Shape, colour plasticity, and habitat use indicate morph-specific camouflage strategies in a marine shrimp

Rafael C Duarte
Martin Stevens
Augusto A V Flores
Shape, colour plasticity, and habitat use indicate morph-specific camouflage strategies in a marine shrimp
10.1186/s12862-016-0796-8R E S E A R C H A R T I C L E Open Access
Background: Colour and shape polymorphisms are important features of many species and may allow individuals to exploit a wider array of habitats, including through behavioural differences among morphs. In addition, differences among individuals in behaviour and morphology may reflect different strategies, for example utilising different approaches to camouflage. Hippolyte obliquimanus is a small shrimp species inhabiting different shallowwater vegetated habitats. Populations comprise two main morphs: homogeneous shrimp of variable colour (H) and transparent individuals with coloured stripes (ST). These morphs follow different distribution patterns between their main algal habitats; the brown weed Sargassum furcatum and the pink-red weed Galaxaura marginata. In this study, we first investigated morph-specific colour change and habitat selection, as mechanisms underlying camouflage and spatial distribution patterns in nature. Then, we examined habitat fidelity, mobility, and morphological traits, further indicating patterns of habitat use. Results: H shrimp are capable of changing colour in just a few days towards their algal background, achieving better concealment in the more marginal, and less preferred, red weed habitat. Furthermore, laboratory trials showed that habitat fidelity is higher for H shrimp, whereas swimming activity is higher for the ST morph, aligned to morphological evidence indicating these two morphs comprise a more benthic (H) and a more pelagic (ST) life-style, respectively. Conclusions: Results suggest that H shrimp utilise a camouflage strategy specialised to a limited number of backgrounds at any one time, whereas ST individuals comprise a phenotype with more generalist camouflage (transparency) linked to a more generalist background utilisation. The coexistence within a population of distinct morphotypes with apparently alternative strategies of habitat use and camouflage may reflect differential responses to substantial seasonal changes in macroalgal cover. Our findings also demonstrate how colour change, behaviour, morphology, and background use all interact in achieving camouflage.

Background

Polymorphism is a common trait in many animal taxa [1,2] and has been a subject of numerous empirical studies testing several evolutionary theories and hypotheses (e.g. [3][4][5]). Aside from facilitating the exploitation of a wider array of habitats [6][7][8][9], polymorphism may also involve a segregation of behavioural traits among morphs, such as related to differences in mating tactics [10,11] or habitat use [12,13]. Morph-specific morphological and behavioural traits can allow individuals to more efficiently gather resources and exploit different niches through the diversification and specialisation of life-history strategies [6,14,15]. Identifying the selective forces responsible for the origins and maintenance of morphs, and unravelling their relative advantages, are important tasks in order to predict population dynamics in varying environments and for understanding evolutionary and developmental strategies [2,16,17].

One of the most longstanding areas where colour and shape polymorphisms have been studied in nature relates to camouflage [4,8,[18][19][20]. Habitat-specific camouflage of colour morphs may be obtained via a number of mechanisms, whereby behavioural and morphological traits of individuals can interact with environmental characteristics to reduce their relative risk of predation [21][22][23]. For instance, individual appearance for camouflage can be either attained through genetic polymorphism [24,25], or through colour change and phenotypic plasticity [26][27][28]. In addition to changes in appearance, camouflage can also be driven by the behavioural preferences of individuals to rest on backgrounds that provide enhanced camouflage [23,29,30]. Evidence of morph-specific behavioural preferences for substrate types has been observed in a variety of taxa, including moths [31], grasshoppers [32] and crabs [7], and we would expect this to be common if morphs have evolved under selection for camouflage against different substrates. Therefore, camouflage in polymorphic species should be driven by both colour change in line with the predominant visual background, and behavioural preferences for individuals to rest on backgrounds that they match.

The degree to which different morphs can exploit alternative (micro) habitats should depend on how effectively individuals can conceal themselves against the background. Therefore, for species living in heterogeneous substrates, different morphs may be effectively concealed in microhabitats with different background colour patterns within the same general environment [17,33,34]. In this case, predation by visual consumers may drive disruptive selection leading to individuals specializing in each of the available backgrounds [35,36], and/or the ability of some individuals to change colour depending on the patch type they live on [26,37,38]. On the other hand, a more generalist fixed strategy may be favoured when optimal colouration is achieved by a compromise in the degree of crypsis obtained in different microhabitats while matching no background very closely [33,39], or through camouflage types that are less restricted to one background type alone (e.g. transparency).

Differential coloration and camouflage strategies may evolve together with both morphological and behavioural traits in polymorphic species [21,32]. For example, colour patterns in Midas cichlid fish are correlated to both body shape and life-style, with golden deeper bodied fish mostly associated to the benthic habitat, and dark slender individuals exhibiting a more limnetic life-style [15]. Also, Dalmatian wall lizards comprise three different colour morphs, with different body and relative head size, which relate to morph-specific trophic niches and cross-habitat distributions [40]. Theory also predicts that morphs with a specialist camouflage strategy would concentrate in habitat patches where concealment is most efficient, increasing substrate fidelity and lowering predation risk [32,41]. Active preference for these patches may lead to exceptionally high population densities, only constrained by habitat carrying capacity [42], favouring high intra-specific competition, with some individuals being displaced to marginal habitat patches [43]. Alternatively, for individuals with a generalist strategy, in which camouflage is less constrained to a limited number of backgrounds, selection may favour a more opportunistic life-style with individuals possessing differential morphology and behaviour [17,44]. A generalist life-style with lower habitat fidelity and increased mobility may allow individuals to reduce competitive interactions and facilitate more efficient resource exploitation and mate searching [45,46]. Strong specialization, coupled to habitat fidelity, and high mobility associated to a more opportunistic use of resources, can be found in different morphs within populations, and their coexistence is apparently mediated by environmental conditions dictating relative fitness of individuals at different frequencies [47,48].

The shrimp Hippolyte obliquimanus is a small gonochoric and polymorphic species [49], very abundant in algal meadows composed of Sargassum furcatum (hereafter Sargassum) and Galaxaura marginata (from now on Galaxaura), the dominant algal species of shallow rocky substrates in the northern coast of São Paulo State, Brazil [9]. Shrimp populations comprise distinct morphs, all belonging to the same species [50], which can be classified as (i) homogeneous individuals (H) with different coloration, most being greenish-brown (H GB ) or pink (H P ), and (ii) striped translucent shrimp (ST), with either longitudinal or transversal colour bands (Fig. 1). Homogeneous shrimp are visually well concealed in both the brown alga Sargassum (H GB ) and the reddish-pink seaweed Galaxaura (H P ), while ST individuals, although found in these same habitats, exhibit less background-specific coloration via the use of transparency (Fig. 1).

The natural distribution of H. obliquimanus individuals between algal habitats is clearly morph-specific [9]. H individuals tend to occupy colour-matching substrates, i.e. greenish-brown shrimp are more abundant in Sargassum, while pink individuals in Galaxaura, and ST shrimp are equally distributed between these macroalgae [9]. While over a period of days and weeks H shrimp may be able to change colour to different substrate types (see below), at any one time they should be restricted to one matching background type alone, and hence we consider them background specialists (but note that over time they may be considered generalists). Mismatching shrimp, i.e. H GB in Galaxaura or H P in Sargassum, are very probably individuals that arrived from a different habitat and had not yet adjusted to local background. In contrast, ST individuals may adopt more generalist background choice behaviour and a camouflage type (transparency) that allows concealment to a range of substrate types. Sex proportions are also different between morphs, with H shrimp being chiefly females and ST mostly males, suggesting that selection for sex-specific traits may be also important in explaining the maintenance of polymorphism in this species [9]. Morph-specific habitat and sex distribution may indicate the existence of behavioural differences between morphotypes [32,51], possibly related to contrasting strategies of habitat use. In the case of H. obliquimanus, cryptic behaviour is expected to be selected in H shrimp, with individuals remaining on colour-matching backgrounds, and a more general life-style is anticipated for transparent ST individuals, which would move more frequently among different substrate types.

In this study, we used a combination of laboratory manipulative experiments, supported by geometric morphometric analyses, to test the hypothesis that colour morphs of H. obliquimanus differ in specific behavioural traits and morphology related to strategies of camouflage and habitat use (namely Sargassum and Galaxaura canopy). We first examined two potential mechanisms by which individuals can enhance crypsis: habitat selection and colour change. We undertook experiments of behavioural habitat selection to test whether morphs actively select the backgroundmatching macroalgal habitat where concealment is more effective. Then, we performed a colour change experiment to investigate if the capacity of colour change differs between morphs and habitats. Because carapace shape can be a proxy for life-style and habitat use in caridean shrimps, with stout forms being an indicative of benthic life-style and more streamlined shapes of a more pelagic behaviour [52,53], we used geometric morphometric analyses and carried out experiments of habitat use to verify whether morphological evidence correlates with behavioural patterns. Together, the results of this study evidenced a link among coloration, morphological, and behavioural traits, illustrating how polymorphism can be advantageous to individuals achieve different camouflage strategies when living in a heterogeneous habitat.

Methods

General procedures

Samples of the macroalgae Sargassum and Galaxaura were collected during the summer and autumn of 2011, 2013 and 2015 by skin diving at rocky bottoms in different sites along the São Sebastião Channel (23°49′38″S; 45°25′ 16″W; São Sebastião, SP, Brazil). Individuals of H. obliquimanus were sorted out from the macroalgae (as in [9]), visually classified as H GB , H P or ST, and used in laboratory experiments to compare morph-specific algal preferences, colour change capacities and behaviour. We validated this visual classification by running a discriminant function analysis (DFA), using the 'lda' function from the package MASS in R [54], on random samples of individuals initially classified as H GB and H P (n = 10), to which colour reflectance values in image RGB colour channels were measured (as described below in 'Colour change and camouflage'). DFA scores for these morphs were discrete and non-overlapping (DFA scores: −5.54 < H GB < −2.46; 2.13 < H P < 5.12) indicating that misclassifications were very unlikely.

Individuals were first acclimated to laboratory conditions for three days and kept in indoor tanks, with their original plant hosts, at ambient temperature and with filtered running seawater and artificial aeration. At the start of the experiments, shrimp were transferred to rectangular plastic aquaria (30 × 20 × 10 cm) and maintained at nearly constant temperature (25°C). In all experiments, the position of aquaria assigned to different experimental treatments was randomly chosen to avoid potential artefacts due to uncontrolled spatial variation of any physical variables within the laboratory room.

Algal preference

General procedures followed standard protocols for multiple-choice tests (e.g. [55,56]). Algae were supplied in equivalent quantities (20 ml) as single clumps anchored to opposite corners of the aquaria (n = 12 for each morph). Fifteen individuals (H GB , H P, or ST) were added to the centre of each aquarium and, after 3 days, algae were carefully enclosed in dip nets and the number of living shrimp counted. As a response variable, we used the difference between the shrimp found at Sargassum and Galaxaura, divided by the total number of shrimp remaining alive at the end of the experiment, to account for mortality (2.8 shrimps ± 0.3). These preference indices were compared among morphs using a 1-way ANOVA. The Student-Newman-Keuls (SNK) procedure was used for a posteriori comparisons. Confidence intervals (95 %) were additionally calculated for each morph.

