Aquaculture 591 (2024) 741147

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Effect of size and shape of artificial pellets on capture success for Octopus
tetricus paralarvae
Stefan Spreitzenbarth a, *, Andrew Jeffs a, b
a
Leigh Marine Laboratory, Institute of Marine Science, University of Auckland, 160 Goat Island Road, 0985 Leigh, New Zealand
b
School of Biological Sciences, University of Auckland, 23 Symonds Street Auckland Central, 1010 Auckland, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: The culture of the paralarvae of merobenthic octopus species generally results in high mortalities with many
Octopus tetricus attempts failing to rear the paralarvae through to juvenile stage. Progress has been made with higher survival for
Feeding behaviour paralarvae cultures, but all successful paralarvae cultures rely heavily on decapod zoeae, which are labour
Paralarvae
intensive and costly to maintain in many instances. The development of an artificial pellet that is easily captured,
Artificial pellet
Size
can be rapidly consumed and fulfils the nutritional needs of octopus paralarvae would greatly advance octopus
Shape paralarvae culture. The aim of this study was to evaluate the effect of shape and size of artificial pellets on
feeding behaviour for early Octopus tetricus paralarvae. This study tested the capture success of six different
artificial pellets in a crossed experiment for size (i.e., small, medium and large), and shape (i.e., spherical or
rectangular). Paralarvae were observed individually for up to 5 min with the number, size, shape and handling
time of pellets captured by 2 days post-hatch O. tetricus paralarvae being recorded. A total of 105 out of 250
paralarvae captured one of the six artificial pellets with no difference between the two provided shapes for small
and large pellets. In contrast, there was a strong preference for smaller artificial pellets with 57 out of 105
paralarvae capturing the small pellets. The handling times ranged from 1 to 485 s across all sizes and shapes of
artificial pellets with shorter mean handling times for rectangular artificial pellets (mean of 13 ± 41 s) compared
to spherical artificial pellets (mean of 31 ± 97 s), regardless of size. Overall, handling times were rather short (<
20 s) for the majority of (91%) of paralarvae that captured artificial pellets. The results show that pellet shape is
mostly irrelevant to early O. tetricus paralarvae, but size is an important factor that must be considered. More­
over, the short handling times suggest a low palatability of the artificial pellets used in the current study, which
will be addressed in future studies.

1. Introduction settling to the seafloor (merobenthic) (Hanlon and Forsythe, 1985;

Global demand for octopus for human consumption has increased
over the past 30 years largely due to rising popularity of Mediterranean
and Asian octopus cuisine (Sauer et al., 2019). The fast growth, high
fecundity, and short lifespan combined with the high market demand
makes octopuses excellent candidates to diversify aquaculture in many
countries. Octopuses are also of high interest to research due to their
unique nervous system, advanced behaviour for invertebrates, including
a sophisticated ability to change colour despite being colourblind
(Hanlon et al., 2011; Young, 1971).
Octopuses have two different life-history strategies which determine
whether hatchlings are benthic from the moment they hatch (hol­
obenthic) or progress through a planktonic paralarval stage before
Uriarte et al., 2011; Young and Harman, 1988). Holobenthic octopus
species have the advantage of high juvenile survival in captivity but
generally produce relatively small numbers of eggs (i.e., a few hundred
in a clutch) while merobenthic octopus species produce hundreds of
thousands of eggs but generally have high paralarvae mortalities when
cultured (Dan et al., 2019; De Wolf et al., 2011; Iglesias et al., 2004,
2007; Joll, 1976; Uriarte et al., 2010; van Heukelem, 1983; Villanueva
and Norman, 2008). Most of the highly sought after octopus species for
human consumption are part of the Octopus vulgaris species complex,
which are all merobenthic species (Sauer et al., 2019).
The development of reliable methods for the culture of merobenthic
paralarvae has been slow because relatively little is known about their
environmental and feeding requirements. Following hatching,

* Corresponding author.
E-mail addresses: stefan.spreitzenbarth@auckland.ac.nz (S. Spreitzenbarth), a.jeffs@auckland.ac.nz (A. Jeffs).

