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Ethogram analysis reveals new body patterning behavior of the tropical arrow squid Doryteuthis plei off the Sao Paulo coast.

Abstract. Squids can express several body patterns, aided by a variety of visual signals that are produced by chromatophore organs. However, for several squid species, body patterning behavior during reproductive activity is still not completely understood. For example, what are the specific patterning changes and other visual signals, how do they appear, and how long do they last? To test the hypothesis that distinct chromatic components appear at different durations on the skin of the tropical arrow squid Doryteuthis plei in the Southern Hemisphere, we identified and described its body patterning behavior. Specimen squids were obtained from off the South Brazil Bight, near the coast of the Sao Paulo shelf. Animals were maintained and monitored in circular tanks for 62 d over six observation periods, from 2011 through 2013. An ethogram was constructed showing 19 chromatic, 5 locomotor, and 12 postural components, or body patterns, associated with reproductive behavior. New chromatic components (i.e., those not yet reported in the North Atlantic D. plei species), particularly those linked to female sexual maturity, were observed. A postural component, the "J-Posture," linked to defenses and alarm, also was noted. The average time spent for "light" components was 32 s. The corresponding "dark" components had an average duration of 28 s. Females displayed the chromatic components related to calm behavior longer than males. However, males appeared to be more dedicated to disputes over resources, and used rapid, miscellaneous visual signaling. In conclusion, new basic types of body patterns are described for D. plei. The repertoire of chromatic components reported in the ethogram is, to our knowledge, the first record for D. plei of the Southern Hemisphere.

Introduction

Squids can express multiple body patterns, each associated with specific behavioral responses. These visual signals are produced by chromatophore organs in the skin that are controlled by hormones and neurotransmitters through specific structures (Hanlon and Messenger, 1996; Messenger, 2001). In most cephalopods, body patterns are created by the simultaneous occurrence of chromatic, postural., and locomotor components. These affect the appearance of the animal and may be acute, with a duration lasting seconds, or chronic, extending for minutes. The composite is produced by groups of the chromatic units constructed from different elements (Hanlon, 1982; Hanlon et al., 1994, 1999; Di-Marco and Hanlon, 1997). The variety of body patterns of each individual is used both for crypsis and communication. The body pattern outline and the spread of chromatophores in the body are important tools for the study of ethology of cephalopods. These characteristics have been used to compare behavioral variation among loliginid squid species (Hanlon, 1982; Hanlon et al., 1994; Hanlon and Messenger, 1996). The pigmentation of cephalopod skin is contained within unique cellular chromatophore organs (Cloney and Florey, 1968). Chromatophores have a unique ability to rapidly change their shape through a specialized neuromuscular control system (Hanlon and Messenger, 1996).

The body pattern dynamics in loliginids have been investigated using captive and field studies in several parts of the world. The patterns are linked predominantly to courtship and mating during reproductive behavior, interspecific association, and competition for resources (Hanlon and Messenger, 1996). The following species have been investigated in previous behavioral studies: Loligo vulgaris reynaudii Orbigny, 1841 (Sauer and Smale, 1993; Sauer et al., 1997; Hanlon et al., 2002), Doryteuthis pealeii Lesueur, 1821 (Griswold and Prezioso, 1981; Hanlon et al., 1999; Sharsha and Hanlon, 2013), Doryteuthis opalescens (Berry, 1911) (Hurley, 1977; Hunt et al., 2000; Hanlon et al., 2004), Loligo spp. (Hanlon, 1998), Sepioteuthis australis Quoy & Gaimard, 1832 (Jantzen and Havenhand, 2003), and Sepioteuthis sepioidea (Blainville, 1823) (Arnold, 1965). There were also behavioral studies of Doryteuthis plei (Blainville, 1823) in captivity in the North Atlantic (Hanlon, 1982; Hanlon et al., 1983) and in the wild, i.e., from a research submersible during night dives (Waller and Wicklund, 1968).

The number of chromatic components described for the genus Loligo is large and complex. Loligo vulgaris reynaudii demonstrates 23 chromatic signals (Hanlon et al., 1994), Loligo forbesi Steenstrup, 1856 uses 17 signals (Porteiro et al., 1990), and D. pealeii has 34 signals (Hanlon et al., 1999). Studies focused on D. plei have reported that the organization of chromatophores and iridophores is not constant, differing and specific in certain regions of the body. For example, larger brown chromatophores are located on the dorsal mantle and small yellow chromatophores appear on the arms or tentacles (Hanlon, 1982). Therefore, the final appearance of a certain chromatic component is not only the result of neural excitation of colored chromatophores, but is also due to the size and distribution (vertical and horizontal) of chromatophores in different parts of the body. According to Hanlon (1982), D. plei displayed 16 chromatic components that were produced through specific static, morphological, and chromatic units.

D. plei inhabits coastal and shelf waters in the Western Atlantic Ocean, from the coast of Florida in the United States (Hixon et al., 1980) to Rio Grande do Sul, Brazil (Perez et al., 2005). This species is an important fishing resource off the Sao Paulo coast and is mostly found at shallow depths (< 30 m) in coastal waters (Gasalla et al., 2005). This squid spawns throughout the year, but its reproductive peaks occur during the summer months (Rodrigues and Gasalla, 2008; Postuma and Gasalla, 2010, 2014). In the South Brazil Bight, many studies have addressed the population biology of D. plei, including growth, reproduction, feeding, and fisheries oceanography (Martins et al., 2006; Martins and Perez, 2007; Gasalla et al., 2010; Postuma and Gasalla, 2010, 2014). However, the behavioral and body patterns of this animal have rarely been described. In this study, we present illustrations and descriptions of behavioral and body patterning. We also provide details of the variety of patterns and duration of each chromatic component observed. It is noteworthy that recent phylogeographic studies suggest that the Brazilian population of D. plei is genetically distinct from D. plei in North America and Central America (Sales et al., 2013).

The aim of the study was to describe the components and body patterns of the squid Doryteuthis plei, which may aid in distinguishing species in the North Atlantic Ocean based on previous work by Hanlon (1982) and Hanlon et al., (1983). To this end, an ethogram of the signals of D. plei, especially those related to reproductive behavior, was constructed. The ethogram is based on quantification of the time and duration of each chromatic component, gender differences, and types of chromatic components (light or dark).

Materials and Methods

Animal capture

Seventy-eight specimens of the tropical arrow squid Doryteuthis plei were obtained using hand jigs and Japanesestyle pound nets ("kaku-ami") off the Ubatuba coast (23[degrees] 51' S; 45[degrees] 08' W) in marine waters less than 10.9 m deep. Additional samples were collected in Sao Sebastiao (23[degrees] 83' S; 45[degrees] 44' W) in 6-m depth. Animals were immediately transported to the laboratories of the research station at the northern coast of Sao Paulo, Brazil, using the research vessel, Veliger II, and the small boat, Nautilus. During transport, animals were held in a 300-1 tank containing local seawater that was constantly aerated by a submersible pump capable of pumping 432 1 [h.sup.-1]. Transport to the laboratory required 5-40 min after each sampling, as the distance ranged from 1.3 to 3.5 nautical miles. All of these steps were taken to minimize the animals' stress and injury during collection and transport (Aguiar et al., 2012; Marian, 2012).