Colour change and camouflage

Previous observations indicated that the capacity of colour change differs between shrimp morphs, with H individuals visually changing their body colour in few days when exposed to an unmatched algal habitat, and ST shrimp being unable to change their coloration in the same period [9]. In this study we restricted further and more detailed analyses of colour change to the H morph. We cannot discard long-term colour shifts in ST shrimp, but because transparent individuals are typically characterized by a much reduced number of colour cells and pigments along the body, as observed in the closely related species Hippolyte varians [57] and Heptacarpus pictus [58], their eventual reorganization would likely respond to a different physiological process [28], acting over longer time-scales (weeks or months [27,28]).

Here, we conducted an experiment to quantify colour change and camouflage in the plastic morph (H), exposing individuals of varying coloration (greenish-brown and pink) to different algal habitats and artificial substrates. By doing this, we aimed to (i) test whether short-term colour changes are possible on these substrates, (ii) examine if the mechanisms controlling colour change in this species depend on visual information or diet by keeping individuals on either artificial or natural substrates, with food resources only available in the latter, and (iii) compare the efficiency of colour alteration to provide camouflage in morphs exposed to colour matched and unmatched backgrounds. Although we acknowledge that it would have been ideal to do so, colour metrics were not quantified before the trials because handling of these small and fragile shrimp could likely alter their behaviour and cause excessive mortality. We therefore used the final colour of shrimp kept against a matching background as their standard in nature. This assumption was tested by comparing hue values (see below) between experimental shrimp on matching backgrounds with shrimp freshly collected in the field (n = 10 for each morph); i.e. experimental H GB on Sargassum vs. natural H GB , and experimental H P on Galaxaura vs. natural H P .

Image analyses

We measured colour for individual algae and shrimp in all experimental treatments using digital image analyses, which provides a powerful and non-invasive approach to quantify animal coloration [59]. A Nikon Coolpix P5000 camera, coupled to a stereomicroscope and a constant white light source of 3200 K colour temperature, was used to obtain all images. Samples were photographed using manual white balancing and exposure settings to avoid colour saturation [59], followed by photographs of one standard grey card (Color Checker Passport, X-Rite), reflecting light equally at 35 % between 400 and 750 nm, using the same camera settings, as required by the sequential method of calibration [60]. Before obtaining colour data, each image was linearised to control for changes in light intensity using a set of six grey references from the colour checker chart (Color Checker Passport, X-Rite), based on the methods described by Westland and Ripamonti [61] and Stevens et al. [59]. This procedure was necessary because many digital cameras show non-linear responses of image values to changes in light levels that need to be corrected before obtaining accurate data. The camera responses were also equalised in relation to the 35 % standard grey card to control for changes in the illuminating light conditions. Finally, images were scaled to reflectance values in red (longwave; LW), green (mediumwave; MW), and blue (shortwave; SW) layers (an image value of 255 on an 8-bit scale is equal to 100 % reflectance [59]).

For each shrimp or algal image, we measured regions of interest (ROIs) and sampled the values of reflectance in the red, green, and blue channels (RGB) using the program ImageJ [62]. For shrimp images, we selected one square (1.5 mm 2 ) on the abdominal region of individuals, between somites 2 and 3, where colour is particularly uniform, and for algal images we selected the entire frond outline (approx. 50 mm 2 ). For shrimp data, we obtained values of colour (hue), which was calculated as the red/green ratio, broadly analogous to the general principle of an opponent colour channels, whereby colour types are encoded by antagonistic neural pathways [63,64] and similar to other past studies [37,65]. Red, grey, and green tones would provide hue values > 1.0, ≈ 1.0 and < 1.0, respectively. The use of this metric does not depend of any specific visual system or predator group [64], allowing us to analyse colour in terms of the physical properties of each shrimp in an intuitive way.

Colour change

We prepared two replicate aquaria for each treatment combination of 'morph' (H GB , H P ) and 'background colour' (brown, pink). Parallel trials were run using 20 ml substrates of either natural (brown Sargassum and pink Galaxaura) or artificial background (assembled stripes of brown and pink plastic tape), summing up 16 experimental units. Artificial substrates matched algal tones as closely as possible, while providing intermediate habitat architecture between the highly intricate Sargassum matrix and the smoother Galaxaura habitat. Seven to eight shrimp were initially added to each of these aquaria, with individuals maintained in artificial substrates supplied pellet shrimp food daily. Air pumps ensured adequate water circulation and aeration. In all treatments, individuals were recovered after 5 days, immediately frozen (a procedure that did not alter their colour), and later photographed to obtain colour values. A few shrimp were lost (possibly owing to mortality) and we had to reduce sample size to the minimum number of individuals found across aquaria (n = 5, for both parallel trials using natural and artificial substrates), ensuring a balanced design. Excess individuals from remaining aquaria were randomly excluded from analyses. To test the ability of individuals to change colour, we compared hue values separately for each experiment (natural or artificial substrates) using a mixed three-factor ANOVA in which factors 'morph' (H GB or H P ) and 'substrate colour' (brown or pink) were fixed and orthogonal, and the factor 'aquaria' , with two levels, was random and nested in the interaction between main factors. The Student-Newman-Keuls (SNK) procedure was used for a posteriori comparisons.

Camouflage

We also aimed to quantify the efficiency of colour change to provide camouflage against both algae. For that, we compared the final colour of shrimp reared in the different experimental treatments to the actual colour of both Sargassum and Galaxaura. We first standardised the reflectance data in the three colour channels (RGB) of shrimp and algae and then converted these values to x and y coordinates in a trichromatic colour space [66]. Colour departures were calculated as the Euclidian distances between coordinates of replicate shrimp and algae. Replicate algal coordinates (n = 20) were randomly split in two groups, to provide independent and balanced distance estimates between algae and shrimp for each morph. We used t-tests, corrected for heteroscedasticity when needed, to compare colour coordinates of each shrimp morph against the colour of both algae, predicting that shrimp colour would be closer to the colour of their rearing background than to the colour of the alternative algal background.

Morphological and behavioural differences between morphs

Intraspecific plasticity of body shape, which substantially affects hydrodynamics, is commonplace in a variety of aquatic invertebrates and fish, and may indicate differential patterns of habitat use and behaviour [15,52,53,67]. Because H and ST morphs were differently distributed between algal habitats and possibly subjected to distinct selective forces [9], we predict that H. obliquimanus individuals will exhibit morph-specific shape, with possible consequences on shrimp behaviour and life-style. Since homogeneous individuals can change their colour in just a few days (see Results), we pooled the H GB and H P categories together in a single group (H) for follow-up comparisons on morphology and behaviour.

Morph-specific shape

We used geometric morphometric analyses to compare carapace shape differences between morphs. Analyses were restricted to males to eliminate any variability owing to sexual dimorphism. Twenty-one H and 25 ST individuals were sorted from samples of Sargassum and Galaxaura collected in the São Sebastião Channel (as in [9]). Shrimp were fixed in 70 % ethanol, stained with rose bengal, and their left carapace side was photographed using a Nikon Coolpix P5000 camera, coupled to a stereomicroscope set at a magnification power of 10×.

Nine landmarks were defined along the margin of the carapace as follows; 1: eye orbit, 2: rostral tip, 3: first dorsal spine, 4: mid-dorsal margin, vertically opposed to landmark 8, 5: posterior dorsal edge, 6: posterior lateral tip, 7: distal ventral margin, vertically opposed to landmark 5, 8: ventral-most point, opposite to landmark 4, 9: ventral insertion point of the antennule. Landmarks were defined using the software tpsDig 2.14 [68], following standardized criteria [69]. Landmark alignment and the acquisition of shape variables, both uniform components (UCs) and relative-warps (RWs), were carried out following the procedures described by Zelditch and coworkers [69], using the software tpsRelw 1.46 [70].

The values of UCs and RWs were separately compared between H and ST individuals, using multivariate analysis of variance (MANOVA). Centroid size (CS), i.e. the square root of the summed squared distances between all landmarks and the carapace centre of gravity (centroid), was used as a size variable and compared between colour morphs with a t-test.

Habitat fidelity and mobility

We compared substrate fidelity and individual mobility between morphs in a simple laboratory experiment. Trials were performed in plastic rectangular aquaria (30 × 20 × 10 cm) provided with a longitudinal flow of 2 l/min containing a single Sargassum clump (40 ml) attached to the upstream end, and 20 shrimps, 10 H GB and 10 ST, at the opposite downstream side. We used only Sargassum as habitat in this experiment because this is the algal type supporting the highest shrimp density in the study area [9], and also because this is the preferred habitat of these colour morphs (see Results). The same experimental setup was replicated five times and, in each trial, all individuals were morph identified (Additional file 1: Figure S1) and monitored using a video camera (Sony HDR-XR250) for 30 min. Five three-minute video samples were selected for analyses, starting at time 1.5 min and taken at every other 3 min intervals, thus providing samples centred at times 3, 9, 15, 21 and 27 min. For each video sample we separated 90 frames (one every 2 s) for analyses. Habitat fidelity was estimated as the percentage of shrimp on algae at frame 45 (at the mid of each sample). In order to quantify mobility, we tracked the position of each shrimp remaining out of algae through all the 90 frames for each period and calculated total travelled distances. These analyses were undertaken using the software ImageJ.

The proportion of shrimp settled on algae was used as a proxy of shrimp habitat fidelity. Between-morph comparisons of these proportions, at different times, were examined using repeated-measures ANOVA because data from the same aquaria are dependent on time. Raw data were used since the sphericity assumption was met (W = 0.089; p = 0.078). Mobility of individuals was first estimated by comparing individual travelled distances between H and ST shrimp using Mann-Whitney tests. Comparisons on ranks did not detect differences between morphs (69.5 < U < 396.5, p > 0.05 for all sampled periods) because most individuals (72 %) moved very little around their initial positions, typically less than 2 cm. Therefore, we proceeded by comparing mobility of the fewer remaining shrimp that did swim considerable distances. Since these were outliers within the whole population (based on an outlier coefficient, k, of 2.0), we first subtracted swimming distances by baseline movement at their respective sampling period, i.e. the upper fence for non-outlying data. These corrected swimming distances were considered independent records and compared between morphs using a t-test.

Results

Algal preference

Shrimp colour morphs exhibited different preferences for algal substrates (ANOVA: F (2,33) = 6.84, p = 0.003). When equal volumes of the two algal types were made simultaneously available to shrimp, both H GB and ST morphs showed higher preference towards Sargassum, compared to H P individuals (SNK tests, p < 0.01). Confidence intervals (95 %) indicate net preference for the brown weed for H GB and ST but not for H P shrimp (Fig. 2).

Colour change and camouflage

Hue values of shrimp held on matching backgrounds are regarded as natural standards, since they did not significantly differ from hue values of respective counterparts in the field (experimental H GB in Sargassum vs. natural H GB : t 18 = 0.52, p = 0.609; experimental H P in Galaxaura vs. natural H P : t 18 = 0.86, p = 0.401). It is thus concluded that homogeneous shrimp (H GB and H P ) exposed to unmatched algal habitats were capable of pronounced colour change over the 5-day periods during which trials were undertaken (Table 1, Fig. 3a).

