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Antennular morphology and contribution of aesthetascs in the detection of food-related Compounds in the shrimp palaemon adspersus Rathke, 1837 (Decapoda: Palaemonidae).

Abstract. Shrimp are an essential ecological component of marine ecosystems, and have commercial importance for human consumption and aquaculture. Like other decapod crustaceans, shrimp rely on chemical senses to detect and localize food resources by means of chemosensilla that are located mainly on the cephalothoracic appendages. Using the shrimp Palaemon adspersus, a model organism with omnivorous feeding behavior, we aimed to provide comparative information on the role of aesthetascs, antennular sensilla, and flicking behavior in food detection. To this end, we examined i) the morphology of antennular sensilla by field emission scanning electron microscopy, ii) the shrimp's sensitivity to a number of food-related compounds (amino acids and sugars) by means of whole-animal bioassays, and iii) the contribution of the aesthetasc sensilla to food detection. Our results showed that, aside from the aesthetascs, only three other main morphotypes of setae with chemoreceptive features were present in the antennules, thus accounting for relatively simple sensillar equipment. Nevertheless, we found broad-spectrum sensitivity of the shrimp to a number of amino acids (i.e., isoleucine, leucine, methionine, phenylalanine, glycine, tryptophan, cysteine, and tyrosine) and carbohydrates (trehalose, maltose, cellobiose, and fructose) that was consistent with the omnivorous or scavenging habits of the animal. Although aesthetasc ablation attenuated flicking behavior in a chemical stimulus-independent manner, success in detection and short-range localization of food did not rely on the presence of aesthetasc sensilla. This finding confirms the existence of a non-aesthetasc alternative pathway for feeding, with functional redundancy in simple generalist feeder models such as shrimp.


As essential ecological constituents of marine ecosystems, shrimp play a crucial role in the intermediate trophic chain (Janas and Bruska, 2010; Seitz et al., 2013). The Baltic shrimp Palaemon adspersus Rathke, 1837 (Decapoda: Palaemonidae), a shallow-water (0-6 m) animal, is a common inhabitant of coasts, lagoons, and estuaries distributed from the Baltic Sea, along the western coast of Norway, the Bay of Biscay, and the British Isles, to the Moroccan coast and the Mediterranean Sea, the Adriatic Sea, the Black Sea and northwest Atlantic-waters (Zariquiey Alvarez, 1968; Lapihska and Szaniawska, 2006; Gonzaiez-Ortegon et al., 2015). The Baltic shrimp is an important commercial species, mainly exploited for human consumption and used as live bait for sport fishing (Manent and Abella-Gutierrez, 2006). The species also represents a valuable live food resource in aquaculture (Holthuis, 1980).

Like other decapod crustaceans, shrimp rely on waterborne chemical cues to produce adequate behavioral responses, from orientation to social communication, detection of predators, sex recognition, and localization of suitable habitats and food resources in their environment (Schmidt and Mellon, 2011 ; Thiel and Breithaupt, 2011). Discrimination of the different stimuli is mediated by peripheral chemoreceptors grouped within a varied array of cuticular sensory organs called sensilla, which are located mainly on the cephalothoracic appendages including antennae, maxillipeds (mouthparts), antennules, and pereiopods (walking legs) (Schmidt and Mellon, 2011; Thiel and Breithaupt, 2011). The biramous antennules of decapods, in particular, are considered the primary sensory organs for olfactory chemoreception and exclusively contain, on the outer flagellum, the aesthetasc sensilla, each innervated by hundreds of olfactory receptor neurons (Schmidt and Mellon, 2011 ). They mediate a number of complex, odor-evoked behaviors such as pheromone-mediated courtship, social recognition, agonistic interactions, aggregation, alarm responses (Gleeson, 1982; Johnson and Atema, 2005; Horner et al., 2008; Shabani et al., 2008; Bauer, 2011), associative learning (Steullet et al., 2002), and, of course, food searching (Steullet et al., 2001).

Palaemonid shrimp have been described as opportunistic omnivores, feeding on a wide variety of benthic organisms (crustaceans, polychaetes, and molluscs), organic detritus, and plant material; but their feeding habits may vary appreciably according to changes in life cycle, age, size, and availability and distribution of food resources in the environment (Gue-rao, 1994; Guerao and Ribera. 1996; Jayachandran, 2001; Kuprijanov et al., 2015). Crustaceans possess chemoreceptor neurons (CRNs) that are well tuned to stimuli of main interest: predatory crustaceans are preferentially sensitive to nitrogen-containing compounds (Zimmer-Faust, 1993; Derby and Sorensen, 2008; Schmidt and Mellon, 2011), while herbivores or omnivores are sensitive to carbohydrates (Trott and Robertson, 1984; Rittschof and Buswell, 1989; Corotto and O'Brien, 2002; Corotto et al., 2007; Solari et al., 2015).