https://doi.org/10.1016/j.aquaculture.2024.741147
Received 25 February 2024; Received in revised form 29 April 2024; Accepted 27 May 2024
Available online 28 May 2024
0044-8486/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S. Spreitzenbarth and A. Jeffs
merobenthic paralarvae typically have limited yolk reserves and are
immediately highly predatory of live prey. More recently, researchers
have made significant advances in octopus paralarvae culture including
achieving paralarvae survival of >80% in Octopus sinensis in Japan (Dan,
pers. com.) and more recently the first large scale O. vulgaris aquaculture
facility has been established in Spain (Kassam, 2023). While the rearing
of paralarvae through to the settlement stage and beyond is being ach­
ieved more frequently, all successful paralarvae culture that has been
reported has principally relied on live prey. Crab zoeae (Portunus tritu­
berculatus) are used as sole prey or partially replacement for Artemia in
Japan (Dan et al., 2019, 2023) and caprellid amphipods (Phtisica marina
and Caprella equilibra) and/or gammarid amphipods, Jassa spp. are used
in Spain (Tur et al., 2020). For research and small-scale culture of
paralarvae, live prey provides a good opportunity to reliably complete
the paralarvae phase of the lifecycle but the production or wild harvest
of zoeae and other live prey are labour intensive and costly, precluding
their use for commercial scale aquaculture (Iglesias and Fuentes, 2014).
A potential solution to this problem could be artificial pellets that are
tailored to meet the needs of octopus paralarvae. Initial experimental
use of artificial pellets and microcapsules with O. vulgaris paralarvae
found that paralarvae rapidly attacked and consumed the artificial
pellets but they provided limited improvement in nutrition and survival
of paralarvae (Navarro and Villanueva, 2003; Villanueva et al., 2002).
Despite the apparent potential for artificial pellets to provide a viable
alternative to provisioning live feed for the culture of paralarvae, min­
imal research in this area has been reported. Artificial pellets offered to
O. tetricus paralarvae showed that paralarvae rapidly attacked and
captured the artificial pellets at a similar rate to live Artemia (Spreit­
zenbarth et al., 2021). Moreover, O. tetricus paralarvae were also
observed to occasionally capture unhatched Artemia cysts that had been
inadvertently included with live Artemia with cultured paralarvae.
These reports indicate that paralarvae of at least two octopus species
attack, capture and partially consume inert particles, including artificial
pellets. However, much work is needed to develop an artificial pellet
that is highly attractive as a prey item, that once captured by paralarvae
can be readily consumed and meets the nutritional requirements of
rapidly growing paralarvae. Determining the characteristics of artificial
pellets that make them highly attractive as a prey item for paralarvae is a
critical first step in developing an effective artificial pellet. Size and
shape of artificial pellets is a fundamental characteristic of prey items in
a visual predator, such as paralarvae, as they typically play a vital role in
triggering predation behaviour which then leads to food capture and
consumption.
Therefore, the aim of this study was to determine whether the suc­
cess of capturing artificial pellets by O. tetricus paralarvae is affected by
the fundamental visual characteristics of size or shape.
2. Material and methods
2.1. Paralarvae source
Adult female O. tetricus were caught from the wild in shallow New
Zealand coastal waters and kept in the laboratory as described by
Spreitzenbarth and Jeffs (2020) for up to three months or until they laid
eggs. Females that started spawning were left undisturbed for three days
after the first egg strings were detected to minimise potential stress for
the female. After three days a total of 20 egg strings were removed and
placed in artificial egg rearing units that create a gentle upwelling and
allow the egg strings to freely tumble against each other. Seawater
temperatures in the adult tank and egg rearing tank were measured daily
before midday, with mean temperatures of 16.0 ± 0.2 ◦ C in the adult
tank and 18.0 ± 1.4 ◦ C in the egg rearing tank until hatch. The egg
rearing unit was fitted with a surface water outflow that transferred
newly hatched paralarvae from the rearing unit into a reservoir which
retained larvae whilst allowing excess water to be drained through a
250 μm mesh. All paralarvae were removed daily from the reservoir and
2
Aquaculture 591 (2024) 741147
if sufficient paralarvae had hatched overnight, they were transferred
into a 2.5 l kreisel tank. Paralarvae within the kreisel tanks were fed
twice per day with live Artemia sp. nauplii instar II (Great Salt Lake
Bohai Bay, Binzhou Marine Science and Technology Co. Ltd.), which
were feed a mixture of 100 ml Diacronema lutheri (1.30 × 107 cells ml− 1)
and 100 ml Tisochrysis lutea (1.09 × 107 cells ml− 1) live algae. The
Artemia nauplii were supplied to the paralarvae cultures at 0.15–0.20
Artemia nauplii ml− 1 from day 0–1 with the last feed being received the
evening before the experiment.