Colour change was very clear in natural algal substrates but not in artificial ones. H GB individuals increased their hue values after being in contact with the red alga Galaxaura, attaining a reddish coloration, and H P shrimp showed the opposite pattern when placed in Sargassum, achieving at the end of the experiment a brownish tone (Fig. 3a). As a result, hue differences between shrimp morphs, within each algal habitat, disappeared at the end of the trial (Fig. 3a; SNK tests, p > 0.05). However, shrimp reared in artificial substrates retained morph-specific hue (thus the significance of 'morph' , Table 1), with no changes toward background colour (Table 1; Fig. 3b). Hue differences between morphs persisted both in brown (SNK test, p < 0.05) and pink (SNK test, p < 0.01) artificial substrates (Table 1).

Although both shrimp morphs were capable of changing colour when exposed to unmatched natural backgrounds, the effectiveness of this change in promoting   Fig. 4a; two sample t-test: Fig. 3 Colour change in homogeneous greenish-brown (GB) and pink (P) individuals when exposed to a algal and b artificial substrates of brown and pink-red coloration for five days. Data from different aquaria, in each combination of 'morph' and 'substrate colour', were pooled. Final shrimp colour (hue) was defined as the ratio between reflectance in the red and green colour channels. Higher hue values correspond to reddish tones. Mean values are denoted by solid black lines and different letters indicate significant statistical differences between groups (p < 0.05) t (18) = 7.16, p < 0.001). Very similar outcomes were observed for H P shrimp. When individuals were placed on Sargassum, they did change colour but ended with values equidistant from both algal types ( Fig. 4b; two sample t-test: t (18) = 0.91, p = 0. 114), indicating poor camouflage. When reared on the matching substrate Galaxaura, the final colour of individuals was again very similar to Galaxaura but distant to Sargassum ( Fig. 4b; two sample t-test: t (18) = 4.63, p < 0.001). , which corresponds to a stouter carapace shape, while ST individuals were mostly distributed along the positive side of the axis, corresponding to a streamlined carapace shape (Fig. 5a). A MANOVA analysis applied to the two uniform components (UCs) further suggested a difference between colour morphs (MANOVA Wilks test: F (2,43) = 4.82, p = 0.013). As RW1, UC1 explained almost all morphological variation between morphs. Such a component refers to uniform contraction/expansion of the whole body, and segregated H shrimp at the negative axis half (carapace dorso-ventrally expanded), and ST individuals at the positive one (carapace dorso-ventrally compressed).

Morph-specific morphological and behavioural patterns

Habitat fidelity and mobility

Substrate fidelity was markedly different between H and ST shrimp over time (repeated-measures ANOVA: F (4,32) = 2.77, p = 0.044; Fig. 5b). At the beginning of the experiment (3 min), the proportion of individuals found on algal clumps was low, but virtually the same for each morph. The number of shrimp using the algal habitat tended to increase through time, but the rate at which they stopped swimming and settled on algae differed between H and ST shrimp. At 9 min, differences were already noticeable, increasing thereafter to statistical significance. At the end of the experiment (27 min), 78 % of H shrimp but only 57 % of ST individuals had settled on algae (Fig. 5b).

Mobility above baseline activity was restricted for a small fraction of the population and decreased from 12 % to 6 % over the experiment (Fig. 5c). Most of these swimming individuals were ST shrimp (61 %). Considering all sampled periods, average mobility was higher in ST (15.2 cm.shrimp −1 .min −1 ) than in H shrimp (5.20 cm.shrimp −1 .min −1 ; two-sample t-test: t (17) = 2.20, p = 0.043). It is also important to note that swimming events over distances larger than 25 cm each minute (n = 5) were only recorded for ST shrimp (Fig. 5c).

Discussion

We report contrasting behavioural and morphological patterns in colour morphs of the shrimp Hippolyte obliquimanus, suggesting a diversification of life-styles between morphs which can be linked to alternative camouflage strategies. Our results indicate that H shrimp are capable of fast colour change, with different colour types concealed in distinct macroalgal habitats. Individuals of this morph are also tightly connected to their benthic habitat, avoiding long-distance swimming away from their host algae, which explains why they concentrate in exceptionally high densities in the brown weed Sargassum [9]. All these features suggest that this morph presents a specialist camouflage strategy, achieved by concealment to a specific background type (at any given point in time, although individuals can change colour over time). In contrast, ST shrimp cannot rapidly adjust their colour to their background environment, and also show low habitat fidelity and substantial swimming activity, indicating a more pelagic life-style. These characteristics are in accordance to their uniform distribution between Sargassum and Galaxaura, the two main vegetated habitats in the study region [9], suggesting a generalist habitat use linked to a camouflage strategy achieved by transparency. It is noteworthy that the results of experiments on behavioural patterns are consistent with morphological analyses, indicating a more benthic life-style for H shrimp and a more pelagic habit for ST shrimp, encompassing an important range of the morphological variation found in caridean shrimp [52,53].

Colour change in H shrimp was observed upon contact with living algal habitats, but not artificial substrates, indicating the process of colour change in this species, and possibly in many other algal-dwelling isopods [71], decapods [7,57] and fish [72], relies, at least in part, on substrate-individual interactions. In fact, some authors have shown that the ingestion of carotenoid pigments can promote colour change in other crustaceans [73,74], typically over a longer period (weeks) than observed in this study. Note that this does not discount a role of visual feedback, and future work should independently change diet and visual appearance to tease apart these effects.

Colour change may be a faster process for small crustaceans shedding thin translucent exuviae (own observations) than large ones, because pigment reorganization in hypodermic colour cells may be readily visible, as observed for another hippolytid shrimp species [57]. Colour change in H shrimp strongly suggests a camouflage strategy by Fig. 4 Colour differences between a homogeneous greenish-brown (GB) and b homogeneous pink (P) shrimp and the algae Sargassum and Galaxaura, for groups of individuals reared for five days in each of these substrates. Colour differences were defined as the Euclidian distance between standardised colour coordinates in a trichromatic reflectance colour space of shrimp and algae. Solid black lines represent mean group values. **p < 0.01; ***p < 0.001; ns: not-significant background matching, whereby individuals' overall body colour, colour pattern, and brightness tend to resemble the general background [75]. However, we observed H shrimp concealed better on the pink Galaxaura than on the brown Sargassum. In Sargassum, H GB and H P ended up with an intermediate body colour pattern, equally distant from the two algal types. In contrast, both shrimp morphs reared in Galaxaura became much better concealed to this substrate than to the alternative Sargassum background. These results were surprising since natural shrimp densities in the brown Sargassum are far higher than in Galaxaura [9]; a difference that could be explained by more efficient camouflage in the former. Our results, however, indicate that this is not the case, and that factors other than colour camouflage alone likely underlie this species distribution in the field. Also, these findings are aligned to ongoing research suggesting better protection against predators in Galaxaura (in prep), highlighting the importance of concealment in the pink weed habitat. Further work on longer term changes in colour than those tested here are also needed.

The Sargassum and Galaxaura canopy constitute the most important habitat types to shrimp in our study area, but the relative value of these habitats for H. obliquimanus is apparently very different [9]. Experiments in the laboratory testing algal preferences showed that H GB and ST individuals actively select Sargassum fronds while H P shrimp did not show any significant preference, indicating that colour camouflage is not an important selective force setting patterns of habitat choices. Strong preference of individuals for Sargassum may be adaptive for several different reasons not addressed in this study. For instance, as a much more physically complex habitat, especially when associated to epiphytic algae (e.g. Hypnea spp. [76,77]), Sargassum would probably supply better shelter from predators and more extensive foraging grounds [78,79] compared to Galaxaura. It is also possible that inconspicuous behaviour coupled to shape resemblance to background details [18,80,81] in the more complex Sargassum would ultimately render superior predator avoidance. More specific research addressing these issues is pending.

Habitat fidelity and mobility further support morphspecific life-styles. Colour-changing shrimp (H) show higher substrate fidelity and lower mobility rates indicating a more specialized habitat use. Although capable of colour alteration towards background matching, moving from one algal habitat to another would likely come at a cost. Settling on non-matching habitat for even a few days, compatible to the time for colour adjustment, may lead to very high predation rates [82][83][84]. Colour change may also carry physiological costs, although these have rarely if ever been quantified [26]. Therefore, at any one time, H morphs may be able to conceal to a specific background type, being considered background specialists. Conversely, ST shrimp may be generally concealed against a wider range of visual backgrounds [85] while moving from one habitat patch to another. Therefore, the transparency of individuals, linked to a higher mobility and lack of substrate fidelity, may eventually promote camouflage by means of a strategy independent (or partially dependent) of background matching, indicating a more generalist type of concealment and habitat use [33,35,39]. Morph-specific lifestyles are supported by natural shrimp distributions [9] and also by geometric morphometrics analyses of carapace shape. The morphological gradient observed overlaps a great deal of the variation for caridean shrimp in general [52,53]. While the more hydrodynamic shape found in ST shrimp clearly resembles the shape of pelagic shrimp species, the stouter H morphology are more akin to benthic species. More streamlined ST shrimp swimming distances within the range of 25 to 45 cm each minute may easily move across different algal habitats, which is not the case of more sedentary and deepbodied H shrimp that were never observed swimming over such distances and tended to settle and remain on algae more frequently. Shrimp morphology, perhaps coupled to behaviour, may also affect camouflage in their algal habitats. Further experimental work is required, however, to examine this issue more closely.

While the different colour types of H and ST individuals may reflect distinct life-styles, we might ask what drives selection for these different approaches. Low dispersal and optimization of resource use can be particularly advantageous in H individuals, which concentrate in habitat patches where shelter is abundant and/or camouflage efficient. Even being a habitat where colour camouflage does not appear to be critical, Sargassum supports high (See figure on previous page.) Fig. 5 Proxy variables indicating different life-styles in homogeneous (H; in grey circles or bars) and striped translucent morphs (ST; in white circles or bars). a Carapace shape differences based on geometric morphometric results. Morphotypes are clearly segregated along the first relative warp axis, from a stout carapace outline representative of H individuals, to a more streamlined shape found in ST shrimp. Percentage values represent the relative warps share of the total morphological variation. Dots along carapace margins show the position of landmarks used in the analysis (see Methods). b Substrate fidelity of morphs, over 30 min experimental trials, expressed as the percentage of individuals settled on algal clumps. Whiskers represent ± 1 SE. *p < 0.05; **p < 0.01. c Morph-specific mobility, expressed as individual average swimming distances (in cm) per minute, over 30 min experimental trials. Measurements for mobile shrimp are outliers (filled circles) from baseline movement of sedentary individuals. Dashed lines represent the upper fence delimiting the non-outlier range (see Methods for details) densities of H shrimp, which exhibit high preference and fidelity to this habitat. The less structured Galaxaura substrate would be important as a secondary habitat to this morph, where colour concealment will be a valuable mechanism to reduce prey detection by visual predators. Based on these assumptions, we may expect strong intraspecific competition in Sargassum habitat, and hence selection for optimal resource use and territorial behaviour, which would possibly lead to displacement of ST individuals to Galaxaura. Density-dependent processes and loss of preferred habitats could be major mechanisms regulating abundance of H individuals. On the other hand, high dispersal potential and a generalist habitat use may be useful traits for ST shrimp. Because ST shrimp are mainly males [9], intense mobility and low substrate fidelity would likely enable males to find more mates in a puresearch strategy, expected for polyginic caridean species such as H. obliquimanus [45,86,87].

