falcata and J. marmorata) which represented 95% of the total number of individuals and the caprelids of the types Phtisica spp. and/or Caprella spp. (preferably Phtisica marina and Caprella equilibra) which represented 5% of the total number of individuals. The gammarideas reached between 2 and 8 mm in length and the caprelids between 4 and 30 mm in length. In the case of the amphipods, the feeding protocol consisted in supplying 3 to 5 prey per paralarva/day, the doses being distributed such that there was always prey available in the tank which gave a range of between 1 to 4 doses/day (5th difference with respect to the prior art).

[0065] From 55 days old, paralarvae were observed which exceeded 6 mg dry weight and which started to migrate to the bottom. These individuals were transferred to a new tank called settlement tank (FIG. 5B) with 400 l volume, square, 50 cm in height, flat bottom, gray and covered with a shadowing net over of its surface. This tank was filled with water from the original tank and included refuges (1 to 2 cm PVC tubes) at the bottom of the tank or hanging cables vertically as well as small stones to enrich the habitat. The density of individuals was 0.1 paralarvae/l. At this point, it was seen that the paralarvae became vulnerable (at least temporarily) and can be attacked by the larger prey, therefore they were only fed with gammarideas of maximum 4 mm in length (6th difference).

[0066] In this settlement tank, a renewal percentage similar to that of the initial tank was maintained from 30 days of age of the paralarvae, i.e. continuous renewal (24 h) which renews approximately 200% of the total volume of the tank each day. The outlet of the water was carried out through a central tube with a net of 300 microns. With respect to the lighting, this was reduced in the initial tank to a range between 40 and 300 lux (69 to 517 W/m.sup.2) at the time where the first paralarvae were observed in pre-settlement (at 55 days and above 6 mg dry weight). Once transferred to the settlement tank, the intensity was maintained around 40 lux (69 W/m.sup.2) with the same photoperiod of the initial tank (14:10) (7.sup.th difference).

[0067] In none of the tests described is the bottom of the tank siphoned of paralarvae. The objective is to reduce the stress on the paralarvae and also to allow the amphipods to colonizer the bottom of the tank since they are detritivores and help to keep the bottom clear of organic remains, preventing the proliferation of possible pathogens (8th difference).

[0068] The amphipods (gammarideas and caprelids) were obtained from rafts and purifiers used in mussel culture of Galician rivers. They were kept in 1000 l tanks similar to those of FIG. 5B (settlement tanks), but without covering and with 10 renewals of their volume per day. The food was based on fish feed and leftovers of mollusks ad libitum. In order to feed the paralarvae, the amphipods were captured by means of two systems. The larger amphipods were obtained by means of siphoning the walls and bottom of the tank and were concentrated in a net. The second collection system consisted in a concentrator using the outlet tube of the water from the tank itself where the prey are stored. A net of 500 microns was placed on the walls of this tube which allowed prey to enter and a net of 200 microns was placed at the bottom of the tube (outlet) which kept them inside the tube. Once the prey are in the interior part of the tube, this tube is removed from the tank and the prey are concentrated into a recipient before being added to the paralarvae tank by means of 1 to 2 l plastic jugs of (9.sup.th difference).

[0069] The data of this test showed a survival greater than 90% at 40 days of life and around 65% at 60 days of life. The growth rate showed an increase in weight of between 5 and 6% per day, reaching 5 mg dry weight at 50 days old (FIG. 4A) and subsequently obtaining paralarvae with dry weights greater than 9 mg between 65 and 75 days old.

Example 4: Control Test: Common Octopus Larval Culture (100 l Tank), Using Artemia Sp., Exclusively as Prey from Day 1 to Day 25 of Life of the Paralarvae

[0070] This test was carried out under the same culture test conditions described in the two previous examples, with the difference being that the volume of the tanks was 100 l instead of 1000 l and three replicas were used instead of just one. The test lasted 25 days. Like in example 2, a control treatment was used, only with Artemia sp., fattened for 7 days with Isochrysis aff. galbana. The growth results of the paralarvae were similar to those of example 2 as can be observed in FIG. 4B with the dry weight of around 1 mg at 25 days of life.