2.2. Manufacturing formulated diets
Six different artificial pellets were manufactured for this experiment
consisting of three different sizes, i.e., small, medium, and large, each of
two different shapes, i.e., spherical and rectangular (Table 1). The small
rectangular shaped artificial pellets were similar in length and double in
width to Artemia adults (~ 1.4 × ~ 0.5 mm), medium rectangular
artificial pellets were similar in length and width to Maja brachydactyla
zoeae, Cancer pagurus zoeae and Paguridae zoeae (~ 2.1 × ~ 0.7 mm),
and large rectangular artificial pellets were similar in length and width
to Crangonidae zoeae (~ 4.9 × ~ 0.9 mm). All these zooplankton species
have been used in feeding and prey studies with O. vulgaris and resulted
in frequent attacks and captures by O. vulgaris paralarvae (Iglesias et al.,
2006; Nande et al., 2017). The heights of all three rectangular shapes
were equal to their width. Spherical shapes were chosen due to the
previously observed capture of small spherical pellet with paralarvae
from O. tetricus (Spreitzenbarth et al., 2021) and O. vulgaris (Navarro and
Villanueva, 2003; Villanueva et al., 2002). The small and medium sizes
of spherical artificial pellets was based on the size of “microdiets”
(1.2–1.9 mm Ø) used by Villanueva et al. (2002) and the incremental
increase from medium to large spherical artificial pellets was set to be an
equal increase as from small to medium, i.e., increase in diameter by
roughly 0.7 mm. A total of 90 samples (n = 15 artificial pellets− 1) were
randomly selected to measure the volume for each of the six artificial
pellets (Table 1). Digital images of all samples were taken with a camera
attached to a microscope (Nikon SMZ1270, 6 MP USB3 camera) and
dimensions, i.e., diameter for spherical artificial pellets and length,
height and width of rectangular artificial pellets, were measured with
image processing software (ImageJ). The measured dimensions of each
artificial pellet where then used to calculate their respective volumes.
All artificial pellets had the same ingredients to avoid any possible
bias due to different composition (Table 2). The ingredients were not
based on meeting nutritional requirements of the paralarvae, but rather
the ability to use the mix of ingredients to form the desired range of
experimental shapes and sizes of artificial pellets and for ensuring their
neutral buoyancy once placed in seawater in the experimental kreisel
tanks. Neutral buoyancy was needed to ensure artificial pellets remained
suspended in the upper region of the water column of the experimental
tank rather than the artificial pellets sinking quickly to the bottom. This
guaranteed proximity between paralarvae and artificial pellets as
Table 1
Physical characteristics of the six artificial pellets used in this study. The di­
mensions [mm] were the theoretical diameter for a spherical pellet and the
length, height and width for the rectangular pellets. Due to each pellet having
slight differences in their dimensions the mean volume [μl] for each pellet size
was calculated (n = 15 samples pellet size− 1). Different lower-case letters
indicate significant differences in volumes between shapes of the same sized
artificial pellets.
Dimensions [mm] Volume [μl] ± SD
Particle Spherical Rectangular Spherical Rectangular
size pellet pellet pellet pellet
Small 1.3 1.6 × 0.9 × 0.9 0.8 ± 0.2a 1.0 ± 0.3a
Medium 1.9 3.0 × 1.1 × 1.1 3.8 ± 0.7a 2.2 ± 0.8a
Large 2.6 4.5 × 1.1 × 1.1 8.6 ± 1.1a 4.9 ± 1.1b
S. Spreitzenbarth and A. Jeffs
Table 2
Ingredients for the fabrication of the six artificial pellets used for this study.