The coexistence within a population of distinct morphs with alternative strategies of habitat use and camouflage, as observed for H. obliquimanus, facilitates diversification on the use of environmental resources [9] and can also have ecological and evolutionary consequences, mainly on population stability over time [5]. The availability of the presumably higher-quality Sargassum habitat in our study region is markedly seasonal, with very high cover during summer and a much reduced density in winter, sometimes collapsing in that season [88]. Temporal variation in Sargassum cover can be a major mechanism controlling H shrimp densities, once individuals show strong specialization for this habitat. Therefore, the existence of an alternative habitat (Galaxaura) and morphs differing in their degree of habitat specialization may allow temporal changes in individual fitness associated with habitat availability and morphs density and frequency. Ongoing research on trophic niche space would further elucidate morph-specific patterns of resource use.

Conclusions

Colour camouflage is a common anti-predator strategy in nature, but few studies investigate complex interactions among colour traits and other morphological and behavioural mechanisms, indicative of general morphspecific life-styles. Our findings illustrate that specific arrangements among morphology, behaviour, and (micro-) habitat use in colour morphs of the algal-dwelling shrimp H. obliquimanus may result in a diversification of camouflage strategies in a species living in a heterogeneous habitat. Colour change ability and high substrate fidelity, associated to a more robust morphology, suggest a specialist camouflage strategy in H individuals. On the other hand, high mobility coupled with a more streamlined morphology and lack of substrate fidelity in ST individuals, indicate a general strategy of camouflage in this morph. Higher mobility of the ST morph, in which more than 70 % of individuals are males [9], may also sustain a pure-search polygynic mating strategy which is predicted for this species. Seasonal changes on macroalgal cover may affect the frequency and fitness of the different colour morphs in the population. Selective mechanisms, such as morph-specific predation by visual consumers through contrasting patterns of habitat use [51,89], would be important forces maintaining the diversification of life-styles and camouflage strategies in this shrimp species.

Additional files

Fig. 1
1Hippolyte obliquimanus colour morphs. Homogeneous (H) individuals characterized by a greenish-brown (H GB : top-left shrimp) or pink (H P : bottom-left shrimp) coloration attaining a good colour match in the brown algae Sargassum furcatum and the red-pink weed Galaxaura marginata, respectively. Striped translucent (ST) individuals bearing longitudinal colour bands (top-right and bottom-right shrimp), showing a general resemblance to both algae

Fig. 2
2Algal preference of homogeneous (greenish-brown; GB and pink; P) and striped translucent colour morphs. f sf and f gm stand for the frequencies of shrimp occupying Sargassum furcatum and Galaxaura marginata fronds at the end of trials. Solid and dashed black lines denote mean values and ± 1 CI (95 %) respectively. Different letters indicate statistical differences among morphs (p < 0.05)

Fig. 5 (
5See legend on next page.) Duarte et al. BMC Evolutionary Biology (2016) 16:218

.
Images extracted from video footage showing the experimental set up used in habitat fidelity and mobility trials (see details in the main text). Homogeneous (H) and striped translucent (ST) morphs of the shrimp Hippolyte obliquimanus were more easily identified in dorsal view, when thin longitudinal stripes of ST shrimps stood clearly out from the bottom of aquaria, contrasting to the solid coloration typical of H shrimp. A lateral view of an ST individual (as the lowermost shrimp in the lower image), showing translucent areas over the abdomen and carapace, could however suffice for morph identification. (PDF 489 kb) Additional file 2: Supporting data. (XLSX 39 kb)

Table 1
1Summary results of the mixed three-way analyses of variance testing the effects of morph type (M; greenish-brown or pink), substrate colour (S C ; brown or pink) and aquaria (nested in the interaction between main factors) in final hue values measured in Hippolyte obliquimanus individuals after being maintained for five days in artificial or algal substratesAlgae
Artificial substrates

Source of variation
df
MS
F
P
MS
F
p

Morph (M)
1
0.170
0.92
0.392
3.750
44.13
0.002

Substrate colour (S C )
1
8.636
46.56
0.002
0.298
3.51
0.134

M x S C
1
0.069
0.37
0.574
0.217
2.55
0.185

Aquaria (M x S C )
4
0.186
1.07
0.388
0.085
0.65
0.632

Error
32
0.174
0.131

C = 0.247; ns
C = 0.240; ns

C Cochran statistic, ns not-significant

AcknowledgementsWe are grateful to Alvaro Migotto for his advice in image acquisition, and two anonymous referees for comments on the paper. We especially thank the technician staff at the Center for Marine Biology for helping in field surveys, and Glauco Machado and Fosca Leite for suggestions on an early manuscript draft.Availability of data and materialsThe datasets supporting the conclusions of this article are included as an additional file (Additional file 2).Authors' contributions RCD and AAVF participated in the design of the study and in the collection of specimens. RCD conducted the experiments, analysed the data and drafted the manuscript with AAVF and MS. All authors wrote and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publication Not required.Ethics approval and consent to participate All experimental procedures used in this study comply with the current laws of Brazilian legislation.• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
Linking color polymorphism maintenance and speciation. S M Gray, J S Mckinnon, Trends Ecol Evol. 22Gray SM, McKinnon JS. Linking color polymorphism maintenance and speciation. Trends Ecol Evol. 2007;22:71-9.

Geographic variation in animal colour polymorphisms and its role in speciation. C A Mclean, D Stuart-Fox, Biol Rev. 89Mclean CA, Stuart-Fox D. Geographic variation in animal colour polymorphisms and its role in speciation. Biol Rev. 2014;89:860-73.

Natural selection in Cepaea. A J Cain, P M Sheppard, Genetics. 39Cain AJ, Sheppard PM. Natural selection in Cepaea. Genetics. 1954;39:89-116.

Selection experiments on industrial melanism in the Lepidoptera. H Kettlewell, Heredity (Edinb). 9Kettlewell H. Selection experiments on industrial melanism in the Lepidoptera. Heredity (Edinb). 1955;9:323-42.

A model of ecological and evolutionary consequences of color polymorphism. A Forsman, J Ahnesjö, S Caesar, M Karlsson, Ecology. 89Forsman A, Ahnesjö J, Caesar S, Karlsson M. A model of ecological and evolutionary consequences of color polymorphism. Ecology. 2008;89:34-40.

Morphological variation and width of ecological niche. L Van Valen, Am Nat. 99Van Valen L. Morphological variation and width of ecological niche. Am Nat. 1965;99:377-90.

Size-related habitat shifts facilitated by positive preference induction in a marine kelp crab. K M Hultgren, J J Stachowicz, Behav Ecol. 21Hultgren KM, Stachowicz JJ. Size-related habitat shifts facilitated by positive preference induction in a marine kelp crab. Behav Ecol. 2010;21:329-36.

Camouflage and individual variation in shore crabs (Carcinus maenas) from different habitats. M Stevens, A E Lown, L E Wood, PLoS One. 9Stevens M, Lown AE, Wood LE. Camouflage and individual variation in shore crabs (Carcinus maenas) from different habitats. PLoS One. 2014;9:1-31.

Morph-specific habitat and sex distribution in the caridean shrimp Hippolyte obliquimanus. R C Duarte, Aav Flores, J Mar Biol Assoc. United Kingdom. Internet]. 1-8. Available fromDuarte RC, Flores AAV. Morph-specific habitat and sex distribution in the caridean shrimp Hippolyte obliquimanus. J Mar Biol Assoc. United Kingdom [Internet]. 1-8. Available from: http://www.journals.cambridge.org/abstract_ S0025315416000230.

The rock-paper-scissors game and the evolution of alternative male strategies. B Sinervo, C M Lively, Nature. 380Sinervo B, Lively CM. The rock-paper-scissors game and the evolution of alternative male strategies. Nature. 1996;380:240-3.

Alternative male mating tactics in a cichlid, Pelvicachromis pulcher : a comparison of reproductive effort and success. E Martin, M Taborsky, Behav Ecol Sociobiol. 41Martin E, Taborsky M. Alternative male mating tactics in a cichlid, Pelvicachromis pulcher : a comparison of reproductive effort and success. Behav Ecol Sociobiol. 1997;41:311-9.

Individual variations in habitat use and morphology in brook charr. P Bourke, P Magnan, M A Rodriguez, J Fish Biol. 51Bourke P, Magnan P, Rodriguez MA. Individual variations in habitat use and morphology in brook charr. J Fish Biol. 1997;51:783-94.

Polymorphic mimicry, microhabitat use, and sex-specific behaviour. M Joron, J Evol Biol. 18Joron M. Polymorphic mimicry, microhabitat use, and sex-specific behaviour. J Evol Biol. 2005;18:547-56.

The ecology of individuals: incidence and implications of individual specialization. D I Bolnick, R Svanbäck, J A Fordyce, L H Yang, J M Davis, C D Hulsey, Am Nat. 161Bolnick DI, Svanbäck R, Fordyce JA, Yang LH, Davis JM, Hulsey CD, et al. The ecology of individuals: incidence and implications of individual specialization. Am Nat. 2003;161:1-28.

Sympatric ecological divergence associated with a color polymorphism. H Kusche, K R Elmer, A Meyer, BMC Biol. 1382Kusche H, Elmer KR, Meyer A. Sympatric ecological divergence associated with a color polymorphism. BMC Biol. 2015;13:82.

The evolution of color polymorphism: crypticity, searching images, and apostatic selection. A B Bond, Annu Rev Ecol Evol Syst. 38Bond AB. The evolution of color polymorphism: crypticity, searching images, and apostatic selection. Annu Rev Ecol Evol Syst. 2007;38:489-514.

Crypsis in a heterogeneous environment: relationships between changeable polymorphic colour patterns and behaviour in a galaxiid fish. K Magellan, E R Swartz, Freshw Biol. 58Magellan K, Swartz ER. Crypsis in a heterogeneous environment: relationships between changeable polymorphic colour patterns and behaviour in a galaxiid fish. Freshw Biol. 2013;58:793-9.

Why habitat architecture and color are important to shrimps living in pelagic Sargassum: use of camouflage and plant-part mimicry. S Hacker, L Madin, Mar Ecol Prog Ser. 70Hacker S, Madin L. Why habitat architecture and color are important to shrimps living in pelagic Sargassum: use of camouflage and plant-part mimicry. Mar Ecol Prog Ser. 1991;70:143-55.

Does variable coloration in juvenile marine crabs reduce risk of visual predation?. A T Palma, R S Steneck, Ecology. 82Palma AT, Steneck RS. Does variable coloration in juvenile marine crabs reduce risk of visual predation? Ecology. 2001;82:2961-7.

Phenotype-environment matching in the shore crab (Carcinus maenas). P Todd, R Briers, R Ladle, F Middleton, Mar Biol. 148Todd P, Briers R, Ladle R, Middleton F. Phenotype-environment matching in the shore crab (Carcinus maenas). Mar Biol. 2006;148:1357-67.

Visual predators impose correlational selection on prey color pattern and behavior. A Forsman, S Appelqvist, Behav Ecol. 9Forsman A, Appelqvist S. Visual predators impose correlational selection on prey color pattern and behavior. Behav Ecol. 1998;9:409-13.

Reduced predation risk for melanistic pygmy grasshoppers in post-fire environments. E Karpestam, S Merilaita, A Forsman, Ecol Evol. 2Karpestam E, Merilaita S, Forsman A. Reduced predation risk for melanistic pygmy grasshoppers in post-fire environments. Ecol Evol. 2012;2:2204-12.