Experimental tank setup

In the laboratory, animals were held in two indoor circular tanks: a 2.3-m diameter, 3000-1 tank with a closed seawater system, and a 1.8-m diameter, 1000-1 tank with a flow-through system (Fig. 1). Both tanks contained gravel and sand substrates. The closed seawater system provided a continuous flow of seawater. A pump with a capacity of 10,000 1 [h.sup._1] was used to circulate water through the sand filter and a UV sterilizing filter. Squids were exposed to ambient light during a 12:12 light:dark photoperiod. During the night periods, observations were aided by a low-intensity LED light (about 50 lumens/watt). Water quality monitoring included temperature, salinity (ppt), and dissolved oxygen, all of which were measured daily with a multiparameter probe. Temperatures ranged from 21.86-28.81 [degrees]C, salinity ranged from 34.71-35.83 ppt, and dissolved oxygen was never greater than 5.00 mg [l.sup.-1]. The mean level of toxic ammonia was 0.020 ppm (range, 0.012-0.035, n = 15), and the mean level of total nitrite-nitrogen (N[O.sub.2]) measured 0.5 mg [l.sup._l] (range 0.02-3.05, n = 15).

Animal care and husbandry

Specimens were monitored for 62 d over six observation periods conducted in November 2011 (15 d), February 2012 (7 d), and March 2012 (14 d) with the closed seawater system; and in November 2012 (13 d), February 2013 (19 d), and November 2013 (10 d) using the flow-through system tank.

The squids' survival period in both systems averaged 7 d; some animals survived up to 19 d in February 2013. Of the population of 78 Doryteuthis plei specimens maintained in captivity, there were 46 females and 32 males. Females were more frequent than males only during February 2012 (19 females, 0 males). Multiple combinations of male/female pairs were acclimated in the tanks (Table 1) for observation and identification of the components of the body patterns related to behaviors. Based on previous studies, we devised a components checklist, including calm and reproductive behaviors (e.g., agonistic [males' dominance over females], courtship with displays of gonads, mating types, and egg-directed behaviors), which was used to categorize behavioral context (Roper, 1965; Flanlon et al., 1983, 1994, 1999, 2002, 2004; Hanlon and Messenger, 1996; DiMarco and Hanlon, 1997; Shashar and Hanlon, 2013).

During the observation period, a male in the tank was considered "dominant" when, having been paired with a female for 10 min, won the disputes with other males considered "intruders." The definition of these terms and of the period were chosen based on a study of fighting tactics of D. plei in the Gulf of Mexico (DiMarco and Hanlon, 1997).

Mean mantle length (ML) for females was 144.23 mm ([+ or -] 26.55 mm, SD; range 65-243 mm), and mean ML for males was 227.84 mm ([+ or -] 45.73 mm, SD; range 99-299 mm). Food was offered ad libitum one to two times per day, and consisted of small fish, frozen or live, plus live crustaceans when available. The species offered during feeding were Sardinella brasiliensis, Anchoa tricolor, and Anchoa sp., measuring 45-89 mm. However, species not described as prey in the diet of D. plei, such as the crustacean Call-inectes danae and fish (the barred grunt Conodon nobilis), also were offered. Observation periods at the 3000-1 tank with the closed seawater system showed a mean daily mortality rate of 22.02% (range 0-33%); observation times at the tank with the 1000-1, flow-through seawater system had a mean daily mortality rate of 21.05% (range 0-47%). A number of factors, including water quality, space confinement, live feed, exposure to light, and low noise and stress, were monitored to ensure the welfare of the study squids, as recommended for cephalopods by Moltschaniwskyj et al. (2007), and the ethical use of animals in applied ethology studies (Sherwin et al., 2003).

Experimental methods

To describe the organization of body patterns, we used the hierarchical classification developed for octopus by Packard and Sanders (1971) and Packard and Hochberg (1977), and reviewed by Hanlon and Messenger (1996). The classification hierarchy follows a top-down flow: (i) body patterns, (ii) components, (iii) units, and (iv) elements.

The chromatic, postural, and locomotor components were described and tabulated in a spreadsheet and compared for time, gender, and chromatic component (light and dark). Body patterns were classified into two categories: (1) chronic patterns and (2) acute patterns. The terminology used to name the components and body patterns was based on studies conducted on the behavior of other loliginids around the world, such as Loligo vulgaris reynaudii (Hanlon et al., 1994) and Doryteuthis pealeii (Hanlon et al., 1999), and on previous studies, particularly of D. plei in the North Atlantic (Hanlon, 1982; Hanlon et al., 1983).

Overall, 96 observations were made from the top of the tank (Fig. 1), averaging 3 h per observation, at a rate of approximately three observations per day, and totaling 530 h over the six observation periods. In addition, 1056 video frames were recorded at an average rate of 15 per day, totaling 28 h, 40 min of video recording time, with a mean filming time of 30 min each (range 1-45 min) (Table 2). The videos were focused on male and female pairs of D. plei, and especially on those that exhibited reproductive behavior. The videos were reviewed six times each, two times looking at one component category, following the methodology described by Hanlon et al. (1999). Observations started with the chromatic component (looking only for chromatic signals), followed by the postural components, and, finally, the locomotor components. Then each component of the body patterns was observed and noted for any squid at a given time during the observation periods. With respect to egg capsule deposition, the first hours of the day (00:00 -06:00 am) were monitored, and observations of egg deposition were noted. Therefore, the observations were focused on a single female. We conducted "focal animal sampling" by following that female as long as possible, filming continuously to record the sequence of behaviors that preceded egg laying, after the methodology of Hanlon et al. (2004).

In analyzing the 1056 videos, we calculated in seconds the mean duration of each chromatic component. The duration of these components did not have a normal distribution (Shapiro-Wilk test); in fact, the duration in seconds was found to violate the criteria for normality. Therefore, the non-parametric Kruskal-Wallis test and post-hoc pairwise comparison tests (Siegel and Castellan, 1988) were applied to assess the influence of gender and type of chromatic component (light or dark) on the duration of each chromatic component. All statistical tests were considered significant when P < 0.05.

Results

Ethogram

An ethogram for Doryteuthis plei, based on our observations and the videos, is shown in Figures 2 and 3. The chromatic, locomotor, and postural components found in this study were used to build up body patterns and represent a segment of the behaviors for this species, especially those related to reproduction. The variation of each chromatic component is shown in Figure 2.

Locomotor components

The hovering position was caused by the interaction of fin movements and siphon fanning. This component was observed in solitary squids and in groups when the squids were calm. Free swimming occurred when the squid moved forward, with the head backward and raised slightly, and the arms somewhat depressed. During this component, the siphon and fins steered the animal during swimming. This posture was observed in calm squids preparing to mate.