Ingredient Fresh-water Sugar1
Proportion [%, by weight] 67.9 9.4
1 2
: Pams Products Ltd., 703 Roberts Line, New Zealand; : Pure Nature, 626 A Rosebank Road, Avondale, New Zealand; : Rogers Distribution Ltd., 46 Honan Place,
New Zealand; 4: GELITA Deutschland GmbH, Uferstraβe 7, Germany: †: based on dehydrated gelatine weight.
O. tetricus paralarvae were previously described to retain in the upper
water layer (Spreitzenbarth and Jeffs, 2022a). A two-step process was
used to manufacture artificial pellets. In the first step a mixture of
freshwater (75% by weight) and sucrose (25% by weight) (Pams Prod­
ucts Ltd., New Zealand) was brought to boil until the sugar fully dis­
solved to form a syrup. The syrup was allowed to cool to room
temperature and then whole milk powder (Pams Products Ltd., New
Zealand) was added (Table 2). Then, sunflower oil (Pams Products Ltd.,
New Zealand) was added to the syrup before guar powder (Pure Nature,
New Zealand) was slowly added to create an emulsified oil solution. This
solution was heated to 60 ◦ C for at least a minute before agar powder
was added. Once thoroughly mixed, the gelatine (GELITA Deutschland
GmbH, Germany) was added and allowed to melt. The mixture was kept
warm (40–50 ◦ C) to avoid gelatine setting. The still warm solution was
slowly dropped from a hypodermic syringe (0.55 × 25 mm, Neolus
needle, Terumo) into a cold sunflower oil bath to create the spherical
artificial pellets. The spherical artificial pellets were separated from the
oil by sieving and excess oil was removed from the surface of the spheres
by carefully rinsing in cold running freshwater. Specifically designed
silicone moulds were used to create the rectangular artificial pellets. For
this the still warm solution was transferred into the silicone moulds
before being placed in a − 20 ◦ C freezer overnight to set. The consoli­
dated artificial pellets were then pressed from the silicone moulds. Once
prepared, all artificial pellets were stored in a refrigerator (4 ◦ C) for up
to three days before being used in feeding experiments with paralarvae.
2.3. Experimental setup and procedure
Experiments were conducted in 2.5 l kreisel tanks, which had a clear
front panel with the remaining components being black in colour. Black
was chosen as the main background colour for the tanks due to previous
observations with O. tetricus paralarvae, where a higher proportion of
paralarvae attacked Artemia when observed in black tanks (Spreitzen­
barth et al., 2021). Kreisel tanks were continuously supplied with
temperature-controlled seawater (19 ◦ C) and a light source (10 W
Colour LED, Orbit Lighting, New Zealand) was placed above the kreisel
tank resulting in a light intensity of ~600 lx (R8140 light meter, REED,
USA) at the water surface.
A total of 250 paralarvae aged 2 dph (days post hatch) were observed
individually by transferring individual paralarvae from the holding
tanks to an experimental kreisel tank with a pipette. Paralarvae mor­
phometrics were not recorded in the current study but a mean total
length of 2.4 ± 0.2 mm and a mean wet weight of 0.9 ± 0.1 mg have
been reported for O. tetricus paralarvae aged 2 dph (Spreitzenbarth and
Jeffs, 2021, 2022b). Equal numbers of all six artificial pellets were then
supplied to each paralarvae. The total density of pellets was 0.24 pellets
ml− 1 (600 artificial pellets in total within a kreisel), with each of the six
artificial pellets contributing 0.04 prey items ml− 1 (100 artificial pel­
lets). Due to the neutral buoyancy of the artificial pellets, they were
quickly dispersed evenly throughout the kreisel tank. Each paralarvae
was observed until it captured one of the six artificial pellets or for a
period of 5 mins after being introduced to the tank if no capture of an
artificial pellet occurred. Thus, the maximum number of captured pellets
per paralarvae was one. A capture of an artificial pellet consisted of the
paralarvae performing a forward movement with increased speed (jet
impulsion) toward an artificial pellet which resulted in the artificial
pellet being within the paralarvae’s arms (Spreitzenbarth et al., 2021). A
record was kept of key characteristics of the feeding behaviour of each
Aquaculture 591 (2024) 741147
Milk powder1 Oil1 Guar powder2 Agar3 Gelatine4
5.1 11.5 1.5 0.4 4.2†
3
paralarva, i.e., no capture or capture and the type of artificial pellet
captured, the time to capture and time subsequently spent handling the
artificial pellet. Handling time commenced when an artificial pellet was
captured, and ended when the pellet was ejected from the paralarvae’s
arms or was fully consumed and was followed for as long as it took for
handling of the pellet to be come to an end, i.e., not confined to 5 min.