Camouflage through behavior in moths: the role of background matching and disruptive coloration. C Kang, M Stevens, J-Y Moon, S-I Lee, P Jablonski, Behav Ecol. 26Kang C, Stevens M, Moon J-Y, Lee S-I, Jablonski P. Camouflage through behavior in moths: the role of background matching and disruptive coloration. Behav Ecol. 2015;26:45-54.

The genetic basis of adaptive melanism in pocket mice. M W Nachman, H E Hoekstra, D Agostino, S L , Proc Natl Acad Sci. 100Nachman MW, Hoekstra HE, D'Agostino SL. The genetic basis of adaptive melanism in pocket mice. Proc Natl Acad Sci U S A. 2003;100:5268-73.

Convergent evolution and divergent selection: lizards at the White Sands ecotone. E B Rosenblum, Am Nat. 167Rosenblum EB. Convergent evolution and divergent selection: lizards at the White Sands ecotone. Am Nat. 2006;167:1-15.

Camouflage, communication and thermoregulation: lessons from colour changing organisms. D Stuart-Fox, A Moussalli, Philos Trans R Soc Lond B Biol Sci. 364Stuart-Fox D, Moussalli A. Camouflage, communication and thermoregulation: lessons from colour changing organisms. Philos Trans R Soc Lond B Biol Sci. 2009;364:463-70.

Reversible colour change in Arthropoda. Kdl Umbers, S A Fabricant, F M Gawryszewski, A E Seago, M E Herberstein, Biol Rev. 89Umbers KDL, Fabricant SA, Gawryszewski FM, Seago AE, Herberstein ME. Reversible colour change in Arthropoda. Biol Rev. 2014;89:820-48.

Color change, phenotypic plasticity, and camouflage. M Stevens, Front Ecol Evol. 4Stevens M. Color change, phenotypic plasticity, and camouflage. Front Ecol Evol. 2016;4:1-10.

Egg-laying substrate selection for optimal camouflage by quail. P G Lovell, G D Ruxton, K V Langridge, K A Spencer, Curr Biol. 23Lovell PG, Ruxton GD, Langridge KV, Spencer KA. Egg-laying substrate selection for optimal camouflage by quail. Curr Biol. 2013;23:260-4.

Microhabitat choice in island lizards enhances camouflage against avian predators. Kla Marshall, K E Philpot, M Stevens, Sci Rep. 6Marshall KLA, Philpot KE, Stevens M. Microhabitat choice in island lizards enhances camouflage against avian predators. Sci Rep. 2016;6:19815.

Recognition of appropriate backgrounds by the pale and black phases of Lepidoptera. Hbd Kettlewell, Nature. 175Kettlewell HBD. Recognition of appropriate backgrounds by the pale and black phases of Lepidoptera. Nature. 1955;175:943-4.

Differential habitat selection by pygmy grasshopper color morphs; interactive effects of temperature and predator avoidance. J Ahnesjö, A Forsman, Evol Ecol. 20Ahnesjö J, Forsman A. Differential habitat selection by pygmy grasshopper color morphs; interactive effects of temperature and predator avoidance. Evol Ecol. 2006;20:235-57.

Animal camouflage: Compromise or specialize in a 2 patch-type environment?. A I Houston, M Stevens, I C Cuthill, Behav Ecol. 18Houston AI, Stevens M, Cuthill IC. Animal camouflage: Compromise or specialize in a 2 patch-type environment? Behav Ecol. 2007;18:769-75.

Detection experiments with humans implicate visual predation as a driver of colour polymorphism dynamics in pygmy grasshoppers. E Karpestam, S Merilaita, A Forsman, BMC Ecol. 1317Karpestam E, Merilaita S, Forsman A. Detection experiments with humans implicate visual predation as a driver of colour polymorphism dynamics in pygmy grasshoppers. BMC Ecol. 2013;13:17.

Optimization of cryptic coloration in heterogeneous habitats. S Merilaita, J Tuomi, V Jormalainen, Biol J Linn Soc. 67Merilaita S, Tuomi J, Jormalainen V. Optimization of cryptic coloration in heterogeneous habitats. Biol J Linn Soc. 1999;67:151-61.

Spatial heterogeneity, predator cognition, and the evolution of color polymorphism in virtual prey. A B Bond, A C Kamil, Proc Natl Acad Sci. 103Bond AB, Kamil AC. Spatial heterogeneity, predator cognition, and the evolution of color polymorphism in virtual prey. Proc Natl Acad Sci U S A. 2006;103:3214-9.

Rockpool gobies change colour for camouflage. M Stevens, A E Lown, A M Denton, PLoS One. 9Stevens M, Lown AE, Denton AM. Rockpool gobies change colour for camouflage. PLoS One. 2014;9:1-8.

Color change and camouflage in juvenile shore crabs Carcinus maenas. M Stevens, A E Lown, L E Wood, Front Ecol Evol. 2Stevens M, Lown AE, Wood LE. Color change and camouflage in juvenile shore crabs Carcinus maenas. Front Ecol Evol. 2014;2:1-14.

Selection for cryptic coloration in a visually heterogeneous habitat. S Merilaita, A Lyytinen, J Mappes, Proc R Soc Lond Ser B Biol Sci. 268Merilaita S, Lyytinen A, Mappes J. Selection for cryptic coloration in a visually heterogeneous habitat. Proc R Soc Lond Ser B Biol Sci. 2001;268:1925-9.

Van Damme R. Morphology, performance, behavior and ecology of three color morphs in males of the lizard Podarcis melisellensis. K Huyghe, B Vanhooydonck, A Herrel, Z Tadic, Integr Comp Biol. 47Huyghe K, Vanhooydonck B, Herrel A, Tadic Z, Van Damme R. Morphology, performance, behavior and ecology of three color morphs in males of the lizard Podarcis melisellensis. Integr Comp Biol. 2007;47:211-20.

Predator and prey views of spider camouflage. M Théry, J Casas, Nature. 415133Théry M, Casas J. Predator and prey views of spider camouflage. Nature. 2002;415:133.

Testing models of source-sink dynamics and balanced dispersal. J E Diffendorfer, Oikos. 81Diffendorfer JE. Testing models of source-sink dynamics and balanced dispersal. Oikos. 1998;81:417-33.

Source, sinks, and population regulation. H Pulliam, Am Nat. 132Pulliam H. Source, sinks, and population regulation. Am Nat. 1988;132:652-61.

Adaptive sympatric speciation of polychromatic "roundfin" sailfin silverside fish in Lake Matano (Sulawesi). F Herder, J Pfaender, U K Schliewen, Evolution (N Y). 62Herder F, Pfaender J, Schliewen UK. Adaptive sympatric speciation of polychromatic "roundfin" sailfin silverside fish in Lake Matano (Sulawesi). Evolution (N Y). 2008;62:2178-95.

Sexual selection. M Andersson, Princeton Universiy PressPrincetonAndersson M. Sexual selection. Princeton: Princeton Universiy Press; 1994.

A trade-off between flight capability and reproduction in males of a wing-dimorphic insect. G A Langellotto, R F Denno, J R Ott, Ecology. 81Langellotto GA, Denno RF, Ott JR. A trade-off between flight capability and reproduction in males of a wing-dimorphic insect. Ecology. 2000;81:865-75.

Density-related migration in planthoppers (Homoptera: Delphacidae): the role of habitat persistence. R F Denno, G K Roderick, K L Olmstead, H G Dobel, Am Nat. 138Denno RF, Roderick GK, Olmstead KL, Dobel HG. Density-related migration in planthoppers (Homoptera: Delphacidae): the role of habitat persistence. Am Nat. 1991;138:1513-41.

Relation between temporal persistence of host plants and wing length in leafhoppers (Hemiptera: Auchenorrhyncha). V Novotný, Ecol Entomol. 19Novotný V. Relation between temporal persistence of host plants and wing length in leafhoppers (Hemiptera: Auchenorrhyncha). Ecol Entomol. 1994;19:168-76.

Hippolyte obliquimanus (Decapoda: Caridea: Hippolytidae): a gonochoric or hermaphroditic shrimp species?. M Terossi, López Greco, L S Mantelatto, F L , Mar Biol. 154Terossi M, López Greco LS, Mantelatto FL. Hippolyte obliquimanus (Decapoda: Caridea: Hippolytidae): a gonochoric or hermaphroditic shrimp species? Mar Biol. 2008;154:127-35.

Sexual ratio, reproductive period and seasonal variation of the gonochoric shrimp Hippolyte obliquimanus (Caridea: Hippolytidae). M Terossi, F L Mantelatto, Mar Biol Res. 6Terossi M, Mantelatto FL. Sexual ratio, reproductive period and seasonal variation of the gonochoric shrimp Hippolyte obliquimanus (Caridea: Hippolytidae). Mar Biol Res. 2010;6:213-9.

Evolution of sex differences in microhabitat choice and colour polymorphism in Idotea baltica. S Merilaita, V Jormalainen, Anim Behav. 54Merilaita S, Jormalainen V. Evolution of sex differences in microhabitat choice and colour polymorphism in Idotea baltica. Anim Behav. 1997;54:769-78.

A morphological approach for relating decapod crustacean cephalothorax shape with distribution in the water column. F Sardà, J B Company, C Costa, Mar Biol. 147Sardà F, Company JB, Costa C. A morphological approach for relating decapod crustacean cephalothorax shape with distribution in the water column. Mar Biol. 2005;147:611-8.

Influence of diel behaviour in the morphology of decapod natantia. J Aguzzi, C Costa, F Antonucci, J B Company, P Menesatti, F Sardá, Biol J Linn Soc. 96Aguzzi J, Costa C, Antonucci F, Company JB, Menesatti P, Sardá F. Influence of diel behaviour in the morphology of decapod natantia. Biol J Linn Soc. 2009;96:517-32.

Modern applied statistics with S. W Venables, B Ripley, SpringerNew York4th edVenables W, Ripley B. Modern applied statistics with S. 4th ed. New York: Springer; 2002.

Habitat architecture and the abundance and body-size-dependent habitat selection of a phytal amphipod. S Hacker, R Steneck, Ecology. 71Hacker S, Steneck R. Habitat architecture and the abundance and body-size-dependent habitat selection of a phytal amphipod. Ecology. 1990;71:2269-85.

Preference-performance relationships and effects of host plant choice in an herbivorous marine amphipod. Agb Poore, P D Steinbenrg, Ecol Monogr. 69Poore AGB, Steinbenrg PD. Preference-performance relationships and effects of host plant choice in an herbivorous marine amphipod. Ecol Monogr. 1999;69:443-64.

Hippolyte varians: a study in colour-change. F W Gamble, F W Keeble, Q J Microc Sci. 43Gamble FW, Keeble FW. Hippolyte varians: a study in colour-change. Q J Microc Sci. 1900;43:589-703.

Color patterns of the shrimps Heptacarpus pictus and H. paludicola (Caridea: Hippolytidae). R T Bauer, Mar Biol. 64Bauer RT. Color patterns of the shrimps Heptacarpus pictus and H. paludicola (Caridea: Hippolytidae). Mar Biol. 1981;64:141-52.

Using digital photography to study animal coloration. M Stevens, C A Párraga, I C Cuthill, J C Partridge, T S Troscianko, Biol J Linn Soc. 90Stevens M, Párraga CA, Cuthill IC, Partridge JC, Troscianko TS. Using digital photography to study animal coloration. Biol J Linn Soc. 2007;90:211-37.