Parallel positioning involved two animals hovering or swimming parallel to each another in the same direction, within one body length of the other. Fin beating a male-specific behavior, occurred during parallel positioning, when two males maneuvered into position to beat their fins against each other. This was a physical and escalating stage with an agonistic context, but it resulted in no obvious physical injury. These encounters lasted up to 10 s in disputes for females during mating.

The chase occurred when one squid (of either sex) vigorously pursued another squid by intraspecific competition or cannibalism in the tank. It occurred especially in moribund squid. This component was often observed during agonistic behavior in males. During the contests, a winner might chase the loser several times, with each chase lasting as long as 40 s. During feeding, squids also chased their prey; chases varied from a few seconds to minutes, when the squid's chase was failing. Escaping and fleeing occurred during intraspecific competition between winners and losers, especially among males during mating.

Jetting consisted of rapid body movements causing the expulsion of water from the siphon, producing rapid jet propulsion. Jetting usually stretched from 0.5 to 1 m in distance and was always performed backwards. Jetting was combined in escapes used to avoid both predators and conspecifics over spawning among females after mating and deposition of an egg capsule into the egg mop (assemblage of egg capsules on the substrate).

The courting pairs component occurred when a male initiated a parallel position to a mature female; agonistic encounters also began with this positioning. However, when squids were placed in the tank, there was no synchronic, parallel swimming. At first, pairs did not swim in the same direction. However, within one day (24 h) of the experiment, they began to do so. Swimming upwards was observed in females when they swam up the tank. This action occurred before male-parallel mating. The female attracted the male to mating by swimming upwards. This movement was observed eight times in the total video footage, lasted an average of 15 s per episode.

Head-to-head mating occurred when a male and female faced each other and the male grasped the female's arms. Spermatophores were placed in a seminal receptacle below the mouth. Male-parallel mating involved a male positioning himself under a female, then grasping her anterior mantle to pass spermatophores into the mantle cavity.

Oviposition consisted of a female taking a single, extruded egg capsule and affixing it to the substrate or to the existing communal egg mop. The female did not hold the egg mop for long periods. A female with a 180-mm mantle was observed sometimes depositing an egg capsule on the substrate. She then positioned herself vertically, that is, at 90 degrees to the substrate, and affixed the egg to it. Afterward, this squid performed a fast backwards movement similar to jetting, and showing an all dark chromatic component.

Bottom sitting occurred when a squid rested on the substrate. This position was observed in tired squids, and was accompanied by the chromatic component of arm spots and bands (see Chromatic components below). This component preceded the death of a squid and could last up to 1 min.

Egg touching consisted of contact with an egg mop by both males and females. Contact ranged from a brief exploratory touch to an embrace of the egg capsule with all of the squid's arms. Females usually laid eggs on the existing egg mop; touching may have been a way of assessing the egg-laying substrate. Males commonly touched eggs; however, touching was often followed by highly aggressive agonistic bouts, suggesting that the eggs provided a visual, tactile, or perhaps chemosensory stimulus.

Chromatic components

Light chromatic components. The clear chromatic component was the most common behavior, recorded 176 times during the entire video footage (Table 3). This component is caused by a retraction of all or a majority of the chromatophores. As a result, the squid's mantle appears almost transparent (Fig. 3A). In clear waters, when the squid was observed against a gravel background, translucency camouflaged the animal. Internal organs, such as the testis in males and the oviducal and nidamental glands in females, were readily visible in our specimens. During feeding, the stomach and the entire digestive system also were seen.

Iridophore splotches appeared on the dorsal mantle and the head. These splotches were a distinctive yellow or golden color, and they helped to produce general camouflage. Three iridescent components are thought to aid in crypsis: (i) iridescent arm stripes, (ii) dorsal mantle splotches, and (iii) dorsal iridophore sheen. This component was observed especially when the squid was calm and floating in the tank. This component occurred 61 times during the total video footage period and lasted from 2 s to 3 min, averaging 46 [+ or -] 36 s (SD) for the dorsal iridophore splotches (Fig. 3B).

Dark chromatic components. The all dark chromatic component usually occurred at night, together with agonistic behavior in males. This coloring is created by expansion of all of the chromatophore cells across the entire mantle, turning the squid entirely dark. Expansion of all of the chromatophore cells produced brown and red coloring (Fig. 3C). All dark (unilateral) coloring occurred six times, and was used during agonistic encounters between males and females (Fig. 3D). This coloring appeared on one side of the mantle, showing the chromatophores expanded in perfect lateral symmetry.

A dark dorsal stripe was observed in calm squids. This stripe is thought to aid in crypsis through counter-shading when viewed laterally, and through disruptive coloration when viewed from above, by covering some of the bright organs, such as the testis, oviducal glands, and ink sac (Fig. 3E). The transverse bands component was observed frequently in groups of large males (Fig. 3F), and appeared in four varieties. The component was found in crypsis behavior through disruptive coloring, as a warning sign when a squid moved close to a possible predator, or when the prey that were offered for feeding were equal to or greater than 50% of the mantle length of the squid. It also occurred in females at the bottom of the tank (Fig. 3G). The most commonly observed pattern was one band (n = 37), followed by a variation that included four bands (n = 31), during the total video footage (Table 3).

The arm spots component was observed at the base of the third arms, the second arms, or both sets of arms. This component was common and had several variations, but we were only able to highlight points on the arms on one side of the body (Fig. 3H). The arm and tentacular stripes components were most readily observed when the tentacles were extended. In most animals, the first or third pair of arms was darkened. The average duration of this component was 55 s (Fig. 31).

The infraocular spot occurred in a circular shape near the eyes, roughly between the eye and the arm spots component. Both components were sometimes expressed simultaneously during alarm situations when coloring was rarely observed. The shaded eye component is a transverse head band of expanded chromatophores, and may aid crypsis as it covers the bright iridescent sclera of the eyes. The fin stripe component expressed during agonistic contests was also observed when the squid was transported to captivity. It was also noted in alarmed squids and especially in large males during fights. Dark arm and head coloring was noted during intraspecific agonistic encounters, but was also seen during mating and care of the egg mop.

The dark fins component was caused by expanded chromatophores in the region above the fins, which darkened them. This component lasted 2 s on average and was rarely observed. The component was most commonly observed in females when displaying alarm behavior.

Females

Females exhibited the light chromatic components for longer periods than males, possibly as a result of their calm or courtship behaviors. The white accentuated oviducal gland of females was similar in appearance to the males' accentuated testis (see Males below), but it differed in shape, position, and frequency of expression (Fig. 3J, Table 3). The accentuated area was an ovate shape on the dorsal lateral region of the mantle. The oviducal glands were observed on the mantle in quick flashes for 2 s or, rarely, for a longer duration, i.e., 1 min.