2.4. Statistical analyses
The data for the measured volume of the samples of six different
artificial pellets was tested for normal distribution and homogeneity of
variances with Shapiro-Wilk and Levene’s test, respectively. Due to the
data deviating from assumptions for parametric statistical methods,
Kruskal-Wallis tests were used to compare the volume of artificial pellets
among the three sizes, the two shapes and their interaction, Dunn’s post
hoc test was used for pairwise comparisons of means when overall sig­
nificant differences were detected (P < 0.05) (Ogle et al., 2023).
Chi-square tests were performed on the raw observation data with
either size of artificial pellets (small, medium or large), shape of pellets
(spherical or rectangular) or the combination of size and type of the
artificial pellet as variables, i.e., small rectangular versus small spherical
pellets, medium rectangular versus medium spherical pellets and large
rectangular versus large spherical pellets. A pairwise comparison was
conducted using chisq.multcomp from the R package RVAideMemoire
with a Bonferroni p-value adjustment (Herve, 2023). All statistical an­
alyses were performed using R (R Core Team, 2023) in conjunction with
RStudio (RStudio Team, 2020). All means are reported with standard
deviation.
2.5. Animal ethics
Experiments were conducted at the University of Auckland, New
Zealand under animal ethics permit No. AEC25053. All research was
carried out in strict accordance with the New Zealand Animal Welfare
Act 1999.
3. Results
The volumes of the artificial pellets were similar between the
spherical and rectangular artificial pellets for the small (P = 0.484) and
medium-sized pellets (P = 0.097) (Table 1). In contrast, the volume of
the large spherical pellets was greater than for the large rectangular
pellets, i.e., means of 8.6 ± 1.1 μl versus 4.9 ± 1.1 μl, respectively (P =
0.004) (H(5) = 88.62, P < 0.001).
Out of 250 paralarvae that were observed, a total of 105 (42%)
successfully captured an artificial pellet within the 5 min observation
period. Time to successful capture of pellets ranged from 5 to 280 s (i.e.,
4.40 min) with no differences in the time to capture (73 ± 67 s) among
pellet shapes (χ2(1) = 1.13, P = 0.288), sizes (χ2(2) = 3.19, P = 0.203) or
their interaction (χ2(5) = 5.93, P = 0.313).
The number of captures was higher for medium sized rectangular
artificial pellets compared to medium spherical artificial pellets with 23
compared to 11 captures (χ2(1) = 4.24, P = 0.040). In contrast, no dif­
ferences in captures were observed between spherical and rectangular
artificial pellets for small and large artificial pellets with 30 compared to
27 captures for small artificial pellets (χ2(1) = 0.16, P = 0.691) and 6
compared to 8 captures for large artificial pellets (χ2(1) = 0.29, P = 0.593)
(Fig. 1). Overall, the number of successful captures was affected by the
3
S. Spreitzenbarth and A. Jeffs Aquaculture 591 (2024) 741147

Fig. 1. Total number of captures by 2 dph old O. tetricus paralarvae for each of the six artificial pellets consisting of three sizes, each in two shapes (n = 105). Grey
bars represent rectangular artificial pellets and white bars represent spherical artificial pellets. Different lower-case letters indicate significant differences between
different shapes within the same size of artificial pellets (P < 0.05). Different upper-case letters indicate significant differences among different sized pellets
regardless of their shape (P < 0.05).