Studying primate color: towards visual system-dependent methods. M Stevens, M C Stoddard, J P Higham, Int J Primatol. 30Stevens M, Stoddard MC, Higham JP. Studying primate color: towards visual system-dependent methods. Int J Primatol. 2009;30:893-917.

Computational color science. S Westland, C Ripamonti, John Wiley & Sons LtdWestland S, Ripamonti C. Computational color science. Chichester: John Wiley & Sons Ltd.; 2004.

National Institute of Health, Bethesda. W Rasband, Imagej, InternetRasband W. ImageJ [online]. [Internet]. National Institute of Health, Bethesda. 1997. Available from: https://imagej.nih.gov/ij/.

Colour vision of domestic chicks. D Osorio, M Vorobyev, C D Jones, J Exp Biol. 202Osorio D, Vorobyev M, Jones CD. Colour vision of domestic chicks. J Exp Biol. 1999;202:2951-9.

Avian vision and egg colouration: concepts and measurements. M Stevens, Avian Biol Res. 4Stevens M. Avian vision and egg colouration: concepts and measurements. Avian Biol Res. 2011;4:168-84.

How to evade a coevolving brood parasite: egg discrimination versus egg variability as host defences. C Spottiswoode, M Stevens, Proc R Soc B Biol Sci. 278Spottiswoode C, Stevens M. How to evade a coevolving brood parasite: egg discrimination versus egg variability as host defences. Proc R Soc B Biol Sci. 2011;278:3566-73.

Animal colour vision-behavioural tests and physiological concepts. A Kelber, M Vorobyev, D Osorio, Biol Rev. 78Kelber A, Vorobyev M, Osorio D. Animal colour vision-behavioural tests and physiological concepts. Biol Rev. 2003;78:81-118.

Shape up or ship out: migratory behaviour predicts morphology across spatial scale in a freshwater fish. B B Chapman, K Hulthén, C Brönmark, P A Nilsson, C Skov, L-A Hansson, J Anim Ecol. 84Chapman BB, Hulthén K, Brönmark C, Nilsson PA, Skov C, Hansson L-A, et al. Shape up or ship out: migratory behaviour predicts morphology across spatial scale in a freshwater fish. J Anim Ecol. 2015;84:1187-93.

TpsDig, digitize landmarks and outlines. Department of Ecology and Evolution. F Rohlf, New York at Stony BrookState University ofRohlf F. TpsDig, digitize landmarks and outlines. Department of Ecology and Evolution, State University of New York at Stony Brook; 2009. Available from: http://life.bio.sunysb.edu/morph/.

Geometric morphometrics for biologists: a primer. M Zelditch, D Swiderski, H Sheets, W Fink, Elsevier Academic PressNew YorkZelditch M, Swiderski D, Sheets H, Fink W. Geometric morphometrics for biologists: a primer. New York: Elsevier Academic Press; 2004.

TpsRelw, relative warps analysis. Department of Ecology and Evolution. F Rohlf, New York at Stony BrookState University ofRohlf F. TpsRelw, relative warps analysis. Department of Ecology and Evolution, State University of New York at Stony Brook; 2008. Available from: http://life.bio.sunysb.edu/morph/.

Color change in a marine isopod is adaptive in reducing predation. K M Hultgren, H Mittelstaedt, Curr Zool. 61Hultgren KM, Mittelstaedt H. Color change in a marine isopod is adaptive in reducing predation. Curr Zool. 2015;61:739-48.

Heterostichus rostratus Girard: genetic versus environmental factors. C Stepien, J Exp Mar Biol Ecol. 100Regulation of color morphic patterns in the giant kelpfishStepien C. Regulation of color morphic patterns in the giant kelpfish, Heterostichus rostratus Girard: genetic versus environmental factors. J Exp Mar Biol Ecol. 1986;100:181-208.

Pigmentation of kuruma prawn, Penaeus japonicus Bate, by various pigment sources and levels and feeding regimes. Y-H Chien, S-C Jeng, Aquaculture. 102Chien Y-H, Jeng S-C. Pigmentation of kuruma prawn, Penaeus japonicus Bate, by various pigment sources and levels and feeding regimes. Aquaculture. 1992;102:333-46.

Astaxanthin deposition in the cuticle of juvenile American lobster (Homarus americanus): implications for phenotypic and genotypic coloration. M Tlusty, C Hyland, Mar Biol. 147Tlusty M, Hyland C. Astaxanthin deposition in the cuticle of juvenile American lobster (Homarus americanus): implications for phenotypic and genotypic coloration. Mar Biol. 2005;147:113-9.

Crypsis through background matching. S Merilaita, M Stevens, Anim. Camoufl. Cambridge. Stevens M, Merilaita SCambridge University PressMerilaita S, Stevens M. Crypsis through background matching. In: Stevens M, Merilaita S, editors. Anim. Camoufl. Cambridge: Cambridge University Press; 2011. p. 17-33.

Temporal variation in Sargassum biomass, Hypnea epiphytism and associated fauna. F Leite, A Turra, Braz Arch Biol Technol. 46Leite F, Turra A. Temporal variation in Sargassum biomass, Hypnea epiphytism and associated fauna. Braz Arch Biol Technol. 2003;46:665-71.

Spatial scaling in the distribution of macrofauna associated with Sargassum stenophyllum (Mertens) Martius: analyses of faunal groups, gammarid life habits, and assemblage structure. M O Tanaka, Fpp Leite, J Exp Mar Biol Ecol. 293Tanaka MO, Leite FPP. Spatial scaling in the distribution of macrofauna associated with Sargassum stenophyllum (Mertens) Martius: analyses of faunal groups, gammarid life habits, and assemblage structure. J Exp Mar Biol Ecol. 2003;293:1-22.

Epiphyte-seagrass relationships with an emphasis on the role of micrograzing: a review. R J Orth, J Van Montfrans, Aquat Bot. 18Orth RJ, Van Montfrans J. Epiphyte-seagrass relationships with an emphasis on the role of micrograzing: a review. Aquat Bot. 1984;18:43-69.

Abundance of mobile epifauna: the role of habitat complexity and predation by fishes. Martin-Smith Km, J Exp Mar Biol Ecol. 174Martin-Smith KM. Abundance of mobile epifauna: the role of habitat complexity and predation by fishes. J Exp Mar Biol Ecol. 1993;174:243-60.

Predator avoidance in seagrass meadows: prey behavior, microhabitat selection, and cryptic coloration. K Main, Ecology. 68Main K. Predator avoidance in seagrass meadows: prey behavior, microhabitat selection, and cryptic coloration. Ecology. 1987;68:170-80.

Why be cryptic? Choice of host urchin is not based on camouflage in the caridean shrimp Gnathophylloides mineri. S Maciá, M P Robinson, Acta Ethol. 12Maciá S, Robinson MP. Why be cryptic? Choice of host urchin is not based on camouflage in the caridean shrimp Gnathophylloides mineri. Acta Ethol. 2009;12:105-13.

Alternative camouflage strategies mediate predation risk among closely related co-occurring kelp crabs. K M Hultgren, J J Stachowicz, Oecologia. 155Hultgren KM, Stachowicz JJ. Alternative camouflage strategies mediate predation risk among closely related co-occurring kelp crabs. Oecologia. 2008;155:519-28.

Evolutionary significance of ontogenetic colour change in animals. C L Booth, Biol J Linn Soc. 40Booth CL. Evolutionary significance of ontogenetic colour change in animals. Biol J Linn Soc. 1990;40:125-63.

Plastic inducible morphologies are not always adaptive: the importance of time delays in a stochastic environment. D K Padilla, S C Adolph, Evol Ecol. 10Padilla DK, Adolph SC. Plastic inducible morphologies are not always adaptive: the importance of time delays in a stochastic environment. Evol Ecol. 1996;10:105-17.

Hidden in plain sight: the ecology and physiology of organismal transparency. S Johnsen, Biol Bull. 201Johnsen S. Hidden in plain sight: the ecology and physiology of organismal transparency. Biol Bull. 2001;201:301-18.

Monogamy in Crustacea and man. W Wickler, U Seibt, Z Tierpsychol. 57Wickler W, Seibt U. Monogamy in Crustacea and man. Z Tierpsychol. 1981;57:215-34.

Sexual system, sex ratio, and group living in the shrimp Thor amboinensis (De Man): relevance to resource-monopolization and sex-allocation theories. J A Baeza, C Piantoni, Biol Bull. 219Baeza JA, Piantoni C. Sexual system, sex ratio, and group living in the shrimp Thor amboinensis (De Man): relevance to resource-monopolization and sex-allocation theories. Biol Bull. 2010;219:151-65.

Can artificial beds of plastic mimics compensate for seasonal absence of natural beds of Sargassum furcatum?. Eas Godoy, R Coutinho, ICES J Mar Sci. 59Godoy EAS, Coutinho R. Can artificial beds of plastic mimics compensate for seasonal absence of natural beds of Sargassum furcatum? ICES J Mar Sci. 2002;59:111-5.

Differential predation on sexes affects colour polymorphism of the isopod Idotea baltica (Pallas). V Jormalainen, S Merilaita, Biol J Linn Soc. 55Jormalainen V, Merilaita S. Differential predation on sexes affects colour polymorphism of the isopod Idotea baltica (Pallas). Biol J Linn Soc. 1995;55:45-68.


Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility
2005 1. 2005

Chartchalerm Isarankura-Na-Ayudhya
Department of Clinical Microbiology
Faculty of Medical Technology
Mahidol University
BangkokThailand

Yaneenart Suwanwong
Department of Clinical Microbiology
Faculty of Medical Technology
Mahidol University
BangkokThailand

Somchai Boonpangrak
Department of Clinical Microbiology
Faculty of Medical Technology
Mahidol University
BangkokThailand

Rachada Kiatfuengfoo
Department of Parasitology
Faculty of Medical Technology
Department of Clinical Microbiology
Faculty of Medical Technology
Mahidol University
BangkokThailand

Mahidol University
2 Prannok Rd10700Bangkok-Noi, BangkokThailand

Virapong Prachayasittikul
Department of Clinical Microbiology
Faculty of Medical Technology
Mahidol University
BangkokThailand

Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility

International Journal of Biological Sciences
142005 1. 2005Received: 2005.10.05; Accepted: 2005.11.21;146 Research paper Corresponding address: Assoc. Prof. Dr. Virapong Prachayasittikul,chimeric green fluorescent proteinzinc ionsco-transformationco-expressionmetal mobility
A significant role of zinc-binding motifs on metal mobility in Escherichia coli was explored using a chimeric metalbinding green fluorescent protein (GFP) as an intracellular zinc indicator. Investigation was initiated by cotransformation and co-expression of two chimeric genes encoding the chimeric GFP carrying hexahistidine (His6GFP) and the zinc-binding motif fused to outer membrane protein A (OmpA) in E. coli strain TG1. The presence of these two genes was confirmed by restriction endonucleases analysis. Co-expression of the two recombinant proteins exhibited cellular fluorescence activity and enhanced metal-binding capability of the engineered cells. Incorporation of the zincbinding motif onto the membrane resulted in 60-fold more binding capability to zinc ions than those of the control cells. The high affinity to metal ions of the bacterial surface influenced influx of metal ions to the cells. This may affect the essential ions for triggering important cell metabolism. A declining of fluorescent intensity of GFP has been detected on the cell expressed of zinc binding motif. Meanwhile, balancing of metal homeostasis due to the presence of cytoplasmic chimeric His6GFP enhanced the fluorescent emission. These findings provide the first evidence of real-time monitoring of intracellular mobility of zinc by autofluorescent proteins.