The lateral mantle spot was expressed as a bold side area of dark chromatophores near the head or in the middle of the mantle. The mantle side spot was observed only when the female was paired with the male during mating, and could indicate receptivity. This component occurred several times and passed relatively quickly after approximately 2 s. This pattern may also indicate the maturity of the animal (Fig. 3K). The shaded oviducal gland component preceded mating and occurred during parallel positioning. It was caused by the selective expansion of chromatophores over the oviducal gland (Fig. 3L).

The red accessory nidamental gland occurred more than 10 times during the entire video footage (Table 3), usually during the daytime, when the females positioned themselves parallel to the males. In Doryteuthis plei, this gland is large and bright, and it can be observed through the mantle either laterally or from below (Fig. 3M). It may also signal female sexual maturity, because it turns red only upon attainment of full maturation.

Males

The accentuated testis component occurred 20 times during the total video footage (Table 3) in mature, mating males with mantle lengths of 200-299 mm (Fig. 3N). It was seen during courtship and parallel swimming immediately prior to or during mating. The accentuated testis appeared when the chromatophores directly above the testis were retracted and the mantle darkened completely. When the mantle is entirely dark, the sexual organ whitens laterally to the mantle and assumes an elongated shape.

The lateral mantle streaks component is produced by longitudinally oriented rows of partly expanded chromatophores (Fig. 30). This phenomenon was observed during agonistic behavior. Shaded testis, caused by the selective expansion of chromatophores over the testis, preceded mating and occurred during parallel positioning.

Frequency and duration of chromatic component expression

Nineteen chromatic (4 light and 15 dark), 5 postural, and 12 locomotor components were identified in our compiled videos and correlated with different body patterns. We observed 923 displays of chromatic components in the skin of squids during the total video footage (Table 3). Most chromatic signals were significantly more frequent during the day than at night (73% of observations; Chi-square test, [[CHI].sup.1] = 3.38, P = 0.05, df = 1). At night, the all dark chromatic components were observed more frequently (Table 3). Data in Figure 4 show the frequency of the chromatic components identified. The clear chromatic component was observed most often (> 13%), and it occurred together with the arm spots (11%) and dark dorsal stripe (7%) components. All dark occurred in 9% of the observations during the total video footage, followed by bands (8.5%). The chromatic components were more evident in the body patterns; however, some components were rarely observed (< 2%). The red accessory nidamental gland, lateral mantle spots, infraocular spots, dark fins, and shaded eye were rare in females.

The average durations of the chromatic components differed significantly (Kruskal-Wallis test, P < 0.05). A pairwise comparison test showed that mean durations were significantly higher in the most enduring components, such as clear, iridophore splotches, and bands (P < 0.05), than in the most short-lived components: arm spots, lateral mantle spot in females, fin stripe, accentuated oviducal gland, lateral mantle streaks, and dark dorsal stripe (Fig. 5, Table 4). The infraocular spot component was seldom displayed; however, it showed a significant difference in time from the accentuated oviducal gland, a fast component. The average time of the chromatic components of light (e.g., clear and iridophore splotches) and dark (e.g., arm spots, lateral mantle spot; see Table 4) also showed a significant difference ([[chi].sup.2] = 13.55, df = 1, P < 0.05). The light components had a longer duration (mean = 32 [+ or -] 25.2 s, SD) than the dark components (mean = 28 [+ or -] 32 s, SD). Significant differences based on gender also were noted (P < 0.05, Table 4, Fig. 6).

Postural component

Drooping arms in a swimming squid is a posture in which all of the arms appear relaxed and suspend downwards. This component preceded catching prey and occurred soon after relaxation. The squid maintains rigid arms, pointing at prey before capturing it. A splayed arm is a posture in which all eight arms are spread and flattened on the horizontal plane. This posture was expressed by both sexes when the chromatophores on the sides of the arms could also be observed. It occurred when a squid was defending a resource such as an egg mop, or when in the presence of another male during courtship or mating. The raised arms posture was a strong signal of alarm, and was used when a rival male was near. It also occurred when the animal was detected by a predator; it assumed a threatening posture by raising its arms to another animal. It was rarely observed in females.

The downward curling position consists of all of the arms and tentacles curled downward at 90 degrees. It is accompanied by four transverse bands on the mantle (Fig. 3G). This position was observed in aggressive encounters and in courtship; it was usually displayed at the bottom of the tank, next to the substrate. Females were more likely to use this position. The J-posture is characterized by raised arms at an angle of about 45 degrees, resembling the letter "J", such that the tips were close to the anterior dorsal margin of the mantle. This position, lasting about 5-7 s, relates to defense and alarm, and was observed in both sexes.

Body patterning

Chronic patterns. Patterns in this category can extend for seconds or minutes. For example, when squids were calm, they had a clear body pattern and the chromatophores were retracted over the mantle. This patterning leads to chromatic components such as the dorsal stripe, arm spots, and the iridophore splotches located on the mantle, fin, and head. These patterns were observed frequently in normal laboratory conditions. Calm animals were usually swimming forwards and backwards, or swimming in place (i.e., the free swimming and hovering locomotor components), at which time the postural component of drooping arms was observed.

The bands body pattern was associated with alert or alarmed behavior, and can be considered a chronic pattern because it occurred frequently for periods ranging from 20 s to 1 min. This pattern can take place when prey or predators are near or when the squid is alone. This body pattern occurred together with other chromatic components (see the male showing a band with arm spots in Fig. 3F; and females with bands, dorsal iridophore sheen, and downward curling in Fig. 3G).

The all dark body pattern was considered chronic because, during the nighttime observation periods, the squids were mostly totally dark or brown (90% of the night observations). In two situations during the daytime observation period, an entire school of squids appeared all dark. Video analysis showed that one squid darkened when it detected a predator, prey, or observer above the tank. This event caused all of the squids in the school to become all dark for 20 s to 2 min. At night, the squids were all dark and moved fast together, using the jetting locomotor component. Jetting occurred when the squid was alone or when prey in a tank was larger than 50% the size of the mantle; at these times, the squid likely believed the prey to be a potential predator. The all dark component was also used for hunting live prey (fish) or as nighttime camouflage.

Reproductive behavior

Acute patterns. These patterns occurred quickly and were linked to intra- and interspecific interactions during reproductive behavior, such as agonistic behavior during fights for a mate or during courtship, mating, spawning, and egg touching. These body patterns occurred for seconds only.

Figure 7 summarizes the behavioral sequence observed during the study. The dashed arrows represent the flow of behaviors over time. After the animals' acclimation period (1 or 2 d), observed behaviors became more complex. During reproductive behaviors, a combination of chromatic components was noted in agonistic activity, and included arm spots and lateral mantle streaks (only males). Mating activity included accentuated testis and oviducal gland, red accessory nidamental gland, mantle spot in females, and shaded testis, observed together with the locomotor components of parallel positioning, fin beating, courting pairs, and oviposition (during the spawning process).