size of the artificial pellet (χ2(2) = 26.46, P < 0.001) with the highest
number of captures (i.e., 57 out of 105) being observed with the smallest
pellets compared to 34 successful captures with the medium pellets (P =
0.048) and 14 with the large pellets (P < 0.001), regardless of shape
(Fig. 1). Likewise, the number of captures was higher with medium
pellets compared to large pellets (P = 0.012), regardless of shape. In
contrast, the shape of the artificial pellets did not influence the number
of captures by paralarvae with 58 paralarvae capturing rectangular
pellets compared to 47 capturing spherical pellets (χ2(1) = 1.15, P =
0.283), regardless of size.
Handling times ranged from 1 to 485 s (8.05 min) across all sizes and
shapes of artificial pellets with nine paralarvae having a handling time
of >60 s, with the remainder having handling times of <19 s (Fig. 2).
The handling time was less for rectangular pellets (mean of 13 ± 41 s)
compared to spherical pellets (mean of 31 ± 97 s) (χ2(1) = 6.64, P =
0.010). Handling times did not vary among sizes of pellets (χ2(2) = 1.02,
P = 0.601) or the interaction of size and shape (χ2(5) = 7.54, P = 0.184).
4. Discussion
Paralarvae of merobenthic octopus species primarily use visual cues
to identify and capture suspended food particles (Villanueva and Nor­
man, 2008). The nature of these visual cues has been subject to minimal
investigation. However, it appears that these cues are reasonably
permissive with a great diversity of live prey of different shapes and sizes
being successfully fed to cultured paralarvae of various species. These
include most commonly Artemia nauplii and adults, various zoeal stages
of a range of decapod species, as well as amphipods, including skeleton
shrimps (Caprellidae) and laterally compressed amphipods (Gammar­
idae) both of which collectively have great diversity in size and
morphology (Carrasco et al., 2003; Dan et al., 2019; Iglesias et al., 2004;
Moxica et al., 2002; Nande et al., 2017; Tur et al., 2020). Physical
movement could be an important visual cue to initiate paralarval food
capture behaviour, as it is for carnivorous fish larvae (Kamler et al.,
1990; Rao, 2003; Wright and O’Brien, 1982). However, reports of
paralarvae capturing inert particles, such as rudimentary artificial pel­
lets or Artemia cysts, microdiets or dead prey suggests the movement of

Fig. 2. Frequency of handling times by 2 dph old O. tetricus paralarvae for the six artificial pellets independent of the three sizes and two shapes (n = 105).

4
S. Spreitzenbarth and A. Jeffs
food particles is not a critical visual cue for triggering capture behaviour
(Boletzky and Hanlon, 1983; Kurihara et al., 2006; Marliave, 1981;
Navarro and Villanueva, 2003; Okumura et al., 2005; Spreitzenbarth
et al., 2021; Villanueva et al., 2002). Consequently, defining the size and
shape of artificial pellets for paralarvae that optimises capture and
subsequent food intake is an important first step toward developing an
effective artificial pellet for more effective mass culture of paralarvae.
The optimal size of artificial pellet is particularly important, as it greatly
influences the amount of nutrient delivered per captured pellet,
increasing exponentially in relation to its average diameter. Unlike most
fish larvae which are limited in prey size by their mouth opening,
octopus paralarvae have the potential to capture prey close to their own
size due to capturing food particles with their arms and suckers and once
captured, consuming the food externally (Carrasco et al., 2006; Hama­
saki et al., 1991; Nande et al., 2017; Villanueva et al., 1995; Villanueva
and Norman, 2008).
All six forms of the artificial pellets were readily captured by the
O. tetricus paralarvae. The proportion of paralarvae capturing pellets in
the current study is similar to a previous study feeding Artemia nauplii
and rudimentary artificial pellets of 0.2–0.4 mm Ø where a mean pro­
portion of 30 ± 10 and 38 ± 15% of paralarvae had at least one suc­
cessful capture within 15 min, respectively (Spreitzenbarth et al., 2021).