Introduction

Zinc is an essential trace element required for the normal growth and viability of organisms [1]. This metal ion plays critical roles in many cellular processes including cofactor of enzymes, nuclear factor and hormone. A large number of enzymes and proteins contain zinc as a structural or catalytic cofactor such as alkaline phosphatase, RNA polymerase, superoxide dismutase and zinc finger proteins. In some eukaryotes, level of intracellular zinc triggers various important cell events. Depleting of zinc ions from the cell leads to cell death and apoptosis meanwhile deficiency of zinc ions in human can cause anemia, loss of appetite, defective of immune function, loss of epithelium integrity and growth retardation [2,3]. Although zinc is not redox active under physiological conditions, excess of zinc ions can cause toxic to cells. Zinc toxicity may mediate via binding to other cation binding sites which subsequently alter the appropriate function of proteins and cofactors. The intracellular level of zinc ions is needed to be regulated to maintain viability and cell function. Precise regulatory systems have been evolved in many organisms to control the uptake, distribution, storage, and detoxification of zinc ions (for recent review see [4]). Several families of integral membrane proteins play role to transport zinc ions across the membranes into and out of the cells. In Escherichia coli, zinc homeostasis is accomplished largely through the transcriptional control of zinc uptake and efflux transporters [5,6]. In mammalian cells, the transmembrane transporters (e.g. ZIP and ZnT families) are involved in the regulatory processes. However, little is known on the molecular mechanism and location of intracellular zinc storage. Recent studies revealed that no persistent cytoplasmic pool (zinc quota) of free zinc exists in E. coli under steady-state growth conditions [7]. In eukaryotic cells, labile zinc level has been estimated at the low-nanomolar range [8]. Monitoring of intracellular zinc level usually requires the fluorescent zinc-specific probes e.g. zinquin, zinpyr-1 and TSQ [9,10,11]. Increasing of the intracellular labile zinc upon up taking from the extracellular medium can be visualized by the fluorescent intensity of cells. Depletion of intracellular pools by metal chelators e.g. TPEN (membrane-permeable) and CaEDTA (membrane-impermeable) reveals a rapid decrease of fluorescence. However, limitation has been on the high background of fluorescence, photobleaching and cell toxicity.

Herein, we explore the feasibility of using metalbinding green fluorescent proteins together with the expression of the zinc-binding motif as tools to study the metal mobility of the cells. The co-expression of the chimeric genes encoding the zinc-binding motif fused to outer membrane protein (OmpA) [12] and the chimeric GFPs [13] were performed in E. coli strain TG1. With an advantage of autofluorescence of the GFP, depletion of zinc ions and metal homeostasis were monitored in real time.

Materials and Methods

Bacterial strain and plasmids

Enzymes and chemicals

Restriction endonucleases and molecular weight marker (λ/HindIII) were obtained from New England Biolabs.

Chelating Sepharose Fast Flow gel was purchased from Pharmacia Biotech, Sweden. All other chemicals were of analytical grade and commercially available.

Co-transformation of chimeric genes

Transformation of chimeric genes into E. coli was performed according to the standard procedure. A plasmid pEVZn, which has previously been proven to express polyhistidine on the cell surface, was firstly transformed into E. coli strain TG1. These cells were grown and treated with 50 mM CaCl 2 to become the competent cells. The plasmid of either pGFPuv or pHis6GFPuv was then co-transformed into the cells. Cells were subsequently spread onto LB agar containing 100 mg/l ampicillin. Transformants those possessed greenish fluorescence (observed under UV transiluminator) were picked and subcultured onto the LB media for isolation. Plasmids were extracted from the cells by Miniprep standard protocol as described by Maniatis. Successfulness of co-transformation was verified via restriction endonucleases analysis.

Verification of co-expression of two recombinant proteins

Cells carrying these two plasmids were grown in liquid LB medium containing 100 mg/l ampicillin at 35°C with shaking for overnight. Cultures were pelleted by centrifugation then washed twice and resuspended in phosphate buffered saline (PBS; 50 mM Na 2 HPO 4 , 0.3 M NaCl, pH 7.4). Cell suspension was loaded onto the Immobilized Metal Affinity Chromatography (IMAC) charged with zinc ions. Unbound cells were removed by washing with phosphate buffer. Binding of cells to the gel was detected by determination of fluorescence emission under UV translumination and fluorescence microscope. Bound cells were eluted with 500 μl phosphate buffer containing 20 mM EDTA. Eluate was collected and subjected to microplate fluorescence reader for fluorescence measurement (excitation at 395 nm and emission at 508 nm).

Effect of zinc ions on fluorescence emission of engineered cells

Cells carrying pEVZn co-transformed with either pGFPuv or pHis6GFPuv were cultivated to allow coexpression between zinc-binding motif on the cell surface and native GFP or His6GFP in the cytoplasm. Cultures were harvested, washed, and resuspended in 5 mM HEPES buffer, 0.85 % (w/v) NaCl, pH 7.1.

Cell suspensions were adjusted to 0.5 OD measured at 600 nm. Aliquots of 50 μl cell suspension were further incubated with various concentrations (50 nM-50 mM) of ZnSO 4 in 96-well microtiter plates. Effect of zinc ions on fluorescent emission of engineered cells was investigated.

The fluorescence intensity was measured at 5, 15, 30, 45, 60, 120, 180, and 240 min using the microplate fluorescence reader.

Results and discussion

On this, we report the first evidence of using the chimeric GFP as an intracellular zinc indicator in E. coli.

Expression of the zinc-binding motif on the surface membrane clearly indicates that limitation of zinc uptake into the cell decreases the fluorescent intensity of the GFP. Meanwhile, the presence of cytoplasmic metal-binding region (His6GFP) plays an active role for metal homeostasis.

Co-expression of chimeric GFP and zinc binding motif

Co-transformation of the two chimeric genes encoding zinc-binding motif fused to outer membrane protein A (OmpA) [12] and chimeric green fluorescent protein carrying hexapolyhistidine (His6GFP) [13] in E. coli strain TG1 was performed. The presence of these two chimeric genes was verified via restriction endonucleases analysis. With respect to the presence of a unique EcoRI site on each plasmid (pEVZn and pHis6GFPuv), the extracted plasmid was cleaved by EcoRI and subsequently analyzed on an agarose gel electrophoresis. As shown in Figure 1, two corresponding bands (3.9 and 3.3 Kb) were observed as compared to the pEVZn and the pHis6GFPuv, respectively (lanes 2, 4 and 7).

To further confirm the co-expression of these two recombinant proteins, cells those exhibited green fluorescence were grown and expression of the zincbinding motif on the cell surface was investigated via binding to the immobilized metal affinity chromatography (IMAC) charged with zinc ions. Binding of engineered cells (TG1/pEVZn + pHis6GFPuv) on the IMAC gel was determined by using UV transiluminator as compared to cells expressing His6GFP intracellularly. As shown in Figure 2, the engineered cells exhibited not only a strong fluorescence as comparable to the control cell (upper panel) but these cells also provided the metal-binding capability (lower panel). Immobilized cells were further visualized via a fluorescence microscope as represented in Figure 3. All these findings inferred not only the success of co-expression but also exploring a feasibility of applying this approach as a tool for verification of metalbinding avidity of the engineered cells. Similar investigations have also been reported by using phase contrast microscopy, electron transmission and fluorescence micrograph or confocal laser scanning microscope [14,15,16].

To further calculate the metal-binding capability of the engineered cells, a linear correlation between fluorescence intensity and amount of by-standing cells on the IMAC gel was determined. Figure 4 showed such the correlation of cells expressing His6GFP intracellularly (Figure 4a) or co-expression of His6GFP and zinc-binding motif (Figure 4b). Both cases demonstrated the linear correspondence with a correlation coefficient very close to 1. In addition, the engineered cells exhibited a bit higher fluorescence emission per se than the cell expressing His6GFP. From these data, calculation could easily be taken by dividing of the fluorescence intensity of cells eluted from the gel by the amount of gel. The amounts of engineered cells and cells expressing intracellular His6GFP immobilized onto the gel were 0.98 × 10 8 cfu/ml and 0.0173 × 10 8 cfu/ml, respectively. This finding suggested that the metal-binding capability of engineered cell was approximately 60-fold higher than those of the control cells. The strong binding affinity is mainly attributable to the presence of zinc-binding motif consisting of several histidine residues throughout the bacterial surface membrane. These basidic amino acids provide more affinity to zinc ions than the polar head group of the membranous material or the peptidoglycan layer of control cells [17].

Location of metal-binding regions on fluorescence responses

To investigate whether location of zinc-binding motif affected intracellular metal mobility, the alteration in fluorescent intensity of engineered cells was monitored. For this purpose, co-transformation between pEVZn and pGFPuv was performed in a similar manner and applied for comparison. All the cells were incubated with various concentrations (50 nM-50 mM) of ZnSO 4 and the fluorescence responses were measured. As shown in Figure 5a, a gradually reduction of fluorescent emission of cells expressing zinc-binding motif on the cell surface and native GFP (TG1/pEVZn + pGFPuv) according to time was observed in the absence of zinc ions.

Low concentration of zinc ions (50 nM) had no significant effect on the fluorescent intensity while contrarily metal at higher concentrations (50 μM -50 mM) enhanced the fluorescence of the cells. In case of cells expressing zincbinding motif on the cell surface together with intracellular His6GFP, the declining of fluorescent emission in the absence of metal ions was not observed (Figure 5b).

More importantly, when various concentrations of zinc ions (50 nM -50 mM) were applied the enhancement effect on the fluorescent intensity was revealed as concentration dependent.

A plausible explanation to the discrepancy observation was the competitive binding to metal ions between the zincbinding motif on the cell surface and intracellular metalbinding region. In case of the native GFP, availability of zinc ions passing through the cellular membrane is limited by binding of the zinc-binding motif on the cell surface. We have previously reported that zinc ions enhanced the fluorescent emission of GFP in vivo [18]. Therefore, depletion of zinc resulted in a reduction of the fluorescent intensity of native GFP. Supportive evidence is that fluorescence emission of native GFP expressed in the TG1 host without expression of the zinc-binding motif on the cell surface is up to 8-10 fold higher than those of the cells carrying pEVZn and pGFPuv (data not shown). Furthermore, an alteration on colony morphology of the engineered cells is detected upon metal deprivation. This is in a good agreement with the reduction of cell viability in the presence of zinc chelator (TPEN) [11].

It is worth to state that there was no significant difference in fluorescent emission between cells expressing His6GFP and cells co-expressing zinc-binding motif in the absence of metal ions (Figure 4). The enhancement effect of zinc ions on the fluorescence emission has readily been observed even at very low amount of metal applied to the cells (Figure 5b). Explanation is that the presence of intracellular polyhistidine may act as a high affinitybinding domain capable of efficiently capturing metals from the environment, which may increase the uptake and in turn modulate the function of metal ions for the cells. Supportive evidences can be taken into accounts i) E. coli strain TG1 expressing His6GFP intracellularly provided more metal accumulation than the others [19], ii) the hexahistidine provided a high affinity toward divalent cations as has been demonstrated by the association to chelating membrane lipid [20,21] and IMAC-Zn 2+ binding [13], iii) the relative mobility of zinc ions across the cell membrane in a similar fashion was observed upon addition of a membrane-impermeable chelator e.g.