Agonistic behavior

In the first days of the observations, a male established dominance and then protected the females from other males in disputes. During agonistic behavior displays, multiple chromatic components were displayed, such as arm spots, lateral mantle streaks (only males), infraocular spot, dark fins, and arms/tentacular stripes together with fin beating, chase (winner), and flee (loser) accompanied by raised arms or splayed arms (Fig. 30). The lateral display patterns were easily observed immediately after the clear body patterning. The displays occurred for a fraction of a second and were repeated two or three times.

The dark flashing pattern occurred in a situation of high stress or an alarm signal during spawning and egg touching, and in response to non-specific threats such as the presence of people around the tank or noises made nearby. A strong alarm signals the squids to darken completely and to assume the J-posture component in both sexes. After mating, the females swim using jet propulsion; their mantle color is completely dark. The jetting is of short duration.

Courtship behavior

The gonads display body pattern was the most frequently shown pattern for reproduction during courtship behavior. Males and females formed pairs, during which the male pursued the female and protected her from other males. Video analysis showed that this behavior occurred 159 times, on an average of two events per day. In most cases (90%), the male began courtship by pairing with a female and moving forwards and backwards in parallel positioning for a lengthy time (> 3 min); the gonads are highlighted between the animals, accompanied by slight touching. However, less frequently, females initiated courting by swimming upwards.

Mating behavior

Two types of mating were observed in our study, and each differed in positioning, duration, and frequency (Table 5). Head-to-head mating was most commonly seen (n = 18), lasting 5-41 s (mean = 17 [+ or -] 10.24 s, SD). In this position, the body pattern was dark, the mate category was "sneakers", and females showed faster displays. Male-parallel mating occurred less often (n = 4), and lasted 10-15 s (mean = 12.2 [+ or -] 2.06 s, SD). During this type of mating, the fourth pair of male arms was totally dark. The mating category was "consorts."

Discussion

Reduction and internalization of the shell is a key trait in the evolution of cephalopods. It has allowed for an active life in the water column and an ability to compete with vertebrates (Packard, 1972). But this lifestyle has also made these animals more exposed and vulnerable to predatory attacks. In response, they have developed sophisticated mechanisms for camouflage that include the use of chromatophores (Messenger, 2001). A system of neurally controlled chromatophores is supremely well adapted for signaling. Many shallow-water cephalopods also use chromatophores to form both inter- and intraspecific visual signals (Hanlon and Messenger, 1996; Messenger, 2001).

This study is the first concerted effort to analyze the behavioral components of Doryteuthis plei in the South Brazil Bight and in the Southern Hemisphere. The behavior of D. plei is complex and presents a variety of chromatic, postural, and locomotor components. Chromatophores can alter visual appearance in response to stimuli (Hanlon and Messenger, 1996). Color changes occurred when a male intruder approached a dominant male to fight for a mating female. During the study, we observed that the light chromatic components (clear and iridophore splotches) had a longer duration than the dark chromatic components, especially those associated with calm behavior. Squid chromatophores are neurally controlled, allowing the animal quickly to select and demonstrate various body patterns. With this quick polymorphism, squids can rapidly hide from predators.

The locomotor components include a variety of movements using the siphon, arms, and fins. However, more attention is required to observe the postural components because they involve the body's position and arms. The locomotor and postural components observed in this study were earlier described for other squids (Hanlon, 1978; Hanlon et al., 1983, 1994, 1999, 2000, 2002; Hanlon and Messenger, 1996; Jantzen and Havenhand, 2003; Buresch et al., 2004; Pham et al., 2009; Shashar and Hanlon, 2013).

Most of the chromatic components observed in this study occurred during diurnal periods, which is easily explained by the difficulty of nighttime observations due to the lack of light in the tank. When the LED light was turned on in the tank, squids kept to the periphery of the light. Several of the 19 chromatic components identified in our study of D. plei in the Southern Hemisphere were identical to those previously described for other loliginids, including Loligo vulgaris (Hanlon et al., 1994), Dory teuthis pealeii (Hanlon et al., 1999), and Doryteuthis opalescens (Hunt et al., 2000). However, in comparison to D. plei in the North Atlantic (see Hanlon, 1982, 1988; Hanlon et al., 1983), a greater variety of chromatic components was observed during our study. Hanlon (1982) describes only 16 chromatic components in D. plei of the North Atlantic (USA). However, the chromatic components that were seen in our study in relation to the sexual maturity of females were not reported by the author studying D. plei of the North Atlantic (e.g., accentuated oviducal gland; (Fig. 3J)); dark arms/head, dark fins, and infraocular spot components also were not mentioned. Possibly, maintenance conditions, such as color, depth, and bottom type of the tank used in this study, influenced the observed patterns of skin coloration of the squids.

Body patterns and behavior

Calm behavior. Calm behavior in this species was scored when squids did not haphazardly strike the tank walls, avoiding significant injury to skin and fin (Hanlon et al., 1983). Through video analysis, we observed that at the beginning of each maintenance period, squids adapted to the tank conditions and showed calm behavior. This state was also easily identified through the clear chromatic component and free swimming. During parallel positioning, a calm state was noted when the postural component was relaxed, with drooping arms; squids appeared to be at ease, and there was no threat in the tank. The clear chromatic component was the most frequently observed display in this study (see Table 3). Our observations support the notion that the D. plei squid mantle is transparent or has pale coloration. Unlike the all dark pattern, this pattern (i.e., clear pattern = calm and all dark pattern = alarmed) has been found for this species (Boycott, 1965; Hanlon et al., 1983) and other loliginids around the world (e.g., Loligo forbesi in Europe, Porteiro et al., 1990; Loligo vulgaris reynaudii in South Africa, Hanlon et al., 1994; Doryteuthis pealeii in Massachusetts, Hanlon et al., 1999; Doryteuthis opalescens on the California coast (USA), Hunt et al., 2000; and Sepioteuthis australis in Australia, Jantzen and Havenhand, 2003).

Alarm behaviors. During the "'dark" (vs. light) observation period, alarm and jetting behaviors were observed through display of all chromatophores on the mantle. This component may also be used as camouflage to catch prey at night. When large or uncommon prey was placed in the tank, it caused a rapid expansion of chromatophores, turning the squid all dark during the day period. However, this pattern is also used intraspecifically during agonistic encounters and between males and females when one squid is alarmed (Boycott, 1965; Hanlon, 1978, 1982; Hanlon et al., 1983, 1994, 1999). Downward curling and J-posture were less frequently observed postures, but downward curling (Fig. 3H) was noted more often than the J-posture. Both postures were observed in males and females, and were related to aggressive behavior or alarm. These two postural components are commonly observed among other squids. The J-posture was reported in D. opalescens (Hunt el al., 2000) and Lolliguncula brevis (Blainville, 1823) (Martins and Perez, 2006). Other equivalent components are found in the following squids: Omithoteuthis antillarum Adam, 1957 (Vecchione and Roper, 1991); the J-curl in Gonatus onyx Young, 1972 (Hunt and Seibel, 2000); arms flexed dorsally in Octopoteuthis megaptera (Verrill, 1885) (Vecchione et al., 2002); upward curl in Sepioteuthis australis (Quoy & Gaimard, 1832) (Jantzen and Havenhand, 2003); and dorsal arm curl in the deep-sea squid Octopoteuthis deletron Young, 1972 (Bush et ai. 2009). This posture was related to deimatic behavior in cephalopods (Hanlon and Messenger, 1996). Downward curling has been reported for D. plei (Hanlon, 1978) and other loliginids (Hanlon et al., 1994, 1999; Hunt et al., 2000; Jantzen and Havenhand, 2003).