Example 5: Common Octopus Larval Culture Test (100 l Tank), Using Artemia sp. from Day 1 to Day 8 and with the Amphipods of the Present Invention Between the Days 9 and 25 of Life

[0071] This test was carried out in parallel and under the same culture conditions described in example 4 with the aim of comparing Artemia sp. with an experimental treatment using the same amphipods of the type Jassa spp. of example 3. The difference is that on this occasion, these amphipods started to be supplied from day 8 of life since this was when it was observed that the majority of larvae ingested this type of prey (the person skilled in the art can easily see this, observing the behavior of the paralarvae or carrying out the protocol explained in the following example in order to know when to start supply the prey). In this case, feeding during the first 8 days consisted only of Artemia sp. (the same type as example 4) and between 9 and 10 days they were fed with a mixture of Artemia sp. (75% of the total of individuals) and amphipods (25%) of the type Jassa spp. of between 1 and 3 mm in length. From 10 days, the larvae started to be fed exclusively with these amphipods under the same conditions as in example 3 (3 to 5 prey/paralarvae/day), until the cultivation was completed. The growth results of the paralarvae in this example were similar to those of example 3 as can be observed in FIG. 4B with a growth rate of 6.7% and survival of 70% at 25 days.

Example 6: Amphipod Capture and Ingestion Test at Different Ages

[0072] In parallel to the rest of the experiments, a new experiment was carried out with the aim of determining with more accuracy the age at which the paralarvae are capable of capturing and ingesting the amphipods of the type Jassa spp. and if there could be differences between lays. With this aim, paralarvae obtained from two different females, which exhibited different initial weights at the time of hatching, were compared. The paralarvae from the first female weighed on average 0.24 mg dry weight, while the paralarvae from the second female had an average of 0.29 mg initial dry weight (both within the normal margins of weight of this species). Both groups of paralarvae were placed in separate 100 l tanks under the same culture conditions as in the previous examples 2 to 5 and at a density of 4 paralarvae per liter. In both cases, amphipods of the type Jassa spp. (20 to 30%) were supplied together with Artemia sp. (80 to 70%) from the first day of life at a density of 3 to 5 prey/paralarvae.

[0073] In both cases, it was observed that all the amphipods were captured. The paralarvae that had captured prey and kept it for 2 to 3 minutes were removed from the tank (including the prey) and they were observed under the microscope (40 times enlargement) to confirm that they had food in their digestive system. The technical details for this observation are found in Nande, M. et al (2017). Prey Capture, Ingestion, and Digestion Dynamics of Octopus vulgaris Paralarvae Fed Live Zooplankton. Frontiers in Physiology, 8, 573. In this example, it was observed that the recently hatched paralarvae of 0.24 mg dry weight attacked the prey from the first day of life, but the ingestion percentage was only 30%, consequently it could be considered too soon to supply said diet. In addition, the paralarvae with 0.29 mg dry weight exhibited an ingestion range of 80% from the first day of life which is considered suitable for starting to supply the amphipods of the type Jassa spp.

[0074] This test reveals that starting to feed using these amphipods can vary from one lay to the next and it is recommendable, although not necessary, to carry out a capture and ingestion test in parallel to optimize the start of feeding with this prey. However, these prey (amphipods) could start to be administered from the first day.

[0075] Comparison of Results Between Control Experiments and Those of the Present Invention.

[0076] Upon comparing the results of the experiments based on the present invention with respect to those known in the prior art, the improvements are clearly significant for all the examples presented (2, 3, 4 and 5) and including tests both in 100 and 1000 l with the method of the present invention. The survival of the paralarvae in the method of the prior art at 30 days old (Garrido et al., 2017) is between 0.14 to 3.77% (the test had a total of 18 tanks), the experiments being completed at this age. In contrast, the conditions of the test in 1000 l tanks of the present invention with amphipods obtained a survival of more than 90% at 40 days and 65% at 60 days old. The culture conditions in 100 l tanks with amphipods in turn obtained a survival of more than 70% at 25 days old. It must also be pointed out that in the 1000 l test, the treatment with Artemia sp. in spite of having a suboptimal diet, exhibited a survival at 40 days of more than 65% which indicates that it is not only the use of amphipods that marks the difference, but that the culture protocol has also improved the survival. With respect to the weight, in the method of the prior art (Garrido et al., 2017), a growth rate with an increase of between 3.9 to 6.4% dry weight per day is obtained, but there are only values up to 15 days old, while the data of the method of the present invention for the tanks with amphipods, a similar increase to the maximums of the prior art (between 5 and 6% dry weight per day) is obtained for the 1000 l tanks and an increase of 6.5 and 7.5% dry weight per day for the 100 l tanks. In the case of the 1000 l tanks, settled larvae with weights greater than 9 mg dry weight between 65 and 75 days old were obtained. FIGS. 4A and 4B show the weight increase in the examples 2, 3, 4 and 5 with the method of the present invention. In this case, the differences between treatments with amphipods and with Artemia sp. can also be observed.