A well-known stereotypical behavior exhibited by decapods is antennular flicking, which represents a sort of "sniffing strategy" aimed at better sampling and detecting chemical cues in the water environment (Schmitt and Ache, 1979; Reidenbach et al., 2008). It consists of rapid, alternating downward and upward movements of the aesthetasc-bearing flagellum, resetting the sensitivity of the fast-adapting chemore-ceptors by continuously exposing them to novel aliquots of the odor-containing fluid. In this respect, triggering or enhancing basal antennular flicking is classically considered an indicator of chemical detection in crustaceans (Derby and Atema, 1982; Daniel and Derby, 1991).

This study aimed to provide information on the role of aesthetascs, antennular sensilla, and flicking behavior in food detection of the previously unexplored shrimp Palaemon ad-spersus. The species was chosen because it represents a simple model organism for decapod crustaceans with omnivorous and/or scavenger feeding habits (Guerao and Ribera, 1996). Knowledge of its feeding behavior is useful for comparisons across taxa, making the Baltic shrimp a valuable model system for comparative evolutionary studies.

Using field emission scanning electron microscopy (FESEM), we first investigated the morphology (typology and topology) of cuticular sensilla on both the medial and lateral flagella of the antennules, with special attention paid to the aesthetasc sensilla. Then we evaluated the detection ability of the shrimp towards a set of pure chemical compounds possibly related to food by studying the rate of antennular flicking in a whole-animal behavioral bioassay. To this end. all amino acids and a number of sugars, such as trehalose, cellobiose, maltose, sucrose, glucose, and fructose, were tested. Finally, we examined the specific contribution of the aesthetasc sensilla in the detection and search strategy of some food-related compounds by means of ablation experiments, a procedure that has been used successfully to assess the functions of che-mosensory organs in other crustaceans (Steullet et al., 2001, 2002). Responses of the shrimp to a highly appetitive commercial pellet food, as well as to glycine and maltose, selected on the basis of the stimulating effect revealed in this study, were evaluated before and after aesthetasc ablation.

Materials and Methods

Animal collection and rearing conditions

All experiments were performed on wild, intermolt, non-ovigerous adult female shrimp Palaemon adspersus, measuring about 20 mm in carapace length. Animals were collected using bottom-dragging nets (66 x 33 cm; 5 mm mesh size) at the Santa Gilla Lagoon in Cagliari, southern Sardinia, Italy. in the spring of 2013-2015. They were kept in a tank with natural, aerated seawater (SW) at 18-19 [degrees]C and 34[per thousand] salinity, and a 12 h light: 12 h dark photoperiod. Animals were fed with a commercial pellet food (Shrimp Natural; SERA, Heinsberg, Germany) three times a week. Uneaten food was always removed within 1 h after it was given.

Electron microscopy

Antennules were prepared for observation using field emission scanning electron microscopy (FESEM). Samples were excised from ice-anesthetized animals and immediately fixed with a mixture of glutaraldehyde (1.25%) and paraformaldehyde (1%) in cacodylate buffer 0.15 mol [l.sup.-1], pH 7.2, at room temperature for 24 h. After rinsing (3x15 min) in a phosphate-buffered saline (PBS) solution, specimens were sonicated for 1 min in Triton X-100 1% to remove from the cuticular surface any material that would impede observation of anatomical details. After they were rinsed in PBS (1 x 15 min), specimens were dehydrated through a graded acetone series and subjected to critical point drying with C[O.sub.2]. Finally, antennules were immobilized on aluminum electron microscopy stubs and coated with a 2-nm thickness of platinum, using an Emitech 575 turbo sputtering apparatus (Quorum Technologies, Lewes, UK). Based on its length, each antennule was divided into the lateral and medial flagella, and each flagellum was subsequently separated into three parts. Observations were carried out with a Hitachi S-4000 FESEM (Hitachi, Tokyo, Japan), operated at 15-20 kilovolts (kV). Images were obtained with Quartz PCI ver. 5 software (Quartz Imaging, Vancouver, BC, Canada). Over 300 images at different magnifications were collected by FESEM. Measurements were obtained on the FESEM images with Image Tool ver. 3.0 for Windows (University of Texas Health Science Center in San Antonio).

Behavioral experiments

Animals were individually exposed to the different stimuli in plexiglass tanks (28 cm long x 18 cm wide x 10 cm high; Appendix Fig. A1), containing about 3.5 1 of seawater (SW) (18-19 [degrees]C; water depth ~7 cm), following the procedure adopted by Kreider and Watts (1998) and Solan et al. (2015). An 8 x 18 cm stimulus delivery compartment was separated from the rest of the tank by a rectangular, opaque plexiglass divider, perforated by about 20 evenly spaced, 3-mm-diameter holes. The holes allowed water and test stimuli to move into the shrimp arena. At the beginning of each test, animals were allowed to acclimatize until becoming motionless. Before the stimulus was supplied, the response of each animal to the same aliquot of SW (blank control) was monitored. Stimuli were added to the tank via the stimulus delivery compartment, and each shrimp was allowed 1 min to respond. Trials were video-recorded for later analysis, using a Samsung SMX-F34 (Samsung, Seoul, Korea) color digital camera mounted above the test tank.