Reproductive behavior in Doryteuthis plei

The reproductive behavior in D. plei includes a variety of skin colorations, movements, and postures (Hanlon et al., 1983; DiMarco and Hanlon, 1997). Shoaling squids have ample opportunity for social communication with conspecifics throughout most of their lives, and some species have established elaborate behavioral sequences, including agonistic, courtship, and mating behaviors (Hanlon and Messenger, 1996).

Agonistic behavior. In our study, the male initiated courtship and immediately established and maintained a dominant relationship over females. Fighting between large males was a conspicuous event during their reproductive behavior. The behaviors also included threats, chases, and fleeing during fin beating; together with the presence of lateral mantle streaks and arm splotch, these behaviors are easily identified. The most noted posture that we observed is splayed arms during agonistic behavior. The locomotor component of fin beating is also easily recognized and represents the escalation of an agonistic encounter by involving physical contact (Porteiro et al., 1990; Hanlon and Messenger, 1996; DiMarco and Hanlon, 1997; Hanlon et al., 2002; Jantzen and Havenhand, 2003; Pham et al., 2009; Shashar and Hanlon, 2013). The courtships are interrupted by large, lone males or intruders, as previously reported (DiMarco and Hanlon, 1997; Hanlon et al., 2002), that engage the paired consorts in agonistic contests, often resulting in successful takeovers. The agonistic behavior of this species was described in detail by DiMarco and Hanlon (1997), who observed various aspects of the behavior mainly in laboratory studies. This behavior occurred similarly in other loliginids (Hanlon et al., 1994, 1999; Hunt et al., 2000).

Courtship behavior. In this study, most squids formed mate pairs (females and males), and the duration of mate pairing lasted for a long period. In the experiment of November 2011, we observed that in the first 2 d of maintenance, squids performed free swimming inside the tank. However, by the third day they had formed pairs. The pairs were generally formed after the agonistic contests. In females, the red accessory nidamental gland and oviducal gland were often visible, as in some species of Loligo (Hanlon et al., 1994, 1999, 2002; Hunt et al., 2000).

Mating behavior. Mating of Doryteuthis plei in this study occurred in two positions and was similar in duration and positioning to what was reported for other loliginids (Hanlon et al., 1994, 1999; Sauer et al., 1997; Jantzen and Havenhand, 2003; Zeidberg, 2009; Sharsha and Hanlon, 2013). The first position was head-to-head mating with a sneaker male, which was observed more often than the male-parallel position with a large consort male. Hanlon (1997) and Hanlon et al. (2002), in studying the behavior of Loligo sp. and Loligo vulgaris reynaudii in South Africa, used the term "sneakers" for the smaller males and "consorts" for the larger males that formed different reproductive disputes during spawning on the seafloor. The male-parallel mating always occurred soon after eggs were deposited on sand at the bottom of the tank. Waller and Wicklund (1968) observed a larger natural spawning shoal in the sea, noting that nearly all squids were paired, and mating in the male-parallel position was followed almost immediately by egg laying (oviposition). Shashar and Hanlon (2013) detailed the multiple mating tactics during copulation in Doryteuthis pealeii.

Egg-directed behavior. The oviposition component was rarely observed in the filming (Le., only twice). Egg deposition of females occurred in a completely darkened lab or in the early hours of the day. Egg depositions that occurred during the night were filmed with the aid of an LED light. The egg mop developed rapidly, and the first paralarvae appeared 10 d after eggs were deposited at the bottom of the tank. This result is similar to findings reported by Roper (1965) for this species. Oviposition was also observed in shallow waters during field studies of this species (Waller and Wicklund, 1968), for D. opalescens in Monterey Bay California USA (Hanlon et al., 2004), and for Loligo vulgaris reynaudii in South Africa (Sauer and Smale, 1993; Hanlon et al., 1994). However, egg touching was observed for long periods among D. plei males and females. Egg touching is common in captivity and can be artificially stimulated by inserting an egg capsule, as described for D. pealeii by Arnold (1962). This action was most common in males guarding an egg mop.

Conclusion

This study reports the first findings of body patterning behavior of Doryteuthis plei in the Southern Hemisphere. Currently, it represents the only ethogram with quantitative analysis of a myopsid cephalopod in South America. Our results showed that most behaviors observed for D. plei are similar to those of other squids around the world, both in captivity and in the field. However, some differences were found between D. plei investigated here and previous studies in the North Atlantic. For example, the chromatic component of the female accentuated oviducal glands was readily observed during pairing or courtship in our study (Fig. 3J). However, the glands were not observed by Boycott (1965) in Bermuda, in natural habitats in the Bahamas (Waller and Wicklund, 1968), or in captivity in Massachusetts (Hanlon, 1982, 1988; Hanlon et al., 1983). We did not observe chromatic components such as infraocular spots or dark arms/head for long periods, as was reported only for Loligo vulgaris (Hanlon et al., 1994) and Doryteuthis pealeii (Hanlon et al., 1999).

Recent phylogeographical studies of D. plei have suggested that the Brazilian population is genetically distinct from D. plei in North America and Central America (Sales et al., 2013). For this reason, behavior is another attribute that can assist in taxonomic identification and phylogenetic analyses (Hanlon, 1988; Hanlon et al., 1999). The genetic description, coupled with detailed behavioral aspects such as those reported in this study, should provide insights into the variability of reproductive behaviors and the potential for differences between the various populations of D. plei in the Atlantic Ocean.

In summary, the squid D. plei has a large repertoire of body patterns, including many combinations of skin coloration, body postures, and swimming movements, that are used specifically for communication during reproductive behavior. Our results showed that the duration of each chromatic component differed significantly, suggesting that these components are connected to behaviors performed during the short life cycle, for example, agonistic behavior or mating. Females displayed the chromatic components for longer durations than the males, which may have been the result of their calm behavior or display of their gonads. The males were dedicated to winning the females in the first days of the observation periods. Head-to-head mating was more frequent and longer lasting than male-parallel mating. Egg-directed behaviors occurred during nighttime periods only.