CaEDTA [10] and iv) the histidine residue plays a major role in many zinc-transport systems [22,23].

All these findings suggest not only the significant role of zinc-binding motif on the metal regulation at the cellular level but also supporting a high feasibility to apply the chimeric GFPs as an intracellular metal indicator in the future [24]. For circumstances, linear response between zinc concentrations and fluorescent intensity of the engineered cells was investigated ( Figure 6). Cells coexpressing the zinc-binding motif and His6GFP provided a linear response ranging from 0.5 μM -5 mM with the correlation coefficient greater than 0.9 (Figure 6b). However, little is known on the enhancement of the fluorescent emission upon addition of zinc or cadmium ions to the cells expressing chimeric GFPs. Further work is required to understand whether the hexapolyhistidine exert their action by mediating direct transfer of the metal ions into the chromophore region as in case of the intra or intermolecular mechanism of copper transfer from the Nterminal histidine rich region to the active site of Cu/Zn SOD [25] or by increasing the local concentration of the metal around the chromophore of the GFP.

Figures

Figure 1 .Figure 2 .
12Restriction Determination of metal-binding capability of engineered cells via fluorescent analysis.Upper panel suspension of cells expressing His6GFP intracellularly (TG1/pHis6GFPuv) and cells co-expressing zinc-binding motif on the cell surface and His6GFP in the cytoplasm (TG1/pEVZn + pHis6GFPuv). Lower panel metal-binding capability of engineered cells immobilized onto the IMAC gel charged with zinc ions.

Figure 3 .
3Metal-binding capability of engineered cells coexpressing zinc-binding motif on the cell surface and His6GFP intracellularly to the immobilized zinc ions on the IMAC gel determined by fluorescence microscope (400 X magnification).

Figure 4 .
4Linear correlation between fluorescent intensity and amount of cells expressing His6GFP intracellularly (TG1/pHis6GFPuv) (a) and engineered cells co-expressing zincbinding motif on the cell surface and His6GFP in the cytoplasm (TG1/pEVZn + pHis6GFPuv) (b).

Figure 5. Effect of various concentrations of zinc ions on fluorescence level of E. coli co-expressing zinc-binding motif on the cell surface and native GFP (a) or His6GFP (b) in the cytoplasm. * Values represented means (n ≥ 2). Symbols: • (no metal); ▼ (50 nM); ▲ (50 μM); ■(50 mM)
Conflict of interestThe authors have declared that no conflict of interest exists.
The biochemical basis of zinc physiology. B Vallee, K Falchuk, Physiol Rev. 73Vallee B, Falchuk K. The biochemical basis of zinc physiology. Physiol Rev 1993; 73:79-118.

Recognition of zinc-deficiency syndrome. A Prasad, Nutrition. 17Prasad A. Recognition of zinc-deficiency syndrome. Nutrition 2001; 17:67-69.

Depletion of endogenous zinc stores induces oxidative stress and cell death in human melanoma cells. E Rudolf, M Cervinka, Acta Medica. 47Rudolf E, Cervinka M. Depletion of endogenous zinc stores induces oxidative stress and cell death in human melanoma cells. Acta Medica (Hradec Kralove) 2004; 47:91-96.

Mammalian zinc transporters. J Liuzzy, R Counsins, Annu Rev Nutr. 24Liuzzy J, Counsins R. Mammalian zinc transporters. Annu Rev Nutr 2004; 24:151-172.

Genome-wide transcriptional response of chemostat-cultured Escherichia coli to zinc. J L Lee, J A Barrett, R K Poole, J Bacteriol. 187Lee JL, Barrett JA, Poole RK. Genome-wide transcriptional response of chemostat-cultured Escherichia coli to zinc. J Bacteriol 2005; 187:1124-1134.

Transcriptional response of Escherichia coli to external zinc. K Yamamoto, A Ishihama, J Bacteriol. 187Yamamoto K, Ishihama A. Transcriptional response of Escherichia coli to external zinc. J Bacteriol 2005; 187:6333-6340.

Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis. C Outen, T O'halloran, Science. 292Outen C, O'Halloran T. Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis. Science 2001; 292:2488-2492.

Measurement of intracellular free zinc in living cortical neurons: routes of entry. S Sensi, L Canzoniero, S Yu, H Ying, J Koh, G Kerchner, D Choi, J Neurosci. 17Sensi S, Canzoniero L, Yu S, Ying H, Koh J, Kerchner G, Choi D. Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 1997; 17:9554-9564.

A decrease in intracellular zinc level precedes the detection of early indicators of apoptosis in HL-60 cells. J Duffy, C Miller, G Rutschilling, G Ridder, M Clegg, C Keen, G Daston, Apoptosis. 6Duffy J, Miller C, Rutschilling G, Ridder G, Clegg M, Keen C, Daston G. A decrease in intracellular zinc level precedes the detection of early indicators of apoptosis in HL-60 cells. Apoptosis 2001; 6:161-172.

Depletion of intracellular zinc from neurons by use of and extracellular chelator in vivo and in vitro. C J Frederickson, S W Suh, J Y Koh, Y K Cha, R B Thompson, C J Labuda, R V Balaji, M P Cuajungco, J Histochem Cytochem. 50Frederickson CJ, Suh SW, Koh JY, Cha YK, Thompson RB, LaBuda CJ, Balaji RV, Cuajungco MP. Depletion of intracellular zinc from neurons by use of and extracellular chelator in vivo and in vitro. J Histochem Cytochem 2002; 50:1659-1662.

Differential regulation of zinc efflux transporters ZnT-1, ZnT-5 and ZnT-7 gene expression by zinc levels: a real-time RT-PCR study. S Devergnas, F Chimienti, N Naud, A Pennequin, Y Coquerel, J Chantegrel, A Favier, M Seve, Biochem Pharmacol. 68Devergnas S, Chimienti F, Naud N, Pennequin A, Coquerel Y, Chantegrel J, Favier A, Seve M. Differential regulation of zinc efflux transporters ZnT-1, ZnT-5 and ZnT-7 gene expression by zinc levels: a real-time RT-PCR study. Biochem Pharmacol 2004; 68:699-709.

Selection of cadmium specific hexapeptides and their expression as OmpA fusion proteins in Escherichia coli. M Mejare, S Ljung, L Bulow, Protein Eng. 11Mejare M, Ljung S, Bulow L. Selection of cadmium specific hexapeptides and their expression as OmpA fusion proteins in Escherichia coli. Protein Eng 1998; 11:489-494.

Construction of a chimeric histidine6-green fluorescent protein: role of metal on fluorescent characteristic. V Prachayasittikul, C Isarankura Na Ayudhya, M Mejare, L Bulow, Thammasat Int J Sc Tech. 5Prachayasittikul V, Isarankura Na Ayudhya C, Mejare M, Bulow L. Construction of a chimeric histidine6-green fluorescent protein: role of metal on fluorescent characteristic. Thammasat Int J Sc Tech 2000; 5:61-68.

Enhanced metalloadsorption of bacterial cells displaying poly-His peptides. C Sousa, A Cebolla, V De Lorenzo, Nature Biotechnol. 14Sousa C, Cebolla A, de Lorenzo V. Enhanced metalloadsorption of bacterial cells displaying poly-His peptides. Nature Biotechnol 1996; 14:1017-1020.

Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Z Xu, S Lee, Appl Environ Microbiol. 65Xu Z, Lee S. Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Appl Environ Microbiol 1999; 65:5142-5147.

Novel Zn 2+ -chelating peptides selected from a fimbria-displayed random peptide library. K Kjaergaard, M Schembri, P Klemm, Appl Environ Microbiol. 67Kjaergaard K, Schembri M, Klemm P. Novel Zn 2+ -chelating peptides selected from a fimbria-displayed random peptide library. Appl Environ Microbiol 2001; 67:5467-5473.

Binding of metals to cell envelopes of Escherichia coli K-12. T Beveridge, S Koval, Appl Environ Microbiol. 42Beveridge T, Koval S. Binding of metals to cell envelopes of Escherichia coli K-12. Appl Environ Microbiol 1981; 42:325-335.

Lighting E. coli cells as biological sensors for Cd 2+. V Prachayasittikul, C Isarankura Na Ayudhya, L Bulow, Biotechnol Lett. 23Prachayasittikul V, Isarankura Na Ayudhya C, Bulow L. Lighting E. coli cells as biological sensors for Cd 2+ . Biotechnol Lett 2001; 23:1285- 1291.

Engineering of chimeric protein for binding to metal ions. C Isarankura Na Ayudhya, Mahidol UniversityBangkok, ThailandPhD ThesisIsarankura Na Ayudhya C. Engineering of chimeric protein for binding to metal ions (PhD Thesis). Bangkok, Thailand: Mahidol University. 2000.

Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy. V Prachayasittikul, C Isarankura Na Ayudhya, T Tantimongcolwat, H J Galla, Biochem Biophys Res Commun. 326Prachayasittikul V, Isarankura Na Ayudhya C, Tantimongcolwat T, Galla HJ. Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy. Biochem Biophys Res Commun 2005; 326:298-306.

Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solidsupported lipid membrane by quartz crystal microbalance and atomic force microscopy. V Prachayasittikul, C Isarankura Na Ayudhya, L Hilterhaus, A Hinz, T Tantimongcolwat, H J Galla, Biochem Biophys Res Commun. 327Prachayasittikul V, Isarankura Na Ayudhya C, Hilterhaus L, Hinz A, Tantimongcolwat T, Galla HJ. Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid- supported lipid membrane by quartz crystal microbalance and atomic force microscopy. Biochem Biophys Res Commun 2005; 327:174-182.

Functional analysis of the Escherichia coli zinc transporter ZitB. S Lee, G Grass, C Haney, B Fan, B Rosen, Anton A Nies, D Rensing, C , FEMS Microbiol Lett. 215Lee S, Grass G, Haney C, Fan B, Rosen B, Anton A, Nies D, Rensing C. Functional analysis of the Escherichia coli zinc transporter ZitB. FEMS Microbiol Lett 2002; 215:273-278.

ZnT7, a novel mamalian zinc transporter, accumulates zinc in the Golgi apparatus. C Kirschle, L Huang, J Biol Chem. 278Kirschle C, Huang L. ZnT7, a novel mamalian zinc transporter, accumulates zinc in the Golgi apparatus. J Biol Chem 2003; 278:4096- 4102.

Chimeric green fluorescent protein-aequorin as bioluminescent Ca 2+ reporters at the single-cell level. V Baubet, H L Mouellic, A K Campbell, E Lucas-Meunier, P Fossier, P Brulet, Proc Natl Acad Sci. 97Baubet V, Mouellic HL, Campbell AK, Lucas-Meunier E, Fossier P, Brulet P. Chimeric green fluorescent protein-aequorin as bioluminescent Ca 2+ reporters at the single-cell level. Proc Natl Acad Sci USA 2000; 97:7260-7265.

A histidine-rich metal binding domain at the N terminus of Cu, Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning. A Battistoni, F Pacello, A Mazzetti, C Capo, J Kroll, P Langford, A Sansone, G Donnarumma, P Valenti, G Potilio, J Biol Chem. 276Battistoni A, Pacello F, Mazzetti A, Capo C, Kroll J, Langford P, Sansone A, Donnarumma G, Valenti P, Potilio G. A histidine-rich metal binding domain at the N terminus of Cu, Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning. J Biol Chem 2001; 276:30315-30325.