The behavioral response was determined by measuring the rate of antennular flicking over a 1 -min interval (flicks/1 min). This time frame was selected on the basis of previous observations on dye diffusion in the experimental tank.

At the end of each stimulation series, the pellet food was tested as a positive control. Shrimp that did not respond to the food were excluded from the data analysis. Animals were not fed for 12 h preceding the experiments.

Aesthetasc ablation

To specifically examine the chemosensory contribution of the aesthetascs in detecting food-related compounds, shrimp were tested before and after selective ablations of the aesthetasc sensilla. For ablations, animals were contained in a pan of fresh SW that was deep enough to cover the animal, and each an-tennule was held in position by a staple pin in such a way that the aesthetasc tufts were easily visible and accessible. Ablations were performed on both lateral flagella using sharptipped forceps under a dissecting microscope, a process that took about 15-20 min. Excision of aesthetascs removed the dendrites of the sensory cells, which may cause unresponsiveness to odors, death and degeneration of the sensory neurons (Harrison et al., 2004). Tests were performed before ablation and were then repeated on the same animals within 2-3 days after aesthetasc removal, to avoid any possible recovery of sensitivity by these sensilla or compensating increase in sensitivity of other sensilla (Hazlett, 1971; Harrison et al., 2004).

Stimuli and supply protocol

The compounds of potential feeding significance--most of which are known to elicit responses from chemoreceptor neurons (CRNs) in other crustacean species (Derby and Atema, 1988; Steullet et al. 2002; Schmidt and Mellon, 2011 ; Solari et al., 2015)--that were tested as stimuli on intact shrimp included the series of 20 amino acids (L-isomer); the disaccharides, trehalose, cellobiose, maltose, and sucrose; and the monosaccharides, glucose and fructose, for a total of 26 compounds. (Amino acids are hereafter abbreviated according to the rules of the International Union of Pure and Applied Chemistry-International Union of Biochemistry [IUPAC-IUB Commission on Biochemical Nomenclature, 1972]). All but the insoluble test chemicals (Sigma-Aldrich, Milan, Italy) were first dissolved in SW at [10.sup.-1] mol [1.sup.-1]. The insoluble compounds such as tryptophan (Trp), cysteine (Cys), tyrosine (Tyr), aspartate (Asp), and glutamate (Glu), were dissolved in SW at [10.sup.-4] mol [1.sup.-1]. Chemicals were then stored frozen as stock solutions.

On the day of the experiments, stock solutions were thawed and serially diluted in SW to be used at three different concentrations: [10.sup.-5], [10.sup.-3], and [10.sup.-l] mol [1.sup.-1] (Trp, Cys, Tyr, Asp, and Glu were diluted at [10.sup.-8], [10.sup.-6], and 1([10.sup.-4] mol [1.sup.-1] concentrations), and, in all cases, were supplied at increasing steps. Ten-ml aliquots of each compound were supplied in about one liter of water contained in the stimulus delivery compartment, where complete mixing was achieved by agitation with a magnetic stir plate, in accordance with Solari et al. (2015). Chemicals thus rapidly reached their final concentrations of [10.sup.-7], [10.sup.-5], and [10.sup.-3] mol [1.sup.-1] ([10.sup.-10], [10.sup.-8], and [10.sup.-6] mol [1.sup.-1] for Trp, Cys, Tyr, Asp, and Glu) and diffused through the holes into the shrimp arena, where they underwent a further dilution up to 1:5. Owing to the procedure adopted for stimulus supply, actual concentrations to which shrimp were exposed and to which they responded likely may have been less than those indicated, that is, the concentration diffusing from the delivery compartment to the shrimp arena. The stirring device was already operating before the shrimp were introduced, and continued for the duration of the experiment.

Therefore, in the present study we used a static system in such a way that each shrimp, after acclimatization, was exposed to a blank stimulation and then to three different increasing concentrations of a same compound, separated by inter-stimulus intervals of at least 3 min. During this stimulation sequence, the water was not replaced (i.e., step-wise stimulations were used, according to Solari et al., 2015).

Each shrimp was supplied with only one chemical. Behavioral assays were performed on 9-15 shrimps for each concentration of amino acids and 9-10 shrimp for each concentration of sugars.

In the aesthetasc ablation experiments, commercial pellet food (n = 11 shrimp), Gly, and maltose (n = 12 shrimp) were used as stimuli. The latter two compounds were selected on the basis of the stimulating effectiveness revealed in the present study, and were tested at [10.sup.-1] mol [1.sup.-1] with the aforementioned procedure. Food was supplied by inserting one pellet (~8 mg in weight) in the stimulus delivery compartment.