Similar proportions of captures (~20–30%) were also observed when
O. tetricus paralarvae aged 2 dph were fed with adult Artemia (~ 1.4 mm
in length) (Good, unpublished data). This further indicates the permis­
sive visual cues used by paralarvae for initiating food capture behaviour.
Likewise, the absence of differences in the frequency of captures be­
tween small spherical and rectangular artificial pellets suggests that less
exacting artificial pellet manufacturing would be suitable for preparing
diets for paralarvae, with standardised aquafeed processes, such as a cut
extruded noodle, being potentially suitable for processing artificial
pellets. This study was limited to two different shapes so as to not
compromise the observer’s ability to distinguish among the different
sizes and shapes when supplying all six types of pellets to the paralarvae
simultaneously. Due to this, further shapes, such as cylindrical shapes
which are commonly used to create artificial pellets for cultures of the
early stages of some marine organisms, could not also be tested but
could be considered in further studies.
The size of artificial pellets proved to be important to initiating the
capture behaviour of paralarvae in this current study, with the smallest
pellets being captured 1.5 times more frequently than medium size
pellets, and 2.5 times more than large pellets. These pellet sizes repre­
sent significant quantities of feed delivered to paralarvae – 0.8, 3.8, and
8.6 μl for small, medium and large pellets, respectively. For comparison,
the mean inner yolk sac volume of newly-hatched O. tetricus paralarvae
was 0.04 ± 0.02 μl and 0.16 ± 0.06 μl (Spreitzenbarth and Jeffs, 2020;
Spreitzenbarth and Jeffs, 2021), which is 8 times, 38 times and 86 times
smaller than the volume of small, medium and large artificial pellets
provided in the current study. Consequently, despite the 1.5 times
reduction in the capture of medium sized artificial pellets, the significant
volumetric difference in terms of potential food intake between small
and medium pellets (4.5 times more) would justify providing both sizes
of pellets at the same time, i.e., a ratio of 3:1 of small to medium pellets
for early O. tetricus hatchlings. Although it has not been tested in this
study, it is likely that paralarvae at later developmental stages will in­
crease the size of prey that they will target, which would enable the
same artificial pellet to be used effectively for culturing paralarvae of a
range of developmental stages. It is also possible that preferences for
shape, size or other characteristics, such as colour, may be become less
permissive as the paralarvae develop, possibly as a result of improving
visual capabilities. Thus, similar experiments should be conducted with
different development stages of paralarvae to identify their optimal
shapes and sizes of artificial pellets.
The generally short handling times (i.e., mean of 21 ± 72 s regardless
of type and shape), of artificial pellets in the current study suggests that
once potential food particles are captured by paralarvae they are rapidly
5
Aquaculture 591 (2024) 741147
assessed for their palatability, with the majority (91%) being rejected
within <20 s after being assessed as unsuitable for ingestion. Most of
these rejections (52%) occurred within the first 1–2 s after capture,
which was visible due to the pellet being thrown away from the arms
within 1–2 s. This motion is best described as the pellet being forcefully
thrown away by the arms resulting in the pellet being several paralarvae
lengths away from the paralarvae. The remaining 48% of paralarvae
manipulated the captured pellets closer to the beak area with their arms
and held them there for a short period (< 2 s and > 20 s) before rejection
occurred. Chemoreceptors on the head region, behind the eyes, mantle
edge and the rim of suckers have been described for O. vulgaris paral­
arvae (Lenz, 1997; Schmidtberg, 1997; Schmidtberg, 1999; Villanueva
and Norman, 2008; Wildenburg, 1997). However, the function of these
chemoreceptors has not been described in great detail, but it could be
assumed that chemoreception plays an important role in initially
assessing suitable prey, especially the chemoreceptors on suckers. This
suggests that an initial assessment of palatability of captured items oc­
curs at, or very shortly after, the first contact with captured particles, i.
e., at the arms (suckers), with a secondary assessment of palatability
near the beak area. Unfortunately, there is no knowledge on chemical
cues that increase capture and ingestion of prey and artificial pellets for
octopus paralarvae, and thus further research is needed to better un­
derstand the impact of this stimulus.