Our evidence supports the theory that the elaborate sensorial system in cephalopods, allowing for rapid chromatophore activity and skin-based communication skills for intra- and interspecific relationships, is complex and highly evolved, even in small-size nektonic species. The particularly ritualized reproductive behavior found in D. plei, with the gonadal displays during courtship and the immediate expansion of some groups of chromatophores and retraction of others, seems to be one of the most complex and interesting body patterning behaviors noted in the marine realm.

Acknowledgments

We thank the crew of the University of Sao Paulo's research vessel "Veliger IF' (Oceanographic Institute) and staff from CEBIMar (Center of Marine Biology) for their collaboration during the fieldwork. The Sao Paulo State Research Foundation (FAPESP; Grant 10/50183-6) and the Brazilian National Council for Scientific and Technological Development (CNPq) (Grants 142333/2011-5 and 141386/2013-4) provided financial support. We would like to thank Silvia De Almeida Gonsalves for help with drawings of the squids. We extend our gratitude to Prof. Daniel Lemos and Ricardo Haruo Ota for their assistance with the maintenance of living squids in the laboratory. This is a contribution of the University of Sao Paulo's Research Cluster on Marine Biodiversity (NP-Biomar).

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FELIPPE A. POSTUMA (*) AND MARIA A. GASALLA

Fisheries Ecosystems Laboratory (LabPesq), Department of Biological Oceanography, Oceanographic Institute, University of Sao Paulo, Praca do Oceanografico 191, Cidade Universitaria, 055080-900 Sao Paulo, SP, Brazil

Received 1 October 2014; accepted 5 May 2015.

(*) To whom correspondence should be addressed. E-mail: felippepostuma@usp.br.

Table 1

Number of Doryteuthis plei females and males with mean mantle length
(mm) (in parentheses), sex ratio with Chi-square test: results during
the six observation periods from 2011 through 2013

Observation period  Females  Males     Sex ratio  Chi-square  df

November 2011        3(144)   7(209)    0.4286     1.600       1
February 2011       19(140)   0        19.000     19.000       1
March 2011           6(162)   5(194)    1.200      0.090       1
November 2012        6(143)   5 (210)   1.200      0.090       1
February 2013        8(160)   7 (281)   1.142      0.060       1
November 2013        4(145)   8(210)    0.555      1.330       1
Total               46(144)  32 (227)   1.516      2.510       1

Observation period  P-value

November 2011        0.21
February 2011        1.30[E.sup.-05]
March 2011           0.76
November 2012        0.76
February 2013        0.79
November 2013        0.24
Total                0.11

Table 2

Video recordings (n) during the six laboratory maintenance periods,
with total video footage and mean, minimum, maximum, and standard
deviation (h:min:s) of each video

Observation period  n     Total video time  mean     min      max

November 2011       462   13:27:20          0:55:55  0:01:26  0:28:33
February 2012       47    1:26:31           0:12:22  0:05:12  0:20:10
March 2012          197   8:53:14           0:38:05  0:05:42  0:38:49
November 2012       189   2:42:02           0:25:32  0:01:12  0:12:10
February 2013       159   1:12:06           0:29:51  0:03:45  0:45:22
November 2013       36    1:02:06           0:19:51  0:02:46  0:35:22
Total               1090  28:43:19          0:30:16  0:01:12  0:45:22

Observation period  SD

November 2011       0:35:21
February 2012       0:05:44
March 2012          0:56:07
November 2012       0:06:34
February 2013       0:21:54
November 2013       0:19:05
Total               0:19:07

Table 3

Time (h:min:s) of expression and frequency of the chromatic components
observed in the video footage of Doryteuthis plei over the six
observation periods from 2011 through 2013

                                   Observation  Component
Chromatic components               frequency    variations

Light chromatic components
Clear                              176
Iridophore splotches                61          Dorsal iridophore sheen
                                                Dorsal iridophore
                                                splotches
                                                Iridescent arm stripes
Accentuated oviducal gland          51
Accentuated testis                  20
Dark chromatic components
All dark                            94          All dark
                                                All dark (unilateral)
Arm spots                          155          Arm spots 1
                                                Arm spots II
                                                Arm spots III
                                                Arm spots IV
Bands                               75          Bands I
                                                Bands II
                                                Bands III
                                                Bands IV
Dark dorsal stripe                  62          Dark dorsal stripe
                                                Dark dorsal stripe II
Lateral mantle streaks              54
Shaded oviducal gland               40
Shaded testis                       32
Infraocular spot                    21
Shaded eye                          14
Fin strip                           14
Dark arms/head                      18
Dark arm stripes/tentacle stripes   10
Lateral mantle spot (f)             11
Dark fins                            8
Red accessory nidamental gland       7

                                                 Time
Chromatic components                   Mean      Min       Max

Light chromatic components
Clear                               -  00:00:32  00:00:01  00:03:07
Iridophore splotches                8  00:00:47  00:00:02  00:01:31
                                   44  00:00:46  00:00:02  00:03:00

                                    9  00:01:00  00:00:03  00:03:00
Accentuated oviducal gland          -  00:00:16  00:00:01  00:01:31
Accentuated testis                  -  00:00:28  00:00:05  00:01:30
Dark chromatic components
All dark                           88  00:00:34  00:00:01  00:03:00
                                    6  00:00:46  00:00:05  00:02:25
Arm spots                          66  00:00:27  00:00:01  00:01:31
                                   11  00:00:12  00:00:01  00:00:55
                                   13  00:00:21  00:00:01  00:01:31
                                   65  00:00:27  00:00:01  00:02:25
Bands                              37  00:01:11  00:00:02  00:03:00
                                    2  00:01:33  00:00:05  00:03:00
                                    5  00:00:17  00:00:01  00:00:55
                                   31  00:00:39  00:00:01  00:02:11
Dark dorsal stripe                 42  00:00:26  00:00:01  00:02:10
                                   20  00:00:30  00:00:01  00:01:31
Lateral mantle streaks              -  00:00:31  00:00:01  00:02:41
Shaded oviducal gland               -  00:00:34  00:00:02  00:02:25
Shaded testis                       -  00:00:35  00:00:01  00:02:25
Infraocular spot                    -  00:00:41  00:00:03  00:02:05
Shaded eye                          -  00:00:09  00:00:03  00:00:18
Fin strip                           -  00:00:41  00:00:05  00:01:30
Dark arms/head                      -  00:00:35  00:00:02  00:01:31
Dark arm stripes/tentacle stripes   -  00:00:39  00:00:01  00:02:55
Lateral mantle spot (f)             -  00:00:19  00:00:03  00:00:55
Dark fins                           -  00:00:50  00:00:02  00:02:00
Red accessory nidamental gland      -  00:00:08  00:00:05  00:00:31

                                   Time
Chromatic components               SD

Light chromatic components
Clear                              00:00:32
Iridophore splotches               00:00:25
                                   00:00:36