In the case of the food, other parameters such as the time it took the shrimp to find the pellet, and the flicking frequency during both the pellet search and the feeding, were also evaluated by simply supplying a food pellet directly in the animal arena.

Data analysis

The effects of the different concentrations of the tested amino acids and sugars on the antennular flicking activity of female shrimp were evaluated by repeated measures ANOVA. For each chemical, post hoc comparisons were performed using Dunnett's test to assess significant differences (P < 0.05) between each stimulus concentration and the relative blank (control) mean. Fisher's least significant difference (LSD) test was used when evaluating significant differences (P < 0.05) between consecutive stimulus concentrations.

Two-way repeated measures ANOVA was also used to analyze differences in the responses of shrimp to food, Gly, and maltose before and after aesthetasc ablation, separately for each chemical stimulus (fixed factor: stimuli [2 levels]; repeated measures factor: ablation [2 levels]). In this case, post hoc comparisons were conducted with the Tukey test, and P-values < 0.05 were considered significant.

Data were checked for assumptions of homogeneity of variance, normality, and sphericity (when applicable). When the assumption of homogeneity of variance was violated, Duncan's test was used for post hoc comparisons (Sollai et al., 2014).

The paired Student's t test with a 95% confidence level (P < 0.05) was used to assess significant differences between intact and aesthetasc-ablated animals in food search time and flicking activity, during both the food search and feeding after the food pellet was found.

Statistical analyses were carried out using STATISTICA for WINDOWS (ver. 7; StatSoft, Tulsa, OK).


Morphology of antennules

The antennules of the Baltic shrimp Palaemon adspersus are sensory organs comprising two slender, filiform flagella, lateral and medial, both of which are implanted on a common rigid peduncle. Each flagellum contains a high number of segments, or annuli, that provide the entire organ with the necessary articulation for flexibility.

The medial flagellum is approximately 16.6[+ or -]0.3 mm long and consists of 70-72 segments that are short and stubby in the proximal third and longer and thinner in the distal portion (Fig. 1 A, B). The medial flagellum houses sensory setae from different non-aesthetasc morphotypes. These setae vary in length, diameter, and robustness, and are hereafter referred to as type 1, type 2, and type 3 sensilla. We found 18-20 robust, long, type 1 sensilla (Fig. 1C, D) that were confined to a number of segments in the basal third of this flagellum. There are two sensilla per segment, each arising from a socket and projecting dorsally at a fixed angle ahead with respect to the flagellar shaft. These setae are also the largest antennular sensilla in both base diameter (36 [+ or -] 3.8 [micro]m) and length (350-400 [micro]m, maximum value 712 [micro]m). They show a marked asymmetry; the setae that protrude toward the animal's midline are larger than those located on the opposite side. They taper gradually to a curved, fluted, beak-like tip, where an apical pore with distinctive chemoreceptive features is present (Fig. 1D). They also appear distally ornamented by cuticular imbrications or irregularities similar to flattened, scale-like segments. Type 2 sensilla (Fig. 1C,E) are much shorter (33.5 [+ or -]2.1 [micro]m) and thinner (base diameter: 3.2 [+ or -] 0.2 [micro]m) than the type 1, even though they also resemble the type 1 sensilla in their curved tip with a fluted, beak-like opening and distal cuticular imbrications. Type 3 sensilla from the medial flagellum are simple setae of smooth appearance and clearly show a rounded apical pore (Fig. 1F, and inset). Type 3 sensilla are 35.6 [+ or -] 0.5 [micro]m long and have a base diameter of 2.4 [+ or -] 0.1 [micro]m. Interestingly, types 2 and 3 sensilla are scattered, featuring a unilateral or bilateral symmetry over the entire flagellum surface, singly or in groups of 3 to 6 sensilla emerging from common sockets.

The lateral flagellum of the antennule (Fig. 2A, B) is comprised of up to 95-98 segments, giving the flagellum a length of about 19.8 [+ or -] 2.5 mm, and shows sensillar equipment comparable in both morphotype and distribution to that of the medial flagellum. The only exception is that it lacks long type 1 sensilla.

A prominent feature of the lateral flagellum is the presence of high-density tufts of aesthetasc sensilla (Fig. 2C-F) arising from a groove in a short, medial collateral branch of the flagellum (about 21-22 segments, 2.5 [+ or -] 0.1 mm in length). Aesthetascs are distributed in multiple rows (Fig. 2C), two rows per segment (one row in the distal margin and the second in the middle of the segment), for a maximum of 40-42 rows, and contain 4-6 sensilla projecting ventrally. Aesthetascs are cylindrical and thin, gradually tapering to a blunt tip that lacks the apical pore (Fig. 2D). Aesthetascs vary in length, depending on their location on the flagellum (up to400 [micro]m long and 15 [micro]m in base diameter), and show a proximal part formed by a few cylindrical elements that are more rigid than the distal ones. Moreover, the distal two thirds of the sensillum has a thin cuticle (0.4-1 [micro]m; Fig. 2E). An interesting feature of the tuft region is the presence of small pores (mean diameter 0.9 [+ or -] 0.1 [micro]m) that open in the canal-like systems just surrounding the groove from which arise the aesthetascs in the same row (Fig. 2F).