An alternative possibility for the general short handling times of
artificial pellets could also be that the surface texture of the artificial
pellet was evaluated by paralarvae as being unsuitable. This is based on
previous observations made with O. tetricus paralarvae that were sup­
plied with dry artificial pellets (i.e., commercial fish larval diets) with
hard exteriors and limited chemical release, which were observed to
have long handling times and subsequently the caecum of the paralarvae
changed colour suggesting full or partial consumption of the captured
particle (Spreitzenbarth et al., 2021). Similarly, O. vulgaris paralarvae
were described to frequently capture and consume moist microdiets,
which were small ovoid pellets ranging from 1.2 to 1.9 mm in diameter
(Navarro and Villanueva, 2003; Villanueva et al., 2002). Future studies
need to evaluate the effect of moist versus dehydrated artificial pellets
and identify whether chemical cues can be used to increase the retention
and ingestion of captured particles.
For the purposes of this study, the composition of the artificial pellets
was based on versatility of fabrication to different shapes and sizes of
pellets rather than meeting nutritional needs. Consequently, carbohy­
drates were the main ingredient (i.e., sucrose) whereas proteins are the
primary source of energy for sub-adult and adult cephalopods (García-
Garrido et al., 2010; Lee, 1994; Morillo-Velarde et al., 2013; O’Dor et al.,
1984; Spreitzenbarth and Jeffs, 2022b). Despite this nine paralarvae
handled and at least partially ingested the supplied artificial pellets. This
was apparent from the pellet being continually positioned against the
beak area for up to 8.03 min after capture combined with the caecum
changing in colour to a slight white. No artificial pellet was completely
consumed which might be due to their relatively large volume. The
longer handling times (> 1 min) in the current study are similar to ob­
servations made with O. tetricus and O. vulgaris paralarvae that received
Artemia as prey. Handling times ranged from 0.20 to 18.18 min for
O. tetricus paralarvae, with most handling times (80%) being within 1.22
to 10.25 min (Spreitzenbarth et al., 2021). Likewise, handling times
ranged from 4.03 to 10.25 min for O. vulgaris paralarvae (Iglesias et al.,
2006). This is a promising result from the current study and suggests that
if palatability and attractiveness can be enhanced for artificial pellets
their consumption would increase.
5. Conclusions
The development of artificial pellets has the potential to greatly
improve the effectiveness of culture of octopus paralarvae, however,
fundamental information about their feeding behaviour requires inves­
tigation if artificial pellets are to be successful. The results of this study
S. Spreitzenbarth and A. Jeffs
indicate that the visual cues leading to capture of artificial pellets by
early paralarvae are permissive, with a wide range of pellet sizes being
readily captured and pellet shape being unimportant. Paralarvae are
able to capture large volume artificial pellets relative to their own body
size, providing an excellent opportunity to maximise food intake for
larviculture. However, the next step toward achieving ingestion of
artificial pellets is improvement of palatability mostly likely through the
inclusion of ingredients that provide chemical cues that stimulate gus­
tatory activity. Overall, these research findings provide an important
first step toward advancing the use of artificial pellets for the rearing of
octopus paralarvae.
CRediT authorship contribution statement
Stefan Spreitzenbarth: Writing – review & editing, Writing – orig­
inal draft, Visualization, Methodology, Investigation, Formal analysis,
Data curation, Conceptualization. Andrew Jeffs: Writing – review &
editing, Writing – original draft, Resources, Project administration,
Methodology, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
We thank the two senior technicians, Rick Johnson and Jimmy
Rapson, for their help with fine tuning the two methods used to produce
the six artificial pellets. We also thank all divers and everyone that
helped with fieldwork, especially Fiona MacKechnie, Jeffrey Good and
Paul Caiger. This research was supported by the Ministry of Business,
Innovation and Employment’s Endeavour Fund (UOAX2208).
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