                                   00:00:48
Accentuated oviducal gland         00:00:21
Accentuated testis                 00:00:30
Dark chromatic components
All dark                           00:00:32
                                   00:00:53
Arm spots                          00:00:28
                                   00:00:15
                                   00:00:27
                                   00:00:29
Bands                              00:00:56
                                   00:02:04
                                   00:00:22
                                   00:00:34
Dark dorsal stripe                 00:00:30
                                   00:00:26
Lateral mantle streaks             00:00:38
Shaded oviducal gland              00:00:35
Shaded testis                      00:00:39
Infraocular spot                   00:00:28
Shaded eye                         00:00:05
Fin strip                          00:00:44
Dark arms/head                     00:00:35
Dark arm stripes/tentacle stripes  00:00:52
Lateral mantle spot (f)            00:00:23
Dark fins                          00:00:38
Red accessory nidamental gland     00:00:08

Table 4

Pairwise comparison test for the average time (s) of the expression of
chromatic components between females and males, light and dark, and
among the 19 components observed from 2011 through 2013 for Doryteuthis
plei in the South Brazil Bight

                                                 Observed    Critical
                      Factors                    difference  difference

Females               Males                       59.830      29.110
Light component       Dark component             69.7656      31.272
Clear                 Accentuated oviducalgland  311.958     163.567
Clear                 Arm spots                  183.532     114.425
Clear                 Fin stripe                 337.264     292.921
Clear                 Dark dorsal stripe         183.417     155.506
Clear                 Lateral mantle streaks     211.561     159.289
Clear                 Lateral mantle spot        384.121     304.739
Iridophore splotches  Accentuated oviducalgland  276.297     167.525
Iridophoresplotches   Arm spots                  147.871     120.014
Iridophoresplotches   Fin stripe                 301.604     295.149
Iridophoresplotches   Lateral mantle streaks     175.900     163.350
Iridophoresplotches   Lateral mantle spot        348.460     306.881
Bands                 Accentuated oviducalgland  320.248     174.072
Bands                 Arm spots                  191.822     128.996
Bands                 Fin stripe                 345.554     298.914
Bands                 Dark dorsal stripe         191.706     166.521
Bands                 Lateral mantle streaks     219.850     170.059
Bands                 Lateral mantle spot        392.410     310.504
Infraocular spot      Accentuated oviducalgland  284.110     251.086


                      Factors                    P-value

Females               Males                      <0.05
Light component       Dark component             <0.05
Clear                 Accentuated oviducalgland  <0.05
Clear                 Arm spots                  <0.05
Clear                 Fin stripe                 <0.05
Clear                 Dark dorsal stripe         <0.05
Clear                 Lateral mantle streaks     <0.05
Clear                 Lateral mantle spot        <0.05
Iridophore splotches  Accentuated oviducalgland  <0.05
Iridophoresplotches   Arm spots                  <0.05
Iridophoresplotches   Fin stripe                 <0.05
Iridophoresplotches   Lateral mantle streaks     <0.05
Iridophoresplotches   Lateral mantle spot        <0.05
Bands                 Accentuated oviducalgland  <0.05
Bands                 Arm spots                  <0.05
Bands                 Fin stripe                 <0.05
Bands                 Dark dorsal stripe         <0.05
Bands                 Lateral mantle streaks     <0.05
Bands                 Lateral mantle spot        <0.05
Infraocular spot      Accentuated oviducalgland  <0.05

The observed differences that were higher than a critical difference
are considered statistically significant at (P < 0.05).

Table 5

Types of mating and their characteristics observed for Doryteuthis plei.


Date        Period   Light  T [degrees]C  Sal ppt  % DO  n (f - m)

13/11/2011  Daytime  N      27.2          35.2     81.9   8(4 - 4)
13/11/2011  Daytime  N      27.4          35.2     77.3   8(4 - 4)
13/11/2011  Daytime  N      27.4          35.2     77.3   8(4 - 4)
13/11/2011  Nightly  Y      27.1          35.1     77.4   8(4 - 4)
13/11/2011  Nightly  Y      27.1          35.1     77.4   8(4 - 4)
13/11/2011  Nightly  Y      27.1          35.1     77.4   8(4 - 4)
13/11/2011  Nightly  Y      27.1          35.1     77.4   8(4 - 4)
13/11/2011  Nightly  Y      27.1          35.1     77.4   8(4 - 4)
13/11/2011  Nightly  Y      27.1          35.1     77.4   8(4 - 4)
14/11/2011  Daytime  Y      26.0          35.4     83.2   7(3 - 4)
17/11/2011  Daytime  Y      24.4          35.5     86.1   4(2 - 2)
17/11/2011  Nightly  N      24.4          35.5     86.1   4(2 - 2)
17/11/2011  Nightly  N      24.4          35.5     86.1   4(2 - 2)
16/03/2012  Nightly  N      27.50         35.00    87.6  10(5 - 5)
17/03/2012  Daytime  N      26.70         34.80    93.3  10(5 - 5)
18/03/2012  Nightly  Y      24.90         34.90    95.1   7(5 - 2)
19/03/2012  Daytime  N      24.90         34.70    97.4   6(4 - 2)
19/03/2012  Nightly  Y      25.90         34.70    90.5   6(4 - 2)
19/03/2012  Nightly  N      25.90         34.80    90.1   6(4 - 2)
20/03/2012  Daytime  N      26.30         34.90    87.3   5(3 - 2)
20/03/2012  Nightly  N      27.00         34.80    90.1   5(3 - 2)

                           Mating        Category of
Date        Mating type    duration (s)  mate

13/11/2011  Head-to-head   15            Sneakers
13/11/2011  Head-to-head   25            Sneakers
13/11/2011  Head-to-head   06            Sneakers
13/11/2011  Male-parallel  15            Consort
13/11/2011  Male-parallel  12            Consort
13/11/2011  Head-to-head   13            Sneakers
13/11/2011  Head-to-head   14            Sneakers
13/11/2011  Head-to-head   08            Sneakers
13/11/2011  Head-to-head   09            Sneakers
14/11/2011  Head-to-head   17            Sneakers
17/11/2011  Head-to-head   20            Sneakers
17/11/2011  Male-parallel  10            Consort
17/11/2011  Male-parallel  12            Consort
16/03/2012  Head-to-head   05            Sneakers
17/03/2012  Head-to-head   10            Sneakers
18/03/2012  Head-to-head   11            Sneakers
19/03/2012  Head-to-head   25            Sneakers
19/03/2012  Head-to-head   41            Sneakers
19/03/2012  Head-to-head   37            Sneakers
20/03/2012  Head-to-head   12            Sneakers
20/03/2012  Head-to-head   21            Sneakers

Day period (daytime, nightly), presence of light (yes, Y; no, N),
temperature (T, [degrees]C), salinity (sal, ppt), percentage of
dissolved oxygen (% DO), duration of mating (s), squid in the tank (n),
number of females (f), and number of males (m).
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Article Details
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Author:Postuma, Felippe A.; Gasalla, Maria A.
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:3BRAZ
Date:Oct 1, 2015
Words:11294
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