Antennular flicking response to food-related compounds

After acclimatization in the experimental tank, animals became virtually motionless. In the absence of stimulation, they displayed only a basal level of antennular flicking. However, in the presence of a stimulating compound, shrimp suddenly started a stereotyped sniffing and search strategy, first characterized by an increase in flicking rate and then associated with a rapid walking phase in the animal arena. These activities mostly culminated in prolonged inspections of the perforated divider in an attempt to access the stimulus delivery compartment from which the shrimp sensed the stimulus was spreading.

A slight decrease in antennular flicking was usually observed during the walking phase, as if the rapid up and down movements of the aesthetasc-bearing flagellum, necessary for resetting chemoreceptor sensitivity, were somehow compensated by the movements of the animal. However, this complex walking pattern was erratic and irregular in most cases, and therefore hard to measure. Mean values [+ or -] SEM of antennular flicking activity in response to the tested compounds at the different concentrations are given in Figures 3 and 4. Specifically, repeated measures ANOVA showed that the amino acids, Ile, Leu, Met, Phe, Gly, Trp, Cys, and Tyr evoked a stimulating effect on the flicking rate (Table 1 ; Fig. 3), although to different extents. Dunnett's post hoc comparisons revealed that Ile, Phe, and Gly were stimulating, compared to the blank controls, at any tested concentrations within the range of [10.sup.-7] to [10.sup.-3] mol [1.sup.-1]; Cys and Met were effective at the two highest concentrations ([10.sup.-8] and [10.sup.-6] mol [1.sup.-1] for Cys; [10.sup.-5] and [10.sup.-3] mol [1.sup.-1] for Met), and Trp was effective only at the highest concentration ([10.sup.-6] mol [1.sup.-1]). Conversely, Leu and Tyr were stimulating only at the lowest concentration ([10.sup.-7] and [10.sup.-10] mol [1.sup.-1], respectively); at higher concentration, flicking rate at both compounds declined to blank levels. All of the other amino acids failed to cause an increase or decrease in blank flicking activity.


Repeated measures ANOVA also showed that among the tested sugars, only the disaccharides, trehalose, maltose, cel-lobiose, and the monosaccharide fructose affected the blank flicking rate (Table 1 ; Fig. 4). As revealed by Dunnett's post hoc comparisons, both maltose and fructose were stimulatory at all concentrations ([10.sup.-7] to [10.sup.-3] mol [1.sup.-1]), cellobiose at the two highest concentrations ([10.sup.-5] and [10.sup.-3] mol [1.sup.-1]), and trehalose only at the highest concentration ([10.sup.-3] mol [1.sup.-1]). No significant changes in flicking rate were elicited when animals were presented with sucrose and glucose, regardless of the concentration used.

Finally, post hoc comparisons also revealed, for each chemical, no concentration-response relationships among consecutive tested concentrations.

Effects of aesthetasc ablation on detection and search for food-related compounds

To better ascertain the specific contribution made by aesthetasc sensilla in detecting food-related compounds, shrimp were tested with the commercial pellet food, Gly, and maltose before and after aesthetasc ablation. Gly and maltose were used because they were the most stimulatory amino acid and sugar, respectively.


Two-way repeated measures ANOVA showed no interaction between the two repeated factors, ablation and chemical stimulus, on flicking response; and this was seen in all tested compounds: food ([F.sub.[1.20]] = 0.0043, P = 0.9479; Fig. 5A), Gly ([F.sub.[1.22] = 0.1016, P = 0.7528; Fig. 5B), and maltose ([F.sub.[1.22]] = 0.0423, P = 0.8387; Fig. 5C). This finding suggests that aesthetasc ablation causes a stimulus-independent decrease in flicking. In fact, following ablation there was a significant reduction in response to all three stimuli, resulting in no difference between pre-ablation blank and post-ablation stimulus; this was also shown in post hoc comparisons. However, despite the drop in flick rate there was still a significant response to post-ablation stimuli compared to post-ablation blank response. This finding suggests that chemosensory-driven flicking activity may be sustained by non-aesthetasc sensilla.

When the food was supplied directly within the animal arena (Fig. 6). the time to locate it did not differ before or after ablation of aesthetascs. This was in spite of the fact that during the search time the ablated animals showed less flicking activity than before ablation.

Finally, once the shrimp found the food pellet they started eating and simultaneously continued flicking the antennules. In this phase, intact animals showed a flicking rate that was higher than that of ablated animals.


Thus, aesthetasc-deprived shrimp showed a reduction in the rate of spontaneous flicking that increased after stimulus presentation. Conversely, a lack of aesthetascs affected neither detection capability nor the time needed for the shrimp to find the stimulus source, at least in the confined experimental environment of this study.


Using the Baltic shrimp Palaemon adspersus as a model shrimp with omnivorous habits, the present morpho-functional and behavioral investigation extends the knowledge of the che-moreceptive features of decapod crustaceans by providing information on the role in food detection of aesthetascs, antennular sensilla, and flicking behavior.

Morphology of the antennules

Our morphological observations revealed the presence of three main, non-aesthetasc morphotypes of setae--in addition to the aesthetasc sensilla--in the antennules. Although variations in general morphology, numbers, and distribution of sensilla within decapod crustaceans are high (Hallberg et al., 1997), the sensillar equipment in the antennules of P. adspersus may be considered relatively simple, from an evolutionary point of view, especially when compared to the complex repertoire of up to 6-10 different sensillum morphotypes described in snapping shrimp (Obermeier and Schmitz, 2004), crayfish (Mellon, 2012), and spiny lobsters (Cate and Derby, 2001). In this respect, the antennules of the shrimp appear similar in their general organization to those of Lysmata shrimp, in which only four types of sensilla were identified (Zhang et al., 2008; Zhu et al., 2011). Because several earlier proposed classification schemes for crustacean sensilla often led to confusion in terminology for small species-specific differences or slight variants in morphotypes (Garni, 2004; see Wortham et al., 2014 for a review), in this study we have conservatively referred to non-aesthetasc sensilla as type 1, type 2, and type 3 sensilla.

Although the type 1 sensillum morphotype is confined to the medial flagellum and is present in a very low number (18-20), it appears, in size and distribution, to be unique to P. adspersus and possibly other palaemonid shrimp; no counterpart has been found in the literature on crustaceans. In fact, based on the type 1 sensillum's exceptional length of up to 600-700 [micro]m, especially for those sensilla located along the medial margin of the flagellum, and for their two-per-segment occurrence, the type 1 sensilla only remotely resemble the long, simple setae described in spiny lobsters (Cate and Derby, 2001). However, simple setae are usually smooth (Cate and Derby, 2001 ; Wortham et al., 2014) and free of microstructures such as the cuticular imbrications and the flattened scale-like segments that we observed in the distal endings of these sensilla. For these characteristics and for their curved, fluted, beaklike tip, the type 1 sensilla are also similar to the denticulate setae described in other decapod shrimp (Bauer and Caskey, 2006), which are much shorter and can be compared to the type 2 sensilla found in large numbers on both flagella. With their curved-beaked tip, the type 1 and type 2 sensilla could also be considered a variant of the long- and short-beaked sensilla of crayfish (Mellon, 2012).


The smooth type 3 sensilla are comparable to the basic morphotype of simple setae that are classically described in crustaceans (Cate and Derby, 2001; Wortham et al., 2014). Interestingly, they appear singly or in mixed groups with type 2 sensilla, as reported in other palaemonids (Bauer and Caskey, 2006). The function of these non-aesthetasc sensilla is still unclear, but they are likely chemosensory, as suggested by the presence--in all three morphotypes--of the apical pore, which is commonly regarded as distinctive of chemoreceptive features (Derby, 1989). These sensilla may also act as bimodal (chemo-mechanoreceptor) sensilla to integrate mechanical and/or hydrodynamic inputs with chemical cues for higher resolution of temporal properties of stimuli and better orientation to the odor source, similar to what has been reported for spiny lobsters and other crustaceans (Cate and Derby. 2001; Mellon, 2007; Schmidt and Mellon, 2011 ).

Finally, our FESEM inspection revealed that the shrimp's aesthetascs, which are densely and exclusively located on the lateral flagellum, resemble the general structure reported in other crustaceans (Hallberg et al., 1997): they are distributed in discrete rows, lack any apical pore, and have a thin cuticle (0.4-1 [micro]m). Thus, the aesthetascs of shrimp may also act as a selective molecular sieve through which appropriate odorants move quickly to enable a prompt response by aesthetasc ol-factory neurons, as has been reported in spiny lobsters (Derby et al., 1997).


Antennular flicking response to food-related compounds

Our behavioral results highlight a broad-spectrum ability of the shrimp to detect compounds related to food, such as amino acids and sugars. The amino acids, Ile, Leu, Met. Phe, Gly, Trp, Cys, and Tyr; the disaccharides, maltose, cellobiose, and trehalose; and the monosaccharide, fructose, all enhanced the antennular flicking response, or the "sniffing strategy," for better sampling and detecting of chemical stimuli in the surrounding water (Schmitt and Ache, 1979; Reidenbach et al., 2008). The sensitivity of the shrimp to amino acids is similar to that of other carnivorous crustaceans (Derby and Sorensen, 2008) that are highly sensitive to small, nitrogen-containing compounds, such as amino acids, amines, nucleotides, and peptides. Such compounds are usually prevalent in the tissues of their animal prey and thus represent good-quality food indicators (Zimmer-Faust. 1993; Schmidt and Mellon, 2011 ). Noteworthy is the observed sensitivity to a number of sugars associated with plants (Ljungdahl and Eriksson. 1985; Mohr and Schopfer. 1995). To date, sensitivity to carbohydrates had been reported in just a few crustaceans with herbivorous or omnivorous feeding habits, mainly crabs (Hartman and Hartman, 1977; Trott and Robertson, 1984; Rittschof and Buswell, 1989) and crayfish (Corotto and O'Brien, 2002; Corotto et al., 2007; Solari et al., 2015). In this context, trehalose may represent an anomalous carbohydrate. Although chemically it belongs to the class of sugars, for the shrimp it represents an indicator of a protein diet; it is a common hemolymph sugar in the body fluid of many invertebrates (Fairbairn, 1958). Such broad sensitivity to different classes of compounds is consistent with the opportunistic omnivorous and scavenging feeding habits of palaemonid shrimp (Guerao and Ribera, 1996).

Overall, our data show that a relatively simple organization of the antennular chemosensory system can provide wide coverage of chemical sensitivity for omnivorous crustaceans. The lack of any concentration-response relationship observed during chemical stimulations also suggests that the sensitivity of the shrimp may not allow resolution of concentration differences as great as 100-fold step concentration increases for the same compound, at least under the experimental conditions (step-wise stimulations) that we adopted. Therefore, the che-moreceptive apparatus may act as a detector of relative rather than absolute intensity of a stimulus by resetting the response threshold to a zero-level in the presence of constant background chemical noise, similar to lobsters (Borroni and Atema, 1988). In other words, this sensory mechanism may be somewhat conserved within decapods.

More investigations are needed to discover whether shrimp are sensitive to a potentially wider range of stimulants. It is known that differences and/or plastic rearrangement in sensitivity may reflect the availability and distribution of food resources in a given habitat. Overall sensitivity can also vary appreciably, depending on changes in life cycle, age, and size of the animal (Guerao, 1994; Guerao and Ribera, 1996).

Effects of aesthetasc ablation on detection and search for food-related compounds

Our results showed that in the shrimp Palaemon adspersus, the lack of aesthetasc sensilla in the lateral flagellum affected neither detection nor searching behavior and short-range localization of food. Their absence attenuated the flicking rate of the antennules. but in a chemical stimulus-independent manner.

These conclusions are supported by the fact that after aesthetasc ablation, the shrimp can still find food, at least under the confined environmental conditions of this study, in the same duration as before ablation. This was true in spite of the fact that a loss of aesthetascs causes a proportional reduction of the flicking rate in both the absence and presence of food or the food-related compounds, Gly and maltose. The former finding concurs with what was previously reported in other decapods such as lobsters, where the aesthetascs allow food odor discrimination, searching, and localization. However, all of these behaviors are also elicited in the absence of aesthetascs, provided that the non-aesthetasc antennular sensilla are intact (Steullet et al., 2001, 2002; Horner et al., 2004). In these animals, such chemosensory information is conveyed to the central nervous system through two anatomically distinct but parallel pathways: the aesthetascolfactory lobe pathway and the non-aesthetasc-lateral antennular neuropil pathway, which are reciprocally connected by local olfactory interneurons (Schmidt et al., 1992; Schmidt and Ache. 1992, 1996a, b). Therefore, the existence of these two antennular pathways for feeding with functional redundancy also may occur in shrimp.

Yet the fact that aesthetasc ablation led to stimulus-independent attenuation of flicking suggests that the chemosensory-driven flicking behavior is sustained exclusively by the non-aesthetasc sensilla. This represents a striking difference between Palaemon adspersus and the spiny lobster, in which flicking behavior towards the chemical stimuli was attenuated completely by ablation of aesthetascs (Daniel et al., 2008). More surprising was the finding that a loss of aesthetascs decreased the flicking rate in the absence of chemical stimulation. This suggests that non-chemical flicking is sustained by the aesthetasc input only partly, and that the integrity of either the aesthetasc or non-aesthetasc pathways is required. In this respect, flicking as a "sniffing strategy" aimed at better sampling and detection of chemicals in the water environment (Derby and Atema, 1982; Daniel and Derby, 1991) is also modulated by hydrodynamic inputs arising from antennular mechanoreceptors (Mellon and Reidenbach. 2011). However, to date, aesthetascs in crustaceans, unlike the bimodal (chemical and mechanical) sensilla present on the antennules, have always been reported to be unimodal; that is, they are exclusively sensors for chemical detection (Steullet et al., 2000). Thus, there is no apparent reason for the lack of aesthetascs to have played any part in the observed reduction in spontaneous flicking--in the absence of stimulation, that is. We hypothesize that aesthetascs are involved in the non-chemosensory circuitry and sustain, at least in part, the basal tonus of the flicking behavior. Further investigation is needed to test this hypothesis.


In this study the Baltic shrimp Palaemon adspersus proved to be a reliable, simple model organism with omnivorous and scavenging feeding habits, useful in providing information about the role of aesthetascs, antennular sensilla, and flicking behavior in food detection. Interestingly, despite the relatively simple sensillar equipment of the antennules, broad-spectrum sensitivity of the shrimp to a number of amino acids and carbohydrates was present, providing wide coverage of chemical sensitivity consistent with its feeding habits.

On the other hand, as we noted, success in detection and short-range localization of food is not strictly dependent on the presence of the aesthetasc sensilla. This finding confirms that a non-aesthetasc alternative pathway, having a functional redundancy with respect to the aesthetasc pathway, is also conserved in simple generalist feeder models like shrimp, similar to carnivorous decapods such as spiny lobsters. Likewise, in shrimp and spiny lobsters, the chemoreceptive apparatus may act as a detector of relative, not absolute, intensity of a stimulus, a sensory mechanism that is somewhat conserved within decapods.

Knowledge of feeding behavior and of the underlying sensory mechanisms in shrimp may be useful for comparative studies across taxa, making the shrimp a valuable model system for the study of other decapods for which similar studies are difficult, if not impossible, such as the deep-sea species.


We are grateful to Dr. Marco Metis, Department of Biomedical Sciences. University of Cagliari, for technical assistance, and Dr. David Nilson for improving the English text. This work was supported by the Fondazione Banco di Sardegna, Italy (Grant no. 2005/2011.1083). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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(1) Department of Biomedical Sciences, Section of Physiology, University of Cagliari, University Campus, S.P. 8, 09042 Monserrato (CA), Italy; (2) Department of Biomedical Sciences, Section of Cytomorphology, University of Cagliari, University Campus, S.P. 8, 09042 Monserrato (CA), Italy; and (3) Department of Life and Environmental Sciences, University of Cagliari, Via Fiorelli I, 09126 Cagliari (CA), Italy Received 12 January 2017; Accepted 30 March 2017; Published online 30 May 2017.

(*) To whom correspondence should be addressed. E-mail:
Table 1
Summary of repeated measures A NO VA on the frequency of antennular
flicking during stimulation with increasing concentrations of different
amino acids and sugars tested in the intact shrimp bioassay

             Compound    F-value                   P-value

Amino acids  Ala         [F.sub.[3.24]] =1.4674    0.2484
             Ile         [F.sub.[3.39]] = 3.2868   0.0306 (*)
             Leu         [F.sub.[3.27]]  = 3.0895  0.0438 (*)
             Met         [F.sub.[3,24]] = 9.0333   0.0003 (*)
             Phe         [F.sub.[3,30]] = 6.8629   0.0011 (*)
             Pro         [F.sub.[3,27]] = 0.7326   0.5416
             Val         [F.sub.[3,30]] = 0.5138   0.6758
             Asn         [F.sub.[3,27]] = 1.3991   0.2645
             Gln         [F.sub.[3,24]] = 0.3629   0.7803
             Gly         [F.sub.[3,27]] = 3.1457   0.0413 (*)
             Ser         [F.sub.[3,24]] = 2.1764   0.1170
             Thr         [F.sub.[3,24]] = 1.1794   0.3383
             Arg         [F.sub.[3,24]]  = 0.5954  0.6241
             His         [F.sub.[3,33]] = 0.3744   0.7719
             Lys         [F.sub.[3,27]]  = 0.6835  0.5698
             Trp         [F.sub.[3,24]] = 6.4299   0.0023 (*)
             Cys         [F.sub.[3,21]] = 10.943   0.0000 (*)
             Tyr         [F.sub.[3,39]]  = 4.4303  0.0089 (*)
             Asp         [F.sub.[3,30]] = 0.5481   0.6532
             Glu         [F.sub.[3,33]] = 1.4574   0.2440
Sugars       Trehalose   [F.sub.[3,27]] = 3.2208   0.0383 (*)
             Maltose     [F.sub.[3,27]]  = 8.0787  0.0005 (*)
             Cellobiose  [F.sub.[3,24]]  = 14.285  0.0000 (*)
             Sucrose     [F.sub.[3,24]] = 0.8066   0.5025
             Glucose     [F.sub.[3,27]] = 1.2814   0.3007
             Fructose    [F.sub.[3,27]] = 6.4997   0.0018 (*)

(*) Significant values (P < 0.05).
Amino acids: Ala, alanine: Arg, arginine; Asn, asparagine; Asp,
aspartate; Cys, cysteine; Gln. glutamine: Glu, gutamate; Gly, glycine:
His, histidine: Ile, Iso-leucine; Leu, leucine; Lys, lysine; Met,
methionine; Phe, phenylalanine; Pro, proline: Ser, serine: Thr,
threonine; Trp, tryptophan; Tyr. tyrosine; Val. valine.
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Author:Solari, Paolo; Sollai, Giorgia; Masala, Carla; Loy, Francesco; Palmas, Francesco; Sabatini, Andrea;
Publication:The Biological Bulletin
Date:Apr 1, 